BAHAGIAN A Pengesahan Kerjasama * Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui kerjasama antara_________________________dengan____________________________

Disahkan oleh : Tandatangan : .. Nama Jawatan (Cop Rasmi) : .. : .. Tarikh : .

* Jika penyelidikan tesis/projek melibatkan kerjasama

Bahagian B Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah Tesis ini telah diperiksa dan diakui oleh: Nama dan Alamat Pemeriksa Luar : Prof. Dr. Hamidi bin Abdul Aziz Pusat Pengajian Kejuruteraan Awam, Kampus Kejuruteraan, Universiti Sains Malaysia, 14300 Nibong Tebal, Seberang Prai Selatan, Pulau Pinang.

Nama dan Alamat Pemeriksa Dalam : Prof. Ir. Dr. Mohd Azraai bin Kassim Timbalan Naib Canselor (Akademik), Pejabat Timbalan Naib Canselor, (Akademik), UTM Skudai. Prof. Madya Dr. Johan Sohaili Fakulti Kejuruteraan Awam, UTM Skudai. Nama Pemeriksa Lain (jika ada) : -

Disahkan oleh Timbalan Pendaftar di Sekolah Pengajian Siswazah:

Tandatangan : .. Nama

Tarikh : .

: ZAINUL RASHID BIN ABU BAKAR

FACULTATIVE ANAEROBIC GRANULAR SLUDGE FOR TEXTILE DYEING WASTEWATER TREATMENT

KHALIDA MUDA

A thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy (Civil Engineering)

Faculty of Civil Engineering Universiti Teknologi Malaysia

JANUARY 2010

ii

I declare that this thesis entitled Facultative Anaerobic Granular Sludge for Textile Dyeing Wastewater Treatment is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.

Signature Name Date

: . : KHALIDA MUDA : 5 January 2010

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Dedicated to my precious love

AKMAL, AIMAN & AMMAR

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ACKNOWLEDGEMENT

In the name of Allah the Most Gracious, the Most Merciful. First and foremost I am truly grateful for the blessings of Allah that gives me the strength to complete this thesis.

I would like to convey my highest gratitude to all my supervisors; Professor Dr. Mohd Razman Salim, Associate Professor Dr. Zaharah Ibrahim and Dr. Azmi Aris for their excellent supervision, encouragement, understanding and patience throughout my study. May Allah bless and reward all of them. Without them, my PhD experience would be a very difficult one. A special thanks to Dr Adibah for allowing me to use all the equipment and facilities at the Faculty of Bioscience and Bioengineering laboratories. To Dr. Arifah and Dr. Robiah of the Mathematics Department, Faculty of Science, thank you very much for assisting me on the statistical analysis. I would also like to express my appreciation to all the laboratory staff; Pak Usop, Ramli, Muzafar and Kak Ros of the Environmental Engineering Laboratory; Kak Fatimah, Awang and Yus of the Faculty Bioscience and Bioengineering laboratories, Encik Jefry of Mechanical Faculty Laboratory. I would also like to extend my thanks to all my seniors; Dr. Ismid, Kak Tim, Kak Farid, Kak Mala, Kak Fauziah, Kak Ngah, Kak Su for the continuous support, advice and encouragement during my study. To my colleagues and juniors, Shamila, Nana, Yati, Isal, Zana, Linda, Muzafar, Norly, Azlan, Zaini, Rosnani, Rosnita, Fairuzah, An and others from IPASA and MP2, thank you very much for their immeasurable friendship, motivation and support. To my dear friend, Aloes, thank you very much for all the support that you have given to me. I am sincerely indebted to my sisters, brothers and other family members especially Kak Hasmah for all the love, support and du'a. Lastly to my precious heroes, Akmal, Aiman and Ammar, thank you very much for all the unconditional love, support, sacrifice and du'a during the hard times. All of you are my reasons to continue striving and overcome all obstacles.

Special thanks to the Ministry of Science, Technology and Innovation (ScienceFund-79137), Ministry of Higher Education (FRGS-78122) and UTM (IRGS-75221) for funding this research.

ABSTRACT

Dye residuals found in textile dyeing wastewater contribute to the difficulty in treating such wastewater. Conventional biological processes failed to treat the wastewater while physico-chemical processes, although successful, are often costly in practice. Sequential anaerobic-aerobic process has been found to succeed in treating the textile wastewater. This study looks at the possibility of developing and applying facultative anaerobic granular sludge (FAnGS) in treating the wastewater in a single reactor under intermittent anaerobic and aerobic conditions. Synthetic textile wastewater which comprised of a mixture of Sumifix Navy Blue EXF, Synozol Red K-4B and Sumifix Black EXA, were used throughout the study. Different sludge and anaerobic granule mixture with the addition of specialized dye degrader microbes customized to treat dyeing wastewater were used at the initial stage. The initial development took place using a 4 L column reactor. Some of the studies were conducted in the same reactor while the remaining was conducted in a smaller scale, all under intermittent anaerobic and aerobic phases. After about 70 days of development, mature FAnGS were developed possessing excellent granules quality. The average size of the FAnGS was 2.3 1.0 mm with average settling velocity of 80 8 m/h resulting in settling velocity index (SVI) of 69 mL/g. Such properties have caused a significant increase in the biomass concentration to 7.3 0.9 g/L, which was observed to be beneficial to the performance of the system. At the end of the development process, the biogranules were able to achieve 94% of COD, 95% of ammonia and 62% of color removal. The oxygen uptake rate (OUR) /specific oxygen uptake rate (SOUR) and specific methanogenic activity (SMA) indicate the presence of facultative, anaerobic and aerobic bacteria within the granules. Six bacteria were identified within the FAnGS which include Bacillus cereus, Pseudomonas veronii, three species of Pseudomonas genus and Enterobacter sp., all are considered in the literature as dye degrader microbes. With the aid of statistical experimental design, subsequent studies showed that the microbial activity of the FAnGS and their performance in removal of organics (in terms of COD) and color were affected by several factors which include substrate concentration, pH, temperature, hydraulic retention time (HRT) and concentration of redox mediator. Interaction effects between the factors were also observed. The magnitude and the direction (positive or negative) of the effects are however dependent on the reacting conditions. Several statistical models describing the relationship between some of the variables were developed. From the study, the highest removal of color (87%) and COD (94%) were achieved by the FAnGS biomass in IFAnGSBioRec system operated with 24 hours HRT with an intermittent of anaerobic (18 hours) and aerobic (6 hours) reactions.

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ABSTRAK

Lebihan baki pewarna dalam air sisa tekstil menyumbang kepada kesukaran dalam olahan airsisa tersebut. Olahan konvensional biologi gagal mengolah airsisa ini manakala olahan kimia-fizikal, walaupun berhasil, melibatkan kos yang tinggi. Olahan berselang seli anaerobik-aerobik telah didapati berjaya mengolah airsisa tekstil. Dalam kajian ini, keupayaan menghasil dan menggunakan enapcemar granul fakultatif anaerobik (FAnGS) bagi mengolah airsisa tekstil dalam satu reaktor dengan fasa anaerobik dan aerobik secara berselang seli diterokai. Air sisa tekstil yg mengandungi campuran pewarna Sumifix Navy Blue EXF, Synozol Red K-4B dan Sumifix Black EXA digunakan sepanjang kajian. Di awal kajian, enapcemar yang berbeza, granul anaerobik dan beberapa mikrob pengurai pewarna dicampurkan dan digunakan dalam mengolah airsisa pewarna. Pembentukan granul dilakukan dalam reaktor berisipadu 4 L. Kesemua kajian yang dijalankan adalah secara berselang seli bagi fasa anaerobik dan aerobik samaada dengan menggunakan reaktor yang sama atau dalam skala yang lebih kecil. Setelah lebih kurang 70 hari, pembentukan FAnGS matang yang memiliki ciri granul yang baik berjaya dihasilkan. FAnGS yang terbentuk mempunyai saiz purata 2.3 1.0 mm dengan halaju enapan 80 8 m/j dan menghasilkan index halaju enapan (SVI) 69 mL/g. Dengan memiliki ciri-ciri tersebut, kepekatan biomas telah meningkat dengan signifikan kepada 7.3 0.9 g/L. Di akhir pembentukan granul, peratus penyingkiran terhadap permintaan oksigen biokimia (COD), ammonia dan warna adalah masing-masing 94%, 95% dan 62%. Analisis bagi kadar pengambilan oksigen (OUR)/ kadar pengambilan oksigen spesifik (SOUR) mengesahkan kehadiran bakteria fakultatif, anaerobik dan aerobik dalam granul yang dihasilkan. Enam bakteria daripada FAnGS dikenal pasti sebagai Bacillus cereus, Pseudomonas veronii, tiga spesis Pseudomonas genus dan Enterobacter sp. Dengan menggunakan kaedah rekabentuk eksperimen, kajian menunjukkan aktiviti mikrob dari FAnGS dan keupayaan menyingkirkan bahan organik (berdasarkan kepada COD) dan warna adalah dipengaruhi oleh beberapa faktor termasuk kepekatan substrat, pH, suhu, masa tahanan hidraul dan kepekatan perantara redox. Kesan interaksi yang wujud antara faktor juga telah diperhatikan. Magnitud dan arah (positif dan negatif) sesuatu kesan adalah bergantung kepada keadaan tindakbalas yang berlaku. Beberapa model statistik telah dibina menghubungkan beberapa faktor yang dikaji. Daripada kajian ini, penyingkiran tertinggi terhadap warna (87%) dan COD (94%) telah dicapai oleh biojisim FAnGS dalam system IFAnGSBioRec yang beroperasi secara tindakbalas olahan berselang seli anaerobik (18 jam) dan aerobik (6 jam) dengan masa tahanan hidraul (HRT) selama 24 jam.

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TABLE OF CONTENTS

CHAPTER

TITLE

PAGE ii iii iv v vi vii xvi xx xxx xxxiii xxxv

DECLARATION DEDICATION ACKNOWLEDGEMENT ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS LIST OF SYMBOLS LIST OF APPENDICES

INTRODUCTION 1.1 1.2 1.3 1.4 1.5 Preamble Objectives of the Study Scope of the Study Significance of the Study Organization of Thesis

1 1 3 4 5 7

BIOGRANULATION TECHNOLOGY IN WASTEWATER TREATMENT 2.1 Introduction

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2.2 2.3

Biogranulation Development of Aerobic Granules 2.3.1 2.3.2 Aerobic Granules from Aerobic Activated Sludge Aerobic Granules Seeded with Anaerobic Granular Sludge

10 12 14 15 18 18 20 21 21 23 24 25 26 27 28 29 29 30 31 33 34 34 35 36 37 38 38 39 39

2.4

Microbial Structure and Diversity of Microorganisms 2.4.1 2.4.2 Microbial Structure Microbial Diversity Size and Morphology Settleability Density and Strength Cell Surface Hydrophobicity Specific Oxygen Utilization Rate Storage Stability Exopolysaccharides Substrate Composition Organic Loading Rate Hydrodynamic Shear Force Feast and Famine Regime Hydraulic Retention Time Presence of Inorganic Composition Concentration of Dissolved Oxygen Slow Growing Organisms Settling Time

2.5

Characteristics of Aerobic Granules 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.5.6 2.5.7

2.6

Factors Affecting the Formation of Aerobic Granules 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 2.6.6 2.6.7 2.6.8 2.6.9

2.6.10 Reactor Configuration 2.6.11 Volumetric Exchange Ratio 2.7 Applications of Aerobic Granule in Wastewater Treatment Systems 2.7.1 High Strength Organic Wastewater

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Treatment 2.7.2 2.7.3 2.7.4 2.7.5 Simultaneous Organic and Nitrogen Removal Phosphorus Removal Phenol Wastewater Treatment Biosorption of Heavy Metals and Nuclear Waste 42 43 44 40

DYE DEGRADATION PROCESS 3.1 3.2 Textile Industry Characteristics of Textile Wastewater 3.2.1 3.2.2 3.3 3.4 Quantity Quality

46 46 48 49 50 52 54 57 59 60 60 62 63 65 67 68 70

Dye and Environmental Problems Treatment of Dyes 3.4.1 3.4.2 3.4.3 Biodegradation of Dyes Bacterial Degradation of Dyes Mechanisms of Biodegradation of Azo Dyes 3.4.3.1 Aerobic Dye Degradation Process 3.4.3.2 Anaerobic Dye Degradation Process 3.4.3.3 Anoxic Dye Degradation Process 3.4.4 Mineralization of Aromatic Amines The Sequential Anaerobic/Aerobic Reactor System 3.5.2 The Integrated Anaerobic/Aerobic Reactor System

3.5

Treatment System for Biodegradation of Azo Dyes 3.5.1

DEVELOPMENT OF FACULTATIVE ANAEROBIC GRANULES 4.1 Introduction

75 75

4.2

Materials 4.2.1 4.2.2 4.2.3 Wastewater Composition Granules Precursor Reactor Set-up Biological Characteristics 4.3.1.1 Morphological and Structural Observation 4.3.1.2 Microbial Activity 4.3.2 Physical Characteristics 4.3.2.1 Settling Velocity 4.3.2.2 Sludge Volume Index 4.3.2.3 Granular Strength 4.3.2.4 Biomass Concentration 4.3.2.5 Sludge Retention Time 4.3.3 4.3.4 Chemical Characteristics Removal Performance 4.3.4.1 Color 4.3.4.2 Chemical Oxygen Demand 4.3.4.3 Ammonia

76 79 81 83 86 86 86 89 91 91 91 92 92 93 94 95 95 95 96 96 99 99 103 106 108 109 110

4.3

Analytical Methods 4.3.1

4.4 4.5

Experimental Procedures Results and Discussion 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 Morphology of Facultative Anaerobic Granular Sludge Cellular Characterization of Facultative Anaerobic Granular Sludge Microbial Activity Size of the Facultative Anaerobic Granular Sludge Settling Velocity of the Facultative Anaerobic Granular Sludge Granular Strength of the Facultative Anaerobic Granular Sludge

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4.5.7 4.5.8 4.5.9 4.6

Biomass Concentration and Sludge Retention Time Mineral and Metal Content Removal Performance

113 114 117 121

Conclusions

EFFECT OF AGGREGATION AND SURFACE HYDROPHOBICITY BY SELECTED MICROBES FROM FACULTATIVE ANAEROBIC GRANULAR SLUDGE 5.1 5.2 5.3 Introduction Materials Analytical Methods 5.3.1 5.4 Chemical Oxygen Demand and Color Removal Experimental Procedures 5.4.1 5.4.2 5.4.3 5.4.4 5.4.5 5.4.6 5.4.7 Isolation Procedure of Bacteria Strain Morphological Characterization Identification of Microorganisms Isolated from Facultative Anaerobic Granular Sludge Specific Growth and Screening for DyeDegrading Bacteria Autoaggregation Assay Surface Hydrophobicity Assay Effect of Substrate Concentration, pH and Temperature on Coaggregation and Surface Hydrophobicity 5.4.8 5.4.9 5.5 2-Level Factorial Experimental Design Response Surface Methodology (Central Composite Design) Results and Discussion 5.5.1 Morphological and Cellular Characterization

123

123 124 126 126 127 127 128 131 131 132 133 134

135 137 139 139

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of Bacteria Isolation from Facultative Anaerobic Granular Sludge 5.5.2 Screening for Dye Degrader and Autoaggregator From Bacteria Strain Isolated from Facultative Anaerobic Granular Sludge 5.5.3 5.5.4 Analysis of the Isolates from Facultative Anaerobic Granular Sludge Effect of Substrate, pH and Temperature on Coaggregation and Surface Hydrophobicity 5.5.4.1 Factorial Analysis: The Main Effect of Substrate on Coaggregation 5.5.4.2 Factorial Analysis: The Main Effect of pH on Coaggregation 5.5.4.3 Factorial Analysis: The Main Effect of Temperature on Coaggregation 5.5.4.4 Factorial Analysis: The Interaction Effect on Coaggregation 5.5.4.5 Factorial Analysis: The Main Effect of Substrate on Surface Hydrophobicity 5.5.4.6 Factorial Analysis: The Main Effect of pH on Surface Hydrophobicity 5.5.4.7 Factorial Analysis: The Main Effect of Temperature on Surface Hydrophobicity 5.5.4.8 Factorial Analysis: The Interaction Effect on Surface Hydrophobicity 5.5.5 5.6 Response Surface Analysis 167 176 Conclusions 164 162 161 159 157 157 156 152 147 141 139

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THE EFFECT OF HYDRAULIC RETENTION TIME ON FACULTATIVE ANAEROBIC GRANULAR

180

SLUDGE 6.1 6.2 6.3 Introduction Materials Analytical Methods 6.3.1 6.3.2 6.3.3 6.4 6.5 Microbial Activity Physical Characteristics Removal Performances 180 182 182 186 186 186 187 188 188 189 196 202 204 207

Experimental Procedures Results and Discussion 6.5.1 6.5.2 6.5.3 6.5.4 6.5.5 6.5.6 Microbial Activity Physical Profile of the Reactor System Effect of Hydraulic Retention Time on Physical Properties of the Granular Biomass Effect of Hydraulic Retention Times on Chemical Oxygen Demand Removal Effect of Hydraulic Retention Time on Color Removal Effect of Hydraulic Retention Time on the Biokinetics Dye of Facultative Anaerobic Granular Sludge during Biodegradation of

6.6

Conclusions

212

EFFECT OF SUBSTRATE AND RIBOFLAVIN ON FACULTATIVE SLUDGE 7.1 7.2 Introduction Materials ANAEROBIC GRANULAR

214

214 215

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7.2.1 7.3 7.3.1 7.4

Granular Precursor Chemical Oxygen Demand and Color Removal

216 216 216 217 218 218

Analytical Methods

Experimental Procedures 7.4.1 7.4.2 Screening for Concentration of Redox Mediator Batch Experiment for Chemical Oxygen Demand and Color Removal Using Facultative Anaerobic Granular Sludge 7.4.3 2-Level Factorial and Central Composite Design Composite Experiment

218 221 221 223 226

7.5

Results and Discussion 7.5.1 7.5.2 Screening for Redox Concentration Factorial Design Analysis of Chemical Oxygen Demand Removal 7.5.2.1 Factorial Analysis: The Main Effect of Substrate Chemical Oxygen Demand Removal 7.5.2.2 Factorial Analysis: The Main Effect of Riboflavin on Chemical Oxygen Demand Removal 7.5.2.3 Factorial Analysis: The Interaction Effect of Substrate and Riboflavin on Chemical Oxygen Demand Removal 7.5.3 7.5.4 Central Composite Design Analysis of Chemical Oxygen Demand Removal Factorial Design Analysis of Color Removal 7.5.4.1 7.5.4.2 Factorial Analysis: Main Effect of Substrate on Color Removal Factorial Design Analysis: Main

228

230

232 234 237 241

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Effect of Riboflavin on Color Removal 7.5.4.3 7.5.5 7.6 Factorial Analysis: Interaction Effect Central Composite Design Analysis of Color Removal Conclusions 257 246 242

CONCLUSIONS AND RECOMMENDATIONS 8.1 8.2 Conclusions Recommendations

259 260 264 267 301-350

REFERENCES Appendices A-G

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LIST OF TABLES

TABLE NO. 3.1

TITLE Characteristics of textile wastewater (Bisschops and Spanjers, 2003 and Dos Santos et al., 2006a)

PAGE 52

3.2

Release of typical pollutants associated with various textile manufacturing processes (Crini, 2006 and Dos Santos et al., 2006a)

53

3.3

Advantages and disadvantages of the current methods of dye removal from industrial effluents (Robinson et al., 2000 and Crini, 2006)

56

3.4

Sequential anaerobic-aerobic treatment system for dye degradation

71-72

3.5

Integrated anaerobic-aerobic sequential treatment system for dye degradation

73-74

4.1

Sequential batch reactor system with intermittent anaerobic/aerobic/anoxic reaction phase treating different types of wastewater

77-78

4.2

List of reagents used in the experiment

79

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4.3 4.4 4.5

List of equipment used in the experiment One complete cycle of the IFAnGSBioRec The OUR levels during the aerobic reaction phase of one complete cycle

80 99 108

4.6 4.7

Characteristics of seed sludge and FAnGS Comparison of mineral content at different stages during the development of FAnGS

112 115

5.1 5.2 5.3

List of reagents used in the experiment List of equipment used in the experiment The variables and their range of high and low values used in the factorial experiment

125 126 136

5.4

Two-level fractional factorial design with three variables (in coded levels) conducted in duplicate (not in randomized order)

136

5.5 5.6

Two-level of CCD experimental run in coded units Morphological and cellular characterization of the twelve isolated bacteria from FAnGS

138 140

5.7 5.8

Characteristics and performance of the isolated bacterial from the FAnGS Taxonomic and phylogenetic characteristic of the isolates from FAnGS

142 148

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5.9

Characteristics of identified selected bacteria strains from FAnGS

149

5.10 5.11

Experimental results for 2-level factorial design analysis The P-values of the estimated main and interaction effects of variables substrates, pH and temperature on to the percentage of coaggregation and surface hydrophobicity after six hours aeration phase

151 152

5.12 5.13

Experimental results for CCD analysis Summary of the P-value of the response surface modeling analysis

168 169

5.14 6.1 6.2 6.3 6.4 6.5

Mathematical models in terms of actual factors Dye degradation process using integrated reactor system Operation steps during single cycle operation Details of experimental condition of the IFAnGSBioRec Oxidation Reduction Potential Biomass concentrations at different stages of the experiment

170 183-184 188 192 192 193

6.6 6.7

Physical properties of the granular biomass at different stage of experiment Profile of COD and color removal percentage at different stages of experiment

197 207

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6.8 6.9

Coefficient of biokinetic parameters Kinetic coefficients of FAnGS at different stages of the experiment

209 210

7.1

Experimental runs of factorial design and CCD in actual and coded values (not in random order)

220

7.2 7.3

Experimental results for factorial design analysis The P-values of the estimated main and interaction effects of substrates and riboflavin for the percentage of COD removal

224 224

7.4 7.5

Experimental results for CCD analysis Summary of the P-value of the response surface modeling analysis

233 234

7.6 7.7

Experimental results for factorial design analysis The P-values of the estimated main and interaction effects of variables substrates and redox mediator for the percentage of color removal

236 237

7.8 7.9

Experimental results for CCD analysis Summary of the P-value of the response surface modeling analysis

246 247

7.10

Mathematical models in terms of actual values

249

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LIST OF FIGURES

FIGURES NO. 1.1 2.1 Outline of the study

TITLE

PAGE 8 13

Design principles of sequencing batch reactor (Jern, 1989)

2.2

Schematic diagram of aerobic granulation developed without any carrier material (Beun et al., 1999)

16

2.3

Granulation development supported by ciliates. A: Formation of floc where the ciliates settle on other organisms or particles (Phase 1). B: Arrow shows the colonization of bacteria on the ciliate stalks (Phase 2). C: Granules grown into bigger sizes with dense core. Zooids of the ciliates stalks completely overgrown by bacterial, die and act as the backbone structure (Phase 3). D: Unstalked free swimming ciliates detach from the biofilm to escape death. Smooth and compact granules are formed. E: The surviving swarming ciliate cells get attached to the matured surface granules (shown by arrow) (Weber et al., 2007)

17

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2.4

The flowchart of the morphological and physical changes of the anaerobic granules in the process of aerobic granule formation in SBR system (Linlin et al., 2005)

18

4.1

Location of textile industry; Ramatex Industry Sdn. Bhd., Sri Gading Industrial Park, Batu Pahat and sewage treatment plant; Indah Water Konsortium Treatment Plant System, Taman Sutera, Skudai.

82

4.2

Schematic layout of the IFAnGSBioRec system (Wang et al. (2004) and Zheng et al. (2005) The IFAnGSBioRec system used in the study Preparation frame work for granule development Characterizations of FAnGS The morphological development of facultative anaerobic granular sludge (scale bar at steady-state equals to 1mm) Pictures were taken using stereo microscope with magnification of 6.3X (a) Granules developed from the activated sludge (b) Granules developed from anaerobic granules patches

84

4.3 4.4 4.5 4.6

85 87 88 100

4.7

Pictures of sludge particles during the initial stage of the experiment (a) and matured FAnGS granules at the 66 days of the experiment (b). Pictures were taken using stereo microscope with magnification of 6.3X (scale bar equals to 1 mm)

102

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4.8

FESEM microstructure observations on mature facultative anaerobic granular sludge under the magnification of 10,000K. (a) Coccoid bacteria tightly linked to one another. (b) Cavities that appear between bacteria clumped inside the granules

104

4.9

The changes on the microbial population during the process development of the FAnGS observed by gram staining procedures under microscopic magnification of 1000K (a) The sludge being dominated by the filamentous organisms. (b) Changes in the domination species within the FAnGS

105

4.10

The profile of dissolved oxygen and oxygen uptake rate in one complete cycle of the IFAnGSBioRec system () Dissolve oxygen, () Oxygen uptake rate (PI and PIII-Anaerobic phase; PII and PIVAerobic phase)

107

4.11

The relationship between the biomass concentrations retained in the reactor with the settling velocity of the FAnGS () Settling velocity; () Biomass concentration

110

4.12

The relationship between the SVI values and settling velocity of the FAnGS () SVI, () Settling velocity

111

4.13

The profile of integrity coefficient representing the granular strength of the FAnGS

112

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4.14

The profile of biomass concentration in the SBR. () MLSS, () MLVSS

114

4.15

The settling velocity profile in relation to mean cell residence time (SRT). () SVI, () SRT

115

4.16

Profile of COD removal during FAnGS development in IFAnGSBioRec system. () Influent COD, () Effluent COD, () COD removal

118

4.17

Profile of Ammonia removal during FAnGS development in IFAnGSBioRec system. () Influent ammonia, () Effluent ammonia, () Ammonia removal

119

4.18

Profile of color removal during FAnGS development in IFAnGSBioRec system. (100 ADMI 1 Platimun-Cobalt). () Influent color, () Effluent color, () Color removal

119

4.19

The removal for COD, ammonia and color in one complete cycle of the SBR system () Color, () COD, () Ammonia

120

5.1

Characterization of microbes isolated from the FAnGS granules

129

5.2

Experimental work for the investigation on the effect of substrate concentration, pH and temperature on the percentage of coaggregation and surface hydrophobicity og the mixed culture

130

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5.3 5.4

Agarose gel electrophoresis of DNA extraction Agarose gel electrophoresis of purified PCR amplification product

144 145

5.5

The pareto chart of the percentage of (a) coaggregation and (b) surface hydrophobicity after six hours of aeration phase (A: substrate; B: pH; C: temperature; : 0.1)

155

5.6 5.7

Main effects plot on the coaggregation Interaction effects plot on the coaggregation process ( Centre point)

158 159

5.8

Main effects plot of variables for the percentage of SHb

161

5.9

Interaction effect plots for the percentage of SHb ( Centre point)

165

5.10

Predicted versus actual data for (a) coaggregation and (b) surface hydrophobicity

171

5.11

(a) Contour and (b) 3D response surface plots representing relationship between pH, temperature and percentage of coaggregation

172

5.12

(a) Contour and (b) 3D response surface plots representing relationship between the concentration of substrate, pH and percentage of surface hydrophobicity

174

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5.13

(a) Contour and (b) 3D response surface plots representing relationship between the concentration of substrate, temperature and percentage of surface hydrophobicity

175

5.14

(a) Contour and (b) 3D response surface plots representing relationship between pH, temperature and percentage of surface hydrophobicity

177

6.1

Experimental analyses on the effect of HRT on granular biomass in treating synthetic textile dyeing wastewater

185

6.2

OUR profile of (a) Stage I (Aerobic phase 2.84 hours), (b) Stage II (Aerobic phase 5.84 hours) and (c) Stage III (Aerobic phase 11.84 hours)

190

6.3

OUR profile of (a) Stage IV (Aerobic phase 11.84 hours), (b) Stage V (Aerobic phase 5.84 hours), (c) Stage VI (Aerobic phase 17.84 hours)

191

6.4

Profile of biomass concentration at different stages of the experiment. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h)

195

6.5

Distribution of size particles at different stages of the experiment. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic

200

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(11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h) 6.6 Profile of sludge volume index throughout the experiment. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h) 6.6 Profile of COD removal performance of the reactor system at different stages of the experiment. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h) 6.7 Profile of COD removal performance of the reactor system at different stages of the experiment. () Influent COD; () Effluent COD, () COD removal. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h) 6.8 Profile of color removal performance of the reactor system at different stages of the experiment. () Influent color, () Effluent color, () Color removal. (100 ADMI 1 Pt-Co). Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and 205 203 191 201

xxvii

Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h) 7.1 Experimental works for the investigation on the effect of substrate concentration and redox mediator on COD and color removal via the aid of experimental design 7.2 Color removal at different concentrations of riboflavin. Absorbance at 600 nm (), absorbance at 542 nm () 7.3 The Pareto chart of COD removal for (a) anaerobic, (b) aerobic and (c) total removal (A: substrate; B: riboflavin; : 0.1) 7.4 Main effect plot of substrate and riboflavin for (a) anaerobic, (b) aerobic and (c) total COD removal 7.5 Interaction plot for the percentage of COD removal for (a) anaerobic, (b) aerobic and (c) total removal (Substrate: ____ 2633.88 m/L; ____ 866.12 mg/L; Centre point) 7.6 The relationship between substrate, riboflavin and percentage of total COD removal after 24 hours of experimental run, (a) Contour plot and (b) Responses surface plot 7.7 Pareto chart of Sumifix Navy Blue EXF removal at (a) 5 and (b) 12 hours (: 0.1; A: Substrate; B: Riboflavin) 238 235 231 229 225 222 217

xxviii

7.8

Pareto chart of Synozol Red K-4B removal at (a) 5 and (b) 12 hours (: 0.1; A: Substrate; B: Riboflavin)

239

7.9

Main effect plot of substrate and riboflavin on the color removal of Sumifix Navy Blue EXF at (a) 5 and (b) 12 hours of experiment under anaerobic condition

243

7.10

Main effect plot of substrate and riboflavin on color removal of Synozol Red K-4B at (a) 5 and (b) 12 hours of experiment under anaerobic condition

244

7.11

Interaction of variables substrate and riboflavin for Sumifix Navy Blue EXF at (a) 5 and (b) 12 hours of the experimental conditions (Substrate: ____ 2366.88 m/L ; ____ 866.12 mg/L; Centre point)

245

7.12

Interaction of variables substrate and riboflavin for Synozol Red K-4B at (a) 5 and (b) 12 hours of the experimental conditions (Substrate: ____ 2366.88 m/L; ____ 866.12 mg/L; Centre point)

245

7.13

Predicted versus actual data for Sumifix Navy Blue EXF removal at (a) 5 hours and (b) 12 hours

250

7.14

Predicted versus actual data for Synozol Red K-4B removal at (a) 5 hours and (b) 12 hours

251

7.15

(a) Contour and (b) 3D response surface plots representing relationship between the concentration of substrate, riboflavin and color removal of Sumifix Navy Blue EXF removal at 5 hours

253

xxix

(Reduced Quadratic Model) 7.16 (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Sumifix Navy Blue EXF removal at 12 hours 7.17 (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Synozol Red K-4B removal at 5 hours 7.18 (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Synozol Red K-4B removal at 12 hours 256 255 254

xxx

LIST OF ABBREVIATIONS

16S rRNA ADMI AnAHR AnFBR ANOVA AO7 AR151 BLASTn CAg CCD COD CR CSTR DB79 DGGE DNA DNT DO EBPR EPA EPS FAD FESEM FAnGS FLAA

16 subunit ribosomal ribonucleic acid American Dye Manufacturing Index Anaerobic-aerobic hybrid reactor Anaerobic fluidized bed reactor Analysis of variance Acid orange 7 Acid red 151 Basic local alignment search tool Coaggregation Central Composite Design Chemical oxygen demand (C-mmoL or mg/L or g/L) Congo red Continuous stirring tank reactor Direct Blue 79 Denaturing gradient gel electrophoresis Deoxyribonucleic acid Dinitrotoluene Dissolved oxygen (mg/L) Enhanced biological phosphorus removal Environmental Protection Act Extracellular polymeric substances Flavin adenine dinucleotide Field-Emission Scanning Electron Microscope Facultative anaerobic granular sludge Flame Atomic Absorption Spectrophotometer

xxxi

FMN FSIH GAO GDP HRT IC IFAnGSBioRec IPC IPPC LOFT MD MG MIDA MLSS MLVSS N&P N/COD NA NAD NCBI OLR ORP OUR P/COD PAO PCR Pt-Co PN POVH PS RB RDR RG

Flavin mononucleotide Fluorescent in situ hybridization Glycogen-accumulating organism Gross domestic product Hydraulic retention time (h or day) Integrity coefficient Intermittent facultative anaerobic granular sludge biological reactor Integrated pollution control Integrated Pollution and Prevention Control Lack of fit test Mixed dye Malachite green Malaysian Industrial Department Authority Mixed liquor suspended solid (mg/L or g/L) Mixed liquor volatile suspended solid (mg/L or g/L) Nitrogen & Phosphorus Nitrogen/Chemical oxygen demand Nutrient agar Nicotinamide adenine dinucleotide National Center of Biotechnology Information Organic loading rate (mg/Lday or kg/m3day) Oxidation reduction potential Oxygen uptake rate (mg/L.h) Phosphorus/Chemical oxygen demand Polyphosphate-accumulating organism Polymerase chain reaction Platinum Cobalt Exoprotein Poly(vinyl alcohol) Polysaccharide Reactive black Rotating disc reactor Residual granules (mg)

xxxii

RSM SBCR SBR SDS SG SHb SMA SOUR SRB SRT STDW STDW SVI TAA TOC UAFB UASB VER

Response surface method Sequencing biofilm configured reactor Sequencing batch reactor Sodium dodecylsulfate Settled granules (mg) Surface hydrophobicity Specific methanogenic activity Specific oxygen uptake rate (mg DO/g VSS.h) Sulfate reducing bacteria Sludge retention time (day) Synthetic textile dyeing wastewater Synthetic textile dyeing wastewater (mL or L) Sludge volume index (mL/g) Total aromatic amines Total organic carbon (C-mmoL or mg/L or g/L) Upflow anaerobic fixed bed Up-flow anaerobic sludge blanket Volumetric exchange rate

xxxiii

LIST OF SYMBOLS

Ce Ci kd M Ox Qe tc tc Vd Ve Ve Ve Vr VT Xd Xe Xe Xr Xvss XVSS1 XVSS2

COD concentration in the effluent (C-mmoL or mg/L or g/L) COD concentration in the influent (C-mmoL or mg/L or g/L) Endogenenous decay rate Biomass concentration (mg VSS/L) Theoretical chemical oxygen demand which is assume as 1.42 mg O2/ mg biomass Effluent flowrate (L/d) Cycle time of SBR operation (d) Cycle time of the SBR operation (d) Manually discharge mixture volume (L) Effluent volume in SBR operating cycle (L) Effluent volume of the SBR operating cycle (L) Working volume of the SBR system (mL or L) Working volume of the SBR system (L) Total working volume in reactor (L) Biomass concentration of manually discharged mixture (g VSS/L) Effluent volatile solid concentration (g VSS/L) Effluent volatile solid concentration (g VSS/ L) Mixed liquor volatile suspended solid in reactor (mg/L) Volatile solid concentration in the reactor system (g VSS/L) Volatile solid concentration at the beginning of operation in SBR reactor (g VSS/L) Volatile solid concentration at the end of cycle operation in SBR reactor (g VSS/L)

xxxiv

Y Yobs

Theoretical biomass yield Observed biomass yield Solid retention time (d) Biomass growth rate

xxxv

LIST OF APPENDICES

APPENDIX A B C D E Data and Calculation

TITLE

PAGE 301 314 319 320 335

Molecular Procedure of 16S Sequence Analysis Morphology of Bacteria Molecular Data Analysis Factorial Design and Response Surface Methodology Data Analysis for Coaggregation and Surface Hydrophobicity Assay

Factorial Design and Response Surface Methodology Data Analysis for COD Removal

341

Factorial Design and Response Surface Methodology Data Analysis for Color Removal

344

CHAPTER 1

INTRODUCTION

1.1

Preamble

Dyes have been one of the most demanding compounds in many industrial sectors with textile industries as the leading and biggest consumers. According to Dos Santos et al. (2003), nearly one million metric tonnes of dyes are annually produced throughout the world with azo dyes representing about 70% of the total production. Dyes are manufactured in such a way to provide long lasting attractive color design to suit a variety of customer needs. Dyes are designed with high stability towards light, heat, and sweat, and resistance to oxidizing agents (Ravi Kumar et al., 1998 and Sun and Yang, 2003). These criteria make the dyes very recalcitrant to degradation, and impose threats to the environment.

With huge consumptions and demand, treatment of textile industrial effluents presents an arduous task. There have been a number of techniques used in treating textile industrial effluent. At present, the main methods in textile wastewater treatment involve physical and chemical processes. However, such methods are often costly. Some treatments, even though capable of removing color, just merely transfer the contaminants from one form to another. The generation of concentrated

2 sludge from coagulation process, for example, would generate another environmental issue which is the disposal of sludge (Pearce et al., 2003). Excessive chemical usage in the textile wastewater treatment process might create secondary pollution problems to the environment. Due to the high cost on operation, maintenance and disposal problems, the present treatment technologies are not favored to be applied at large scale for textile industries (Ghoreishi and Haghighi, 2003). Furthermore, according to the Integrated Pollution Control (IPC) regulations, any decoloration systems involving destruction technologies that prevail as transferal of pollution from one part of the environment to another is prohibited (Willmott et.al., 1998).

The biological treatment process has been a major unit in wastewater treatment plants. However, a variety of chemicals are used in the textile industry and due to stringent effluent requirements by the authority, conventional biological treatment seems to be ineffective in treating wastewater. Furthermore, dyes are known for their complex chemical structure and mostly are of synthetic origin. Due to the recalcitrant nature of these compounds, the conventional treatment system fails to remove sufficient color and other pollutants that are present in the textile wastewaters (Stolz, 2001; Pandey et al., 2007; van der Zee and Cervantes, 2009).

Studies have shown that complete mineralization of dye compounds requires both anaerobic and aerobic biological treatment approaches (Melgoza et al., 2004). The former will cause the cleavage of the azo bond. The latter performs complete mineralization of the dye compounds to form harmless and stable byproducts.

Nowadays, there is a trend to use microorganisms in the form of aggregates as compared to suspended cells. These aggregates perform degradation process either through cell-to-cell interaction or in combination with other particulates, forming biofloc known as granules. The granular system is endowed with some characteristics of good settling ability, high concentration of microorganisms with strong and compact structure and high biomass retention that could withstand significantly higher organic loading (Morgenroth et al., 1997 and Moy et al., 2002).

3 Having such characteristics, the granular system has great advantages over conventional activated sludge.

Granules consist of millions of microorganisms that clump together with anaerobic microorganisms occupying the inner layer of the granules and with aerobic microbes at the outer layer. With the presence of both types of microorganisms within the granules, the granular system can be used for complete degradation of textile wastewater. However, studies on the applications of granular systems treating textile wastewater are apparently lacking. capability of the treatment system. Hence, more research needs to be conducted in this area to provide a better understanding on the mechanisms and

The aim of this study is to develop an effective bioprocess that is able to treat textile wastewater. The study is focused on the use of facultative anaerobic granular sludge as the treatment process. The development of facultative anaerobic granular sludge and identification of factors affecting their effectiveness in degradation process is the emphasis in this study.

1.2

Objectives of the Study

The specific objectives of this study are: i. To develop facultative anaerobic granular sludge (FAnGS) under intermittent anaerobic and aerobic reaction mode in a sequential batch reactor system with the use of synthetic textile dyeing wastewater.

4 ii. To characterize the physical, chemical and biological properties of the developed FAnGS and to identify the most suitable mixed bacteria consortia isolated from the FAnGS that are capable of being an aggregator and dye degrader.

iii.

To characterize the aggregation and surface hydrophobicity properties of the selected mixed bacteria consortia as a function of substrate concentration, pH and temperature.

iv.

To study the effect of hydraulic retention time with variation of intermittent reaction mode on the performance of FAnGS in terms of chemical oxygen demand (COD) and color removal.

v.

To investigate the effect of substrate concentration and riboflavin as the redox mediator on the performance of FAnGS in terms of COD and color removal.

1.3

Scope of the Study

This study covers the design and application of a laboratory-scale reactor system identified as Intermittent Facultative Anaerobic Granular Sludge Biological Reactor (IFAnGSBioRec). The design and operation of the reactor system are based on the sequential batch reactor system. The FAnGS is developed using synthetic textile dyeing wastewater containing a mixture of three dyes namely Sumifix Black EXA, Sumifix Navy Blue EXF and Synozol Red K-4B.

5 The matured FAnGS is characterized for its physical, chemical and biological properties. The microorganisms present in the FAnGS are identified through the application of a molecular technique and are used for further detailed investigation with respect to the aggregation and surface hydrophobicity, the important aspects of the initial mechanism that takes place in the development of FAnGS. The performance of the FAnGS in treating textile dyeing wastewater are investigated based on COD and color removal. The effects of different substrate and redox mediator concentration as well as variation of hydraulic retention times on the COD and color removal are also explored. The performance of the FAnGS in COD and color removal has also been studied with the variation on substrate and redox mediator concentration in synthetic textile dyeing wastewater (STDW). In addition to the IFAnGSBioRec, some of the experiments are conducted in serum bottles. Some of these experiments also involve the use of statistical experimental design.

1.4

Significance of the Study

Biogranular systems either anaerobic or aerobic granules have been studied for the degradation of different types of wastewater (Beun et al. 1999; Moy et al., 2002; Arrojo et al., 2004; Lemaire et al., 2007 and Chen et al. 2008a). The applications of granular system for the treatment of textile wastewater have been reported by many researchers (Razo-Flores et al., 1997; Tan et al., 2000; van der Zee, 2001a; and Dos Santos et al., 2003). However, most of the research on the degradation of dye stuff in textile wastewater is focused on the applications of anaerobic granular system. Apparently, the use of FAnGS for the dye degradation process appears to be missing. The importance of this study is therefore listed as follows;

i.

The study provides the design and procedural input of a compact laboratory-scale reactor system known as IFAnGSBioRec, fabricated

6 specifically for the formation of facultative anaerobic granular sludge for treating textile wastewater.

ii.

The study provides the procedures for the formation of FAnGS and its physico-chemical and biological characteristics.

iii.

The study provides details in relation to the effect of substrate, pH and temperature on aggregation and surface hydrophobicity of the mixed bacteria culture selected from FAnGS. The findings would provide knowledge on suitable conditions for the development of the FAnGS, customized for degradation of textile wastewater.

iv.

The study provides information regarding the most suitable combination time for anaerobic and aerobic reaction phases in the IFAnGSBioRec cycle tailored for degradation of textile dyeing wastewater.

v.

The study also provides the biokinetic parameters including biomass growth rate (), endogenenous decay rate (kd), observed biomass yield (Yobs) and theoretical biomass yield (Y) in relation to changes of HRT during textile dyeing degradation by the FAnGS.

vi.

The study also provides the effect of using different concentrations of substrate and redox mediator in relation to dye degradation by the FAnGS.

7 1.5 Organization of Thesis

The thesis is presented in eight chapters. Chapter 1 provides the overview of problems generated from the textile wastewater contamination and setbacks in effluent treatment. The chapter also points out the importance of biological treatment in degrading the dyes from the textile wastewater. The literature review is divided into two parts i.e. Chapter 2 and 3. Chapter 2 mainly discusses on the outline of the granulation process including the theoretical features of granules development, factors affecting the granulation process and also the applications of granular systems. Chapter 3 highlights the mechanisms involved in dye degradation process and the biological treatment system used for textile wastewater treatment.

Chapters 4, 5, 6 and 7 present the works that have been conducted in this study. Chapter 4 presents the study on the development and characterization of the FAnGS and also the performance of the FAnGS reactor system on COD and color removal. Chapter 5 focuses on the initial stage of the granulation process by looking into the aggregation and surface hydrophobicity of the selected mixed bacteria isolated from the FAnGS. Chapter 6 presents the effect of hydraulic retention time on the performance of COD and color removal. Chapter 7 focuses on the applications of redox mediator to enhance color removal by the FAnGS. Lastly, Chapter 8 presents the conclusions of this study. Figure 1.1 gives the flowchart illustrating the overall outline of the experimental work for this study. This chapter also provides recommendation for future research exploration in relation to the findings of this study.

8
Study on Facultative Anaerobic Granular Sludge for Textile Wastewater Treatment

Development of facultative anaerobic granular sludge for textile wastewater treatment (Refer Chapter 4)

Study on the effect of substrate concentration, pH and temperature on coaggregation and surface hydrophobicity (Refer Chapter 5)

Study on the effect of hydraulic retention time on facultative anaerobic granular sludge (Refer Chapter 6)

Study on the effect of substrate and riboflavin on facultative anaerobic granular sludge (Refer Chapter 7)

Factorial Design (Refer 5.4.8)

Central Composite Design (Refer 5.4.9)

Factorial Design (Refer 7.4.3)

Central Composite Design (Refer 7.4.3)

Data Analysis
Characterization of Facultative Anaerobic Granular Sludge Coaggregation (Refer 5.5.4.25.5.4.4 and 5.5.5) Surface Hydrophobicity (Refer 5.5.4.65.5.4.8 and 5.5.5) Biokinetic parameter (Refer 6.5.6) Removal Performance

Biological Characteristic Morphological & Structural (Refer 4.5.14.5.2) Microbial Activities (Refer 4.5.3 and 6.5.1) Characterizatio n of Microbes (Refer 5.5.15.5.3)

Physical Characteristic Settling Velocity & SVI (Refer 4.5.5)

Chemical Characteristic Mineral & Metal Content (Refer 4.5.8)

Removal Performance COD Removal (Refer 5.5.9 & 6.5.4)

COD Removal (Refer 7.5.27.5.3)

Color Removal (Refer 7.5.47.5.5)

Granular Strength (Refer 4.5.6)

Ammonia Removal (Refer 5.5.9)

Granular Biomass & SRT (Refer 4.5.7 & 6.5.2-6.5.3)

Color Removal (Refer 5.5.9 and 6.5.5)

Conclusions Figure 1.1 Outline of the study

CHAPTER 2

BIOGRANULATION TECHNOLOGY IN WASTEWATER TREATMENT

2.1

Introduction

The technology of cell immobilization has been used for decades in the environmental engineering and bioengineering fields (Liu and Tay, 2002). Microbial immobilization can be classified into three different categories which are biofilm, entrapped microorganisms and microbial aggregates. Biofilms are formed when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to all kinds of material such as plastics, polymers, ceramics, rocks, basalts, activated carbon or any other solid surface (Costerton et al., 1995 and Kwok et al., 1998).

Entrapped or encapsulation of the microorganisms is another form of microbial agglomeration where microbes can be trapped in hydrophobic gels or other types of gels such as polyacrylamide, chitosan, alginate, agar cellulose acetate and polyvinyl-alcohol (Kim et al., 2000). These gel substances confine the migration and maintain high concentration of microorganisms in the reactor system. The performance of the reactor system would be expected to reach a higher removal efficiency due to the presence of high cell concentration.

10 Granular sludge, also regarded as the special case of biofilm formation, has been successfully developed either through anaerobic conditions (Lettinga et al., 1980; Schmidt and Ahring, 1996; Zhang et al., 2007a) or aerobic conditions (Morgenroth et al., 1997, Bao et al., 2009; Shi et al., 2009). Microbial granulation is a self-immobilization community that can be formed with or without support material. It is estimated that about 900 anaerobic granular sludge systems have been successfully operated all over the world (Alves et al., 2000).

2.2

Biogranulation

Granules, defined as discrete macroscopic aggregates consist of dense microbial consortia packed with different bacterial species amounting to millions of organisms per gram of biomass (Weber et al., 2007). They are formed through selfimmobilization microorganisms which involves cell to cell interactions inclusive of biological, physical and chemical processes. According to Calleja (1984), microbial granulation is gathering of cells to form a fairly stable, contiguous, multicellular association under physiological condition.

The granulation system is first recognized in an up-flow anaerobic sludge blanket (UASB) system as anaerobic granular sludge. Since then, extensive investigations have been carried out by many researchers for the past two decades via the innovative upflow sludge bed (USB) type reactor (Bachman et al., 1985 and Lettinga et al., 1997). The application of anaerobic granulation system is relatively well known through the successful demonstration particularly in removing biodegradable organic matter from municipal and industrial wastewater (Lettinga et al., 1980; Fang and Chui, 1993; Schmidt and Ahring, 1996).

11 The success of granulation systems is related to their capacity of good settling property of the biomass without the need of a biomass carrier. This would allow high solids retention time and process stability with simple and low-cost equipment (Ahn and Richard, 2003). Large size and high density of microorganisms have led to rapid settling capacity which simplifies the separation of treated effluent from the biomass (Liu and Tay, 2004). Such characteristics enable the granular sludge to handle high liquid flows with long biomass residence times and minimal suspended solids released in the effluent (Wirtz and Dague, 1996).

Since the microorganisms within the granules are closely clumped together, this generates syntrophic associations which occur due to optimum distances between microbial associates at appropriate substrate levels. Such condition enables high and stable performance of metabolism activities (Batstone et al., 2004).

Granules also consist of extracellular polysaccharides substances (EPS) produced by the microorganisms within the granules that help to strengthen the granular structure. The presence of EPS covers the granular structure and acts as protection shield to the microbes against shock loading and toxic compound that may be present in the wastewater (Tay et al., 2005a). With such characteristics, granulation system can also be regarded as an efficient device in the removal process of xenobiotic from wastewater (Bathe et al., 2004 and Wuertz et al., 2004).

Despite the successful performance of anaerobic granular sludge systems, attention is later diverted to the development and application of aerobic granulation. This is due to several drawbacks that have been observed in the application of the anaerobic, including long start-up period, operations at relatively high temperatures and are not suitable for nutrient removal and low strength organic wastewater (Liu and Tay, 2004).

12 2.3 Development of Aerobic Granules

Development of aerobic granules has been first reported in a continuous aerobic up-flow sludge blanket reactor by Mishima and Nakamura (1991). The granules are claimed to exhibit very good settling property. Aerobic granules are compact, regular and of smooth rounded shape with an apparent outer surface which can easily be differentiated from the loose, fluffy and irregular flocs of conventional suspended sludge. Some researchers have also claimed that aerobic granules are an extension and a special case of biofilm formation (El-Mamouni et al., 1995).

Aerobic granulation system has been used for organics, nitrogen, phosphorus and toxic substances removal, especially of the high strength wastewater (Moy et al., 2002; Arrojo et al., 2004; Qin and Liu, 2006; Yi et al., 2008; Kishida et al., 2009). Bacteria normally do not aggregate naturally to each other due to repulsive electrostatic forces via the presence of negatively-charged protein compounds of the cell wall (Voet and Voet, 2004). However, under selective environmental condition, microorganisms are capable of attaching to one another and thus form aggregates. These aggregates, consisting millions of microorganisms with different functioning roles are responsible for degrading a mixture of organic compounds within the wastewater as well as removing the nutrients.

Most research and reports on aerobic granulation are developed in sequencing batch reactor (SBR) systems (Morgenroth et al., 1997; Beun et al., 1999; Hailei et al., 2006; Chen et al., 2008a; Chen et al., 2008b; Kim et al., 2008). The reaction phase can be in the condition of anaerobic, aerobic or anoxic with or without mixing depending on the purpose of the treatment process. Figure 2.1 shows the steps involved in one complete cycle of the SBR system.

13

Fill

React Idle

Aeration/mixing Draw Settle

Decanting

Figure 2.1 Design principles of sequencing batch reactor (Jern, 1989)

Aerobic granulation involves multiple-step processes engaged with both physicochemical and biological forces that make significant contributions in the granules development (Calleja, 1984; Linlin et al., 2005; Weber et al., 2007). Aerobic granules can be developed purely from activated sludge, as described by Beun et al. (1999) and they can also be developed using anaerobic granules as the seed sludge (Linlin et al., 2005). The mechanisms for development of aerobic granules from activated sludge are slightly different from the development of granules seeded with anaerobic granules. This is discussed in the following sections.

14 2.3.1 Aerobic Granules from Aerobic Activated Sludge

Aerobic granulation is not a standalone process but resulted from the integration of many aspects such as physical, chemical and biological processes and interaction with the surrounding environment. Liu and Tay (2002) explain the The first stage of the involvement of different types of physicochemical and biological forces that are responsible for the development of aerobic granules. granulation process is the cell-to-cell or cell-to-solid surface interaction initiated by diffusion of mass transfer, hydrodynamic forces of the surrounding areas, thermodynamic effects, gravitational force, as well as the competency of cells to move towards one another (Pratt and Kolter, 1998). In the second step several physical (for instance, the Van der Waals forces, surface tension, hydrophobicity, opposite charge attractions, thermodynamic of surface free energy, bridges by filamentous bacteria), chemical and biochemical (cell surface dehydration, cell membrane fussions and signals among microbial communities) attractive forces are involved in stabilizing the multicell links that are formed in the earlier step (Bossier and Verstraete, 1996 and Tchobanoglous et al., 2004). The third step is the maturing stage which involves the production of substances that facilitate the interaction of cell-to-cell and results in the development of highly organized microbial structure. During this stage there are changes in the mechanisms of metabolite production such as higher production of extracellular polymer, growth of cellular cluster, metabolite change and environmental-induced genetic effects. hydrodynamic shear forces (Chisti, 1999a). The final step in aerobic granulation involves shaping of the three dimensional aerobic granules by

Beun et al. (1999) has also described the path of aerobic granules formation. Immediately after inoculation, the reactor system is found to be dominated by bacteria and fungi. Mycelial pellets that are formed by the dominating fungi manage to retain in the reactor due to the good settling ability. Bacteria which do not hold this characteristic are discarded with the effluent. With the shear force imposed by aeration, the filaments are detached from the surface of the pellets. The pellets grow bigger and bigger until they manage to reach up to 5-6 mm in diameter. With time,

15 when the pellets have grown too big, they will be defragmented. The matured pellet start to rupture into smaller pieces when there is limitation of oxygen to penetrate into the inner parts of the pellets. The fragment of mycelial pellet acts as the immobilized matrix for the bacteria to grow and form new colonies. At this stage, the bacteria can be considered big enough to settle at faster speed and able to escape washout. The bacterial colonies grew larger and developed granules. When the granules are formed, the whole system is governed by bacterial growth. Figure 2.2 illustrates the steps in the development of aerobic granules, as explained by Beun et al. (1999).

Weber et al. (2007) have illustrated three consecutive phases of granular mechanism development with the involvement of several eukaryotic organisms. Microscopic analysis has revealed that eukaryotic organisms play a key role in aerobic granule formation seeded with sludge from municipal wastewater treatment plants. Most frequently seen, stalked ciliates of the subclass Peritrichia and occasionally, the fungi, are found to be involved in the granulation process development. Figure 2.3 shows the development of aerobic granules with the ciliates as the main foundation (Weber et al., 2007).

2.3.2 Aerobic Granules Seeded with Anaerobic Granular Sludge

Development of aerobic granules using anaerobic granular sludge as seeding material has been demonstrated by Linlin et al. (2005). Through microscopic observation, the mechanisms involving morphological and physical changes of the anaerobic granular sludge into the formation of aerobic granules in the SBR system is demonstrated in a flow chart shown in Figure 2.4. During the initial stage, the anaerobic granular seeds disintegrate into irregular smaller flocs and debris when exposed to hydrodynamic shear force during aerobic conditions. Some of these flocs and debris are washed out. The remaining flocs and debris act as a precursor that

16 initiates the growth of new aerobic granules. The hydrodynamic shear force also help in shaping the formation of the structural community of the microbial aggregates during the maturing stage (Di Iaconi et al., 2006). The optimal combination of the shear force and the growth of the microorganisms within the aggregates govern the stable structural formation of the granules (Chen et al., 2008a). The morphology of these aerobic granules is slightly different as compared to the aerobic granules developed without the presence of anaerobic granular seed sludge. Small patches of defragmented anaerobic granular seeds are clearly observed within the developed aerobic granules.

Inoculation

Pellet formation

Shear force

Granules of bacteria coloni

Lysis

Oxygen limitation

Colonisation of bacteria

Figure 2.2 Schematic diagram of aerobic granulation developed without any carrier material (Beun et al., 1999)

17

Figure 2.3

Granulation development supported by ciliates. A: Formation of floc

where the ciliates settle on other organisms or particles (Phase 1). B: Arrow shows the colonization of bacteria on ciliate stalks (Phase 2). C: Granules grow into bigger sizes with dense core. Zooids of the ciliate stalks completely overgrown by bacteria, die and act as the backbone structure (Phase 3). D: Unstalked free swimming ciliates detach from the biofilm to escape death. Smooth and compact granules area formed. E: The surviving swarming ciliate cells get attached to the matured surface granules (shown by arrow) (Weber et al., 2007)

18

Anaerobic granular seeding; regular shape, black color; D=1.1mm

Day 7

Anaerobic granular seed shrunk and disintegrated due to aerobic condition

Day 21

Yellow colored granules; dominancy by aerobic microbes, SS increased and recombined

Day 35

Steady stage granules D=1.2 mm

Day 50

Small aerobic granules appear with filamentous dominancy

Day 42

Figure***: The

Settling time decreased to 5 min; most SS were washed out

Figure 2.4

The flowchart of the morphological and physical changes of the

anaerobic granules in the process of aerobic granule formation in SBR systems (Linlin et al., 2005)

2.4

Microbial Structure and Diversity of Microorganisms

The structural and diversity of microorganisms have been one of the main focuses in a granulation study. A variety of micro-scale techniques and instrument together with molecular biotechnological approaches have been applied by researchers in order to obtain a better understanding on the interaction and mechanisms involved in the process and development of granulation systems.

2.4.1 Microbial Structure

The microscopic structure of aerobic granules have been examined using a wide range of micro-scale techniques including scanning and transmission electron

19 microscopy, confocal laser scanning microscopy combined with fluorescent in situ hybridization (FISH) and specific fluorochromes. Different arrangement either by reactor configuration or substrate utilized as the sole carbon source in media preparation or even different microorganisms specifically used in the granulation process reveal different microbial structures of the aerobic granules (Toh et al., 2003; Weber et al., 2007; Lemaire et al., 2008a).

The structure of an aerobic granule consists of different layers occupied by different types of microorganisms or substances depending on its individual function in the granulation development (Tsuneda et al., 2004; de Kreuk et al., 2005; Abreu et al., 2007). Usually the outer layer of the aerobic granule will be conquered by aerobic or obligate aerobic microbes. For example, ammonium-oxidizing bacterium Nitrosomonas spp. has been found mainly at a depth of 70 to 100 m from the granule surface (Tay et al., 2002a). At the deeper area of the aerobic granules where oxygen could not penetrate, anaerobic bacteria Bacteroides spp. is found 800 to 900 m below the granules surface (Tay et al., 2002b).

Most of the structures of the aerobic granules contain channels and cavities covering thickness areas of 900 m from the surface of the granules. The pore structures assist and create pathways for the exchange of nutrient, metabolites and oxygen moving into and out of the inner parts of the granules to the surrounding areas. However, at the depth of 300 to 500 m from the granules surface, the pores are denser (Tay et al., 2003). The pores at the depth of 400 m below the granule surface are filled with polysaccarides. Due to the dense structure, the movement of oxygen and nutrient are obstructed and has resulted in death to the microorganisms at the core of the aerobic granules. The layer of the dead microbes is located at 800 to 1000 m (Toh et al., 2003).

The optimal diameter for aerobic granules is less than 1600 m in order to obtain full utilization of the aerobic microbes. This is twice the distance from the granule surface area before reaching the anaerobic region within the aerobic granules

20 (Tay et al., 2002c). Mushroom-like structures are observed to be present in the development of aerobic granules mediated with high substrate N/COD ratios (Liu et al. 2004a). The observation of thick layers of differential mushroom-like structure has been reported earlier by Costerton et al. (1995) in biofilms of mixed bacterial communities. Complex microbial community distributes themselves to increase accessibility towards nutrients, which increases survivality and stability of its microstructure (Watnick and Kolter, 2000). A matured granule comprises of two separate layers which are a dense core zone and a fringe zone. The core zone is consisted of a mixture of dense rods and cocci bacterial cells and EPS. While, the fringe zone is represented with a loose structure that comprises of bacteria and stalked ciliates of fungal filaments (Weber et al., 2007).

2.4.2 Microbial Diversity

Molecular biotechnology has been used in the investigation of microbial diversity developed within granular biomass (Jang et al., 2003; Lemaire et al., 2008a; Zhu et al., 2008). Nitrifying, denitrifying, glycogen-accumulating bacteria and phosphorus-accumulating bacteria are among the identified bacteria that can be present in the aerobic granules operating under different experimental conditions (Tsuneda et al., 2003a; Meyer et al., 2006; Lemaire et al., 2008a).

Different types of microbes are observed to dominate within granules which are closely related to the composition of the culture media. Jiang et al. (2004a) has used denaturing gradient gel electrophoresis (DGGE) analysis techniques to study the microbial diversity of the aerobic granules. There was a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of beta-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. By using Fluorescence in situ hybridization identified by Lemaire et al. (2008a),

21 Accumulibacter spp. (a polyphosphate-accumulating organism, PAO) localized at the outermost 200 m region of the granule while Competibacter spp. (a glycogenaccumulating organism, GAO) dominated in the granule central zone, the area that could not be penetrated by the oxygen molecules.

2.5

Characteristics of Aerobic Granules

Aerobic granules are known for their characteristics that represent their outstanding features required for excellent stability and high efficiency performance of a reactor system making it an innovative modern technology for the wastewater treatment industry.

2.5.1 Size and Morphology

The size of a granule is an important parameter that can influence the performance and stability of the reactor system. Granules with bigger diameter can easily be defragmented under high shear force resulting in high biomass washout. Meanwhile, granules that are too small cannot develop good settling property which may end up with higher suspended substances in the effluent. Granules with bigger sizes will be developed in the SBR system supplied with low superficial air velocity while smaller granular sizes will be observed formed in systems aerated at higher superficial air velocity (Chen et al., 2007). Different granular sizes ranging from as small as 0.3 mm to as big as 8.8 mm in diameter possessing different granular characteristics were reported by various researchers (Dangcong et al., 1999; Tay et al., 2003; Zheng et al., 2005).

22 According to Chisti (1999a), the size of the suspended biosolids is controlled by the hydrodynamic shear force of the reactor system. The size of the aerobic granule varies depending on the balance between the growth and shear force imposed by superficial air velocity that give the hydrodynamic shear force on the newly developed granules. The observed growth of microbial aggregates is the net result of the interaction between growth and shear forces (Yang et al., 2004a). The usual reported average diameter of an aerobic granule is in the range of 0.2 mm to 5 mm (Liu et al., 2003a). Bigger size of aerobic granules has been reported with size of 710 mm (Morgenroth et al., 1997 and Wang et al., 2004). Based on the biological viability and physical properties, Toh et al. (2003), suggested that for the optimal performance and economic purposes, the operational size range for effective aerobic SBR granular sludge should be in the diameter of 1.0-3.0 mm.

The usual structure of the aerobic granule is normally spherical in shape with smooth surface areas (Peng et al., 1999; Zhu and Wilderer, 2003; Adav and Lee, 2008a). The morphology of the granules can be influenced by the type and concentration of substrate used in the media compositions. Based on the electron microscope (SEM) observations, glucose-fed granules appeared with fluffy outer surface due to the predominance of filamentous bacteria growth. On the other hand, the acetate-fed granules showed compact microstructure and smooth outer surface. The non-filamentous and rodlike bacteria were observed dominating the acetate-fed granules that are tightly linked together (Tay et al., 2001a).

Since difference type of microorganisms may predominate at different substrate concentration levels, using different concentrations of substrate in granules development may influence the structural and morphology of the developed granules. The growth rate of filamentous organisms is shown to be higher at lower substrate concentrations as compared to floc forming organisms (Schwarzenbeck et al., 2005). Fluffy and loose morphology, mainly occupied by filamentous bacteria are observed in granules cultivated at low organic loading rate (OLR). However, the granular structures evolved into smooth irregular shapes with fold, crevices and depressions at higher loading rate. The irregular structures are thought to allow better diffusion and

23 penetration of nutrients into the internal part of the granules and the same goes for the metabolites excreted out from the granules to the surrounding areas (Moy et al., 2002).

2.5.2 Settleability

Settleability of a granular sludge shows the aptitude of the granule to settle down within a specified period of time. Good settleability properties are indicated by fast and clear separation between the sludge biomass and the effluent. It can be represented by the settling velocity and sludge volume index (SVI) of a particular granule. The settling velocity of aerobic granules is in the range of 30 to 70 m/h depending on the size and structure of the granules. The settling velocity of the aerobic granules is comparable to the anaerobic granules. to those of aerobic granules. Settling velocity of activated sludge flocs is in the range of 8 to 10 m/h which is three times lower than Good settleability profile of aerobic granules is desirable in wastewater treatment plants as good settling properties facilitate a high percentage of sludge retention in the reactor system. Superior characteristic of settleability assist to maintain the stability performance of the reactor system, show high removal efficiency and can be used for wastewater with high hydraulic loading (Beun et al., 2000 and Tay et al., 2001b).

Sludge volume index represents the volume of 1 g of sludge that can settle within 30 min (Tchobanoglous et al., 2004). The SVI of conventional bioflocs is very much higher as compared to the SVI of the aerobic granules indicating very poor settling property. The bioflocs with average diameter around 70 m have the SVI value of 280 ml/g which is mainly dominated by filamentous bacteria (Tay et al., 2001b). The SVI value of flocs in an activated sludge system is observed to be above 150 mL/g (Crites and Tchnobanoglous, 1998). Granular sludge, on the other hand, has SVI of lower than 100 mL/g (Peng et al., 1999, Liu et al., 2003b and Qin et

24 al., 2004a). Most of the sludge biomass will be retained in the clarifier and can avoid washout.

2.5.3 Density and Strength

In environmental engineering, the density of microbial aggregates is frequently used to describe the strength and compactness of the microbial interaction. The observed density of microbial aggregates is the consequence of balance interaction between cells (Liu and Tay, 2004). The density of the aerobic granule is reported to be in the range of 32.2 to 110 g VSS/L (Beun et al., 2002; Arrojo et al., 2006; Di laconi et al., 2006). The biomass density of detached bioflim particles from the biofilm airlift suspension reactor is 15 g/L particles, lower compared to 48 g/L of the biomass density of aerobic granules developed in the sequential batch airlift reactor. Both of the reactor systems are operated at the same organic loading rate and same superficial air velocity (Beun et al., 1999).

The specific gravity of aerobic granules is in the range of 1.004 to 1.065 (Etterer and Wilderer, 2001, Liu et al., 2004a; Yang et al., 2004a). It is observed that when the aerobic granules grow bigger the compactness of the granules decreases revealing less solid and loose architectural assembly. In other words, granules with smaller sizes are more compact as compared to larger aerobic granules (Toh et al., 2003). Liu et al. (2004a) reported that, as the specific growth rate reduce from 0.1/day to 0.04/day, smaller aerobic granular size but with higher specific gravity (1.065 to 1.015) are formed. This indicates a compact and stronger formation of microbial structure.

Granules which can withstand high abrasion and shear force are considered as granules that possess high physical strength. The physical strength of the granules is

25 expressed as the integrity coefficient, an indirect quantitative measurement on the ability of the granules to endure the hydrodynamic shear force often imposed on to the granules during the reactor operations (Ghangrekar et al., 2005). This is measured by placing the granules in a conical flask subjected to 200 rpm agitation speed for 5 minutes. The parts that are loosely attached within the granules will be detached and known as the residual of the granules. The ratio of residual granules to the total weight of the granular sludge represents the integrity coefficient of the granules. A good granular strength is indicated with the integrity coefficient of lower than 20.

2.5.4 Cell Surface Hydrophobicity

It has been reported that the formation of biofilm and anaerobic granules are very much affected by the changes in the physico-chemical properties of cell surface (Zita and Hermansson, 1997 and Kos et al., 2003). When the bacterium approaches another bacterium, there will be energy involved as the crucial force (hydrophobic interaction) in the formation of the adhesive connection (Liu et al., 2004b).

Cell adhesion process is governed by three important forces which are electrostatic force, Van der Waals force and polymeric interaction (Azeredo and Oliveira, 2000). Since all bacteria cells have negative surface potential, electrostatic force caused repulsion between cells. Meanwhile, the Van der Waals force and polymeric interaction are the attractive forces. However, the Van der Waals force is considered as an independent environmental factor so the adhesion of cell is governed more by the electrostatic force and polymeric interaction. The polymeric interaction is enhanced by the presence of the EPS. Any changes in the EPS production and composition will be reflected by alteration of the physicochemical characteristic of the cellular surface including surface charges, hydrophobicity and other properties (Wang et al., 2006a).

26 Aerobic granulation can be regarded as a microorganism-to-microorganism self-immobilization process, in which cell hydrophobicity could be used as a decisive parameter in determining the microbial interaction and the structural compactness of aerobic granules (Liu et al., 2004b). Cell hydrophobicity is an important affinity force in cell self-immobilization which governed the mechanisms of cell adhesion (Daffonchio et al., 1995 and Kos et al., 2003). The cell hydrophobicity is believed to be the main triggering force in the initial stage of the biogranulation process and strengthen the cell-to-cell interaction (Liu et al., 2003b). Lin et al. (2003) reported that the formation of heterotrophic and nitrifying granules show nearly two fold higher of cell surface hydrophobicity as compared to the bioflocs.

2.5.5 Specific Oxygen Utilization Rate

Measurements on the activity of certain enzymes or specific products of the bacterial metabolism are among methods available to evaluate the activity of the activated sludge (Lazarova and Manem., 1995). Specific oxygen utilization rate (SOUR) is a useful parameter that can be used as an indicator of microbial activity of the microorganisms. The effect of any alteration on the physical and chemical conditions of the reactor system on microbial activity can be represented by measuring the SOUR. The value can be regarded as an important parameter that can be used to assist the permissibility of substance loading rate most importantly onto treatment of toxic chemicals such as phenol or any petrochemical substances. The SOUR values measured for aerobic granules have been reported by many researchers.

The values vary depending on various aspects such as biomass density of the microbes involved, types and concentration of substrates used as well as the conditions of experiment (Zhu and Wilderer, 2003; Ergurder and Demirer, 2005a; Liu and Tay, 2007a; Chen et al., 2008b). Granules that contain high concentrations

27 of Ca2+ seem to give lower SOUR values as compared to the non-Ca2+-accumulated granule. It has been suggested that the presence of too much of Ca2+ might give a negative effect on the bioactivity of the granules (Ren et al., 2008). Increase in the hydrodynamic shear force in terms of superficial air velocity will significantly increase the SOUR level by increasing the respiration activities of the microorganisms (Tay et al., 2001a). This may be due to the fact that the high hydrodynamic shear force causes increment on rate of the oxygen transfer between the granules and the liquid interface (Chisti, 1999b). Linear correlation is observed on the biochemical reaction between the oxygen consumption that represents the bioactivity of microbial metabolisms with the production of carbon dioxide. At high metabolism rates where high oxygen utilization occurred, less cell mass is produced and more of the substrate is converted into carbon dioxide (Tay et al., 2004).

The SOUR values are inversely related to the settling time imposed by hydraulic selection pressure onto the microorganisms in the reactor system (Qin et al., 2004b). Changes in the hydraulic selection pressure are able to regulate the respiratory activity of the microorganisms. The SOUR of microorganisms is also affected by the long storage of aerobic granules under anaerobic conditions. The SOUR value of granular sludge decreased after a long storage (Zhu and Wilderer, 2003).

2.5.6 Storage Stability

The condition during the storage of granules is another important aspect that needs to be considered. Without proper storage, granules may lose its stability and microbial activities within the granules may deteriorate. These may affect the characteristics and performances of the granules. The obvious changes that could be observed after a long storage in tap water are the changes in color of the aerobic granules which turn from brownish-yellowish (fresh aerobic granules) to gray and

28 black. However, aerobic granules stored in phosphate buffer saline solution experienced less color changes. It is expected that the color changes are due to the anaerobic metabolisms generated from stored aerobic granules (Ng, 2002 and Tay et al., 2002c).

Granules may lose its microbial activity and stability when stored for an extended period and are also closely related to the storage temperature. The granules experience an endogenous respiration and disintegration of the granules during storage at high temperatures and without any supplement of external carbon sources. Long storage periods at cool temperatures were reported to cause decrease in the granular strength as compared to fresh aerobic granules. The strength of glucose-fed and acetate-fed granules both reduced by 7-8% after four months stored at 4oC (Tay et al., 2002c and Liu and Tay, 2004).

2.5.7 Exopolysaccharides

The extracellular polysaccharides substances (EPS) are metabolic products secreted by microorganisms in the form of sticky material (Liu et al, 2004c). EPS consists of a variety of organic substances such as polysaccharide (PS), exoprotein (PN), deoxyribonucleic acid (DNA), humic acid, uronic acid and other materials (Matthew and John, 1997 and Wang et al., 2005a). They act as a buffering stratum for cells against a harsh exterior environment. Under nourished conditions, EPS would serve as carbon and energy source (Liu et al., 2002 and Zhang and Bishop, 2003). EPS are thought to act as the glue that holds the bioflocs together to form bigger aggregates (Matthew and John, 1997). They are responsible to mediate both the cells cohesion and adhesion, and play a crucial role in maintaining structural integrity of the biofilm matrix. The networking between cell and EPS would form aggregates that led to the formation of biofilm (Nielsen et al., 1997 and Zhang et al., 2007b). The aggregates combine through several binding interaction such as specific

29 proteinpolysaccharide interactions, hydrophobic interactions, hydrogen bonding, and ionic interactions (Zhang et al., 2007a).

2.6

Factors Affecting the Formation of Aerobic Granules

The formation of aerobic granules can be affected by many factors and conditions. Factors that have been identified to influence the granular formation include substrate composition, OLR, hydrodynamic shear force, feast and famine regime, feeding strategy, SRT, concentration of dissolved oxygen, reactor configuration, settling time and volumetric exchange ratio. Of all the listed factors, the major selection pressures responsible for the successful aerobic granular formations are the settling time and volumetric exchange ratio (Liu et al. 2005a). Unsuitable adjustment on the values for the settling time and the volumetric exchange ratio will lead to the failure of granules formation.

2.6.1 Substrate Composition

Different substrate composition used as the source of energy in the aerobic granules development resulted with different granular structures and microbial diversity found within the granules. The microstructure and species diversity of aerobic granules are closely related to the type of carbon source used. Glucose-fed aerobic granules exhibit a filamentous structure, while acetate-fed aerobic granules are dominated by the rodlike bacteria with very compact structure. Formation of aerobic granules is a process independent of the characteristics of the feed wastewater (Beun et al., 1999; Moy et al., 2002; Jiang et al., 2002; Arrojo et al., 2004).

30 The size of developed granules is also affected by the type of substrate presence in the media used as the feed solution. Amongst the four substrates (i.e glucose, glucose with acetate acid, acetate acid and ethanol), ethanol-fed granules appeared to be the largest and most stable granules as compared to others (Erguder and Demirer, 2005b).

Aerobic granules developed with the presence of nitrogen and carbon sources have resulted with the co-existing of heterotrophic, nitrifying and denitrifying microniches within the granules. The activities exhibited by the different The nitrifying microniches are found governed by the substrate N/COD ratio.

activity is significantly enhanced with the increase of the substrate N/COD ratio, while the heterotrophic activity is decreasing (Yang et al., 2004b). More compact granular structure is developed with high substrate N/COD ratio. The Kagg value that represents the equilibrium position of a microbial aggregation process and eq, the density of aerobic at equilibrium, shows an increasing trend as the substrate N/COD ratio increases (Liu and Tay, 2004).

2.6.2 Organic Loading Rate

Aerobic granules can be cultivated in a wide range of organic loading rates (2.5 -15 kg COD/m3day) demonstrating that the level of organic loading rates have insignificant effect on the formation of the aerobic granules (Moy et al., 2002; Liu et al., 2003d; Yang et al., 2004b). However, different concentrations of the OLR greatly influenced the characteristic of the formed granules.

At OLR of 1.68 kg COD /m3day, smaller granules are developed containing mainly bacteria microcolonies with minimal settling velocity of 9.6 m/h. When the OLR is increased to 4.2 kg COD/m3day, denser and more compact granular structure

31 with improved settling velocity are observed. The granular structure of higher OLR is dominated by bacteria with different types of morphotypes (Li et al., 2006a). Liu et al. (2003b) reported, increase in organic loading from 3 to 9 kg COD/m3day resulted in an increase of the mean granular size from 1.6 to 1.9 mm. However, the physical strength of aerobic granules decreased as the organic loading rate is increased. This is associated with the increased in the biomass growth rate that has caused reduction in the strength of the three-dimensional structure of the microbial community (Liu et al., 2003a). Tay et al. (2004) observed when the OLR was lower than 1-2 kg COD /m3day, the development of granules would be a failure. However, at too high OLR (more the 8 kg COD /m3day), unstable granules with destruction on granular strength and structural integrity would appear. Having OLR set at about 4 kg COD /m3day, stable granules are developed, characterized by high removal performance (99% of soluble COD removal) and good settleability properties with SVI of 24 mL/g MLVSS (Tay et al., 2004).

From the perspective of microbiological surface properties, a higher ratio of extracellular protein to polysaccharides showed more percentage of surfaces hydrophobic and less negative surface charge. This condition is suitable for granulation development. At higher OLR (4-12 g COD/Lday), decrease of the protein secretion and increase in the polysaccharides concentration in the sludge EPS have been observed indicating a low extracellular protein to polysaccharides ratio. This condition is inappropriate for granules formation. COD/Lday (Zhang et al., 2007b). This explains why the disintegration of granules occurred when the OLR increases between 10-12 g

2.6.3 Hydrodynamic Shear Force

Through observation, diverse characteristic with respect to the physical changes on the granular structure is developed under different pressure imposed by

32 the hydrodynamic shear force. Aerobic granules can be formed at hydrodynamic shear force in terms of superficial upflow air velocity of above 1.2cm/s in a SBR column. When the system is operated with superficial air velocity of less than 0.3 cm/s, it is only filled with bioflocs (Tay et al., 2001c).

Stable and robust granular structure for long-term reactor operation could be achieved in the system operated with high hydraulic shear force (2.4-3.2 cm/s). Granules developed at higher hydraulic shear force will be smaller in size but more regular, rounded and compact. However, large-sized filamentous granules with irregular shape and loose structure can lead to poor performance, and operation instability can occur in systems run with low hydraulic shear force (0.8-1.6 cm/s) (Chen et al., 2007). In terms of equilibrium size and size-dependent growth rate, the growth of aerobic granules are inversely related to shear force imposed to microbial community, while a high organic loading favors the growth of aerobic granules, leading to large size granules (Yang et al. 2004b). The compactness of the granular structure formed under high superficial air velocity is due to the excretion of the EPS and reduction of surface free energy (Beun et al., 1999 and Liu et al., 2004d).

The hydraulic shear force not only influences the physical structure of the formed granules but also affect the metabolic behavior of the microbes within the granules. When the superficial gas velocity increases, the respiration activity (SOUR) and the ratio of sludge polysaccharides to sludge-proteins are also increased (Tay et al., 2001c). The changes on the bioactivity among the microorganisms under high shear force are directed with the formation of larger, compact and stable granules. Mild shear force at agitation rates of 400-600 rpm exerts biological floc with higher surface hydrophobicity, larger floc size and lower sludge volume index, indicating a favorable condition for settleable floc growth (Liu et al., 2005b). From an engineering point of view, hydrodynamic shear force can be manipulated, as a control parameter, to enhance the microbial granulation process (Liu and Tay, 2002). However, Liu and Tay (2002) have added further that the hydrodynamic shear force is not a primary inducer for the aerobic granulation in the SBR.

33 2.6.4 Feast and Famine Regime

Sequential batch reactor which operates with intermittent feeding strategy can cause the microorganisms within the reactor to experience periodic starvation through feast and famine regimes. Under periodic starvation conditions, the microorganisms become more hydrophobic and high cell hydrophobicity that facilitates microbial aggregation (Bossier and Verstraete, 1996 and Liu et al., 2004b).

Periodic feast and famine regimes can be regarded as a kind of selection pressure for the microbes that could cause alteration of the cell surface properties and lead to more of cell aggregation. According to Liu et al. (2005b), high feast-famine ratio feeding applied to SBR systems may influence the characteristic of the developed granules that lead to the formation of dense and compact aerobic granules.

Prolonging famine regime means an increase in the starvation period. Under starvation conditions, bacteria become more hydrophobic and facilitate more microbial adhesion and aggregation. Since aggregation is an effective strategy of cells against starvation, utilizing prolong starvation treatment would improve the efficiency of bioaugmentation. The starvation phase has caused a decrease in surface negative charge from 0.203 to 0.023 meq/ g VSS and an increase in the relative hydrophobicity from 28.8 to 60.3% of aerobic granules. The EPS, especially protein concentrations, are well correlated with surface charge and relative hydrophobicity. It is concluded that a reasonable amount of EPS should be controlled to form and maintain aerobic granules and starvation is important in initiating the aerobic granulation (Li et al., 2006b). However, according to Liu et al. (2007b), the starvation phase in aerobic granulation is not a prerequisite since granules are developed in SBR systems operated with 1 hour cycle time operation. However, prolonged starvation times exhibited more stable granule formation. The starvation time of a system may need to be controlled in a reasonable range.

34 2.6.5 Hydraulic Retention Time

Hydraulic retention times of 4 to 6 hours have resulted in a good granulation process while higher HRTs (i.e 24 hours) could not support granulation. HRTs in the range between 2 to 12 hours are favorable for formation of stable aerobic granules with good settling properties and activity (Pan et al., 2004). At shorter HRTs (i.e 1.5 hours), the granulation process speeds up due to strong hydraulic selective pressure. However, the structures of the granules are fluffy and exhibit poor settling ability that demonstrated very unstable granules (Liu and Tay, 2008). Granules cultivated at HRT ranging between 6 to 12 hours, possess high percentage of cell hydrophobicity as compared to the granules developed with HRT of 24 hours (Liu et al., 2003c).

2.6.6 Presence of Inorganic Composition

The presence of divalent ions are reported to enhance microbial aggregation. Ca2+ probably acts as a constituent of extracellular polysaccharides or proteins used as linking materials (Grotenhuis et al., 1991a).

The additional Ca2+ has accelerated the aerobic granulation process and produced better settling and strength of aerobic granular sludge properties, and also exhibited higher polysaccharide contents (Bruus et al., 1992). Augmentation with 100 mg of Ca2+/L, speed up the granulation development from 32 days to 16 days. The Ca2+ binds to the negatively-charged groups present on the bacterial surface and the EPS to form a strong and sticky non deformable polymeric gel-like matrix (Jiang at el., 2003).

35 Ca2+-rich granules have successfully developed after 3 months operation in SBR systems supplied with media-rich Ca2+ (40 mg/L). The granules exhibited higher granular strength with increasing granular size. Calcium-fed granules with calcium content from 89.8 to 151 mg/g SS show compressive strength of 0.16-0.42 N/mm2. However, high accumulation of Ca2+ has reduced microbial activity (Ren et al., 2008).

Aerobic granules are also capable of absorbing inorganic compounds. The increased absorption of Ni2+ is observed with the increase of pH from pH 2 to pH 6 with the maximum absorption occurring at pH 6. Large quantities of K+, Mg2+ and Ca2+ are released when Ni+ is being absorbed into the granules indicating an ion exchange mechanism that take place (Xu et al., 2006).

2.6.7 Concentration of Dissolved Oxygen

The concentrations of DO in the reactor for aerobic granules development is not considered as the decisive parameter. This is because aerobic granules can be developed at DO concentrations as low as 0.7-1.0 mg/L and as high as 6 mg/L (Yang et al., 2003; Qin et al., 2004a; Tsuneda et al., 2004).

Aerobic granules developed under low DO concentrations (0.5 -2.0 mg/L) produce sludge with poor settling properties and high turbidity in the effluent. Deterioration on settling properties of the sludge is associated with excessive growth of filamentous bacteria and the formation of porous flocs (Martins et al., 2003 and Liu and Liu, 2006). High oxygen concentrations are required to obtain stable granules (de Kreuk and van Loosdrecht, 2004).

36 The percentage of dissolved oxygen that enables penetration into the granules depends on the size of the granules. The diffusivity of dissolved oxygen becomes a major limiting factor for metabolic activity when the size of the granules is more than 0.5 mm (Li and Liu, 2005).

Different types of substrate used to feed the granules formation exhibit different properties with respect to oxygen diffusivity. Acetate-fed granule sizes between 1.28-2.5 mm show oxygen diffusivity coefficients of 1.24-2.2810-9 m2/s while phenol-fed granule sizes between 0.42-0.78 mm exhibit oxygen diffusivity coefficients of 2.50-7.6510-10 m2/s. Oxygen diffusivity declines as the diameter of the granules increase. The diffusivity of oxygen for acetate-fed granules is proportional to the granular size. Meanwhile, for phenol-fed granules the diffusivity is proportional to the square of the granule diameter. The different patterns of oxygen diffusivity among the two types of granules is due to higher secretion of extracellular polymer content by phenol-fed granules, yielding lower oxygen diffusivity (Chiu et al., 2006).

2.6.8 Slow Growing Organisms

Slow growing organisms can be used to improve the stability and removal efficiency of the aerobic granular sludge as seen in the SBR system supplemented with low oxygen concentrations (Beun et al., 2002). Slow growing bacteria with a low growth yield are more capable to grow as granules than fast growing aerobic heterotrophic bacteria. Selection of slow growing bacteria can be achieved by having a long anaerobic feeding period followed by an anaerobic reaction phase (Brdjanovic et al., 1998 and de Kreuk and van Loosdrecth, 2004).

37 By having substrate concentrations with a high ratio of N/COD, the nitrifying population will be enriched. This would enhance the slow growth rate of the aerobic granules. At this condition, a smaller size of matured granules are observed with strong structure and good settleability as compared to the granules generated with high growth microbial rates (Liu et al., 2004a).

2.6.9 Settling Time

Settling time is considered as one of the most decisive factor in the formation of aerobic granules in a SBR system (Liu et al., 2005a). For a successful formation of aerobic granules, short settling time is compulsory. If the settling time is not short enough, the dominancy of granular biomass will not happen.

Aerobic granules are successfully cultivated and become dominant in SBR systems operated with 5 minutes settling time. Whereas, a mixture of aerobic granules and suspended sludge is observed in a reactor system operated at settling times longer than 10 minutes (Qin et al., 2004b). When the SBR system is operated with 2 to 5 minutes settling time, complete granular biomass is formed in the reactor. These granules contain higher EPS protein with improved cell surface hydrophobicity (McSwain et al., 2005 and Qin et al., 2004b).

A short setting time exerts a major hydraulic selection pressure that would select good settling bioparticles for granulation. Short settling time allows more sludge floc to retain in the reactor and overcome the presence of bioparticles. This will result in a successful development of the granulation process (Lin et al., 2003; Wang et al., 2004; Hu et al., 2005).

38 2.6.10 Reactor Configuration

The configuration of the reactor gives an impact on the liquid flow pattern and able to cause microbial aggregates in the reactor. In a column type reactor, allowing the air or liquid upflow, creates a relatively homogenous circulation flow and localized vortex along the reactor height (Beun et al., 2002 and Liu and Tay, 2002). This pattern forces the microbial aggregation to adopt a regular granular shape that has minimum free surface energy. The SBR should have a high H/D ratio to improve selection of granules by the difference in settling velocity (Beun et al., 1999).

Successful development of stable aerobic granules is observed in an airlift reactor operated at superficial air velocity of 1.2 cm/s. The reactor is designed with an additional draft tube placed in the reactor column. Such configuration is believed to be able to increase the shear forces built up in the reactor system compared to a bubble column reactor configuration (Liu et al., 2007b).

2.6.11 Volumetric Exchange Ratio

The volumetric exchange rate (VER) and settling time are the most important factors that determine the successful development of the aerobic granules (Liu et al., 2005c). The fraction of aerobic granules in the total biomass is proportional to the VER. The reactor is dominated by aerobic granules when the VER of the reactor system is designed with higher percentage of about 60-80%. However, when the VER is 40% or less, a mixture of aerobic granules and suspended sludge will be present. The production of EPS is stimulated significantly by high VER (Wang et al., 2006b and Zhu and Wilderer, 2003).

39 2.7 Applications of Aerobic Granules in Wastewater Treatment Systems

Aerobic granules are a rapidly emerging technology in biological wastewater treatment. The applications of aerobic granules improve process efficiency by allowing high and stable biodegradation conversion rates, efficient biomass separation and high microorganism accumulation within the aggregates. Aerobic granular sludge systems have been reported capable of treating high-strength organic wastewater and could be used to remove a wide range of pollutants. Biogranulation is capable of accommodating a wide range of treatment capacities with varying loading rates, wastewater composition and treatment goals. Biogranules could be used for a specific treatment target by developing specific granules for specific treatment. .

2.7.1 High Strength Organic Wastewater Treatment

One of the prominent characteristics of aerobic granular sludge is the ability to retain high concentrations of biomass. Such ability enables the aerobic granular sludge system to withstand and treat high strength organic loading wastewater. The feasibility on treating high strength organic loading by using this system has been demonstrated by several researchers such as Moy et al. (2002), Jiang et al. (2004b), Eckenfelder et al. (2006) and Chen et al. (2008a). An average of 3.3 mm of aerobic granules was developed with biomass density of 11.9 gVSS/L granule in systems supplied with organic loading substrate of 7.5 kg COD/m3day. When higher organic loading is applied, a balance between the hydrodynamic shear force and the level of organic loading rate used is very important for the production of more compact granules and stability of the reactor system (Buen et al., 1999).

40 Moy et al. (2002) has successfully cultivated glucose-fed aerobic granules at higher organic loading rates (615 kg COD/m3day). At OLR of 15 kg COD/m3day, the performance of the reactor system showed more than 92% of COD removal. The COD removal rate was observed high at various organic loading rates (6 to 12 kg COD/m3day) indicating high granular bioactivity with good reactor performance (Chen et al., 2008a).

Jiang et al. (2004) reported that as the concentration of phenol loading increases from 1.0 to 2.5 kg phenol/m3day, an obvious influence on the structure, activity and the metabolism of the aerobic granules was observed. The granular system shows complete phenol degradation at all different phenol concentration levels used in the study. The structure, activity and the rate of metabolisms of the granules, increased and at a peak when the concentration of phenol loading is increased from 1.0 to 2.0 kg phenol/m3day. Compact granules with good setteability properties are sustained at the loading rate until 2.0 kg phenol/m3day. However, those characteristics start to decrease when the loading is increased to more than 2.0 kg phenol/m3day. The granular structure starts to weaken when the phenol loading is increased to 2.5 kg phenol/m3day, due to the toxic effect of the phenol compound.

2.7.2 Simultaneous Organics and Nitrogen Removal

Nitrification and denitrification for complete mineralization of nitrogen compound is observed to be carried out by aerobic granules. Arrojo et al. (2004) had successfully showed the simultaneous removal of organics and nitrogen with removal efficiencies of 80% and 70% respectively. This has been conducted at high organic and nitrogen loading rates of 7 g COD/ (Ld) and 0.7g NH4+-N/(Ld), respectively, of a dairy wastewater. Beun et al. (2001) had also reported on the application of aerobic granules in the nitrification and denitrification processes. The ammonia oxidizing bacteria responsible for the nitrification process has been found

41 localized at the upper layer of the granules and down to 300 m into the granular thickness. The denitrification process takes place at the inner core of the granules where the oxygen could not penetrate. (2003). The coexistence of heterotrophic and nitrifying populations in aerobic granules has also been reported by Jang et al.

Qin and Liu (2006) have successfully used microbial granules cultivated under alternating anaerobic and aerobic reaction phases in the SBR system. The system shows an effective percentage removal for organic carbon (95-97%) and complete conversion of ammonia to nitrogen gas (99-100%). The results give an indication on the coexistence of heterotrophic, nitrifying and denitrifying populations in the microbial granules.

The activities of the nitrifying and denitrifying populations are very much affected by the N/COD ratio and the levels of dissolved oxygen. High N/COD ratios enhance the performance of the nitrifiers. However, the activities of the heterotropes population within the granules are reduced with the increase in the N/COD ratio. A sufficient level of dissolved oxygen concentration is required for a sufficient mass transfer between the liquid and granules during denitrification (Yang et al. 2003). The successful cultivation and performance of nitrifying organisms within the granular structure show that the aggregates are able to act as an effective protection for the sensitive nitrifying population. This shows that granulation systems are adaptable to treatment for different types of wastewater compositions.

2.7.3 Phosphorus Removal

Removal of phosphorus by aerobic granules has been studied by several researchers including Cassidy and Belia (2005), De Kreuk et al. (2005) and Lemaire

42 et al. (2007). Aerobic granules can be designed to treat different types of pollutants. This could be achieved through incorporating specific degrader microbes during the development of the aerobic granules. Lin et al. (2003) had successfully developed aerobic granules containing phosphorus-accumulating microbes by applying substrate P/COD ratio ranging from 1/100 to 10/100 in a SBR system. Phosphorusaccumulating microbial granules are developed with an attempt to improve the problems associated with phosphorus removal by conventional biological treatment. Phosphorus-accumulating microbial granules are reported capable of adsorbing phosphorus in the range of 1.9% to 9.3% by weight of the granules. There would be an uptake of soluble organic carbon and release of phosphate during the anaerobic stage followed by rapid phosphate uptake in the aerobic stage. This typical profile of soluble COD and PO4-P could be an indication for the typical P-accumulating characteristics. The accumulated phosphorus decreased with an increase in the substrate P/COD ratio. A 2.5% of influent P/COD ratio resulted in an accumulation of 6% of P in the granules. The same percentage of accumulated P has been reported by Cassidy and Belia (2005) with the use of influent P/COD ratio of 2.8%. Over 98% of COD and P removal and over 97% removal of N and VSS are reported in treating abattoir wastewater by using aerobic sludge granules.

De Kreuk et al. (2005) has claimed that at low oxygen concentrations (20%), simultaneous COD, N and P removal could occur since heterotrophic growth was able to develop inside the granules. Accumulibacter spp (a polyphosphateaccumulating organism, PAO) and Campetibacter spp (a glycogen nonpolyphosphate-accumulating organism, GAO) are incorporated in the development of aerobic granules in a SBR system with alternating aerobic and anaerobic reaction periods by Lemaire et al. (2008b). The PAO spp. dominated the 200 m of the outer region of the granule while the Campetibacter spp. dominated in the core zone of the granule. This aerobic granule is able to demonstrate a good phosphorus and nitrogen removal.

43 2.7.4 Phenol Wastewater Treatment

Biological degradation of phenolic wastewater is generally preferred due to lower cost and the possibility of having complete mineralization process. Degradation at low concentrations of phenol was successful but dealing with high concentrations of phenol-containing wastewater exerted toxicity effect by the substrate itself. High concentrations and fluctuations of phenol load cause breakdown of the activated sludge processes (Watanabe et al., 1999) and death to the phenoldegrading bacteria. However, phenol degradation by using aerobic granules displays an excellent degradation performance (Chou et al., 2004, Chou and Huang, 2005; Tay et al., 2005a).

Chou et al., (2004) has reported the percentage of COD removal of phenolcontaining wastewater was high with an average removal of 93.9% when the system operated at 25oC. The COD removal is higher and reached 97.9 to 98.2% when the temperature is increased from 30 to 40oC. Tay et al., (2005b), reported the phenol degradation rate of aerobic granular biomass was not affected by the increase of phenol loading rate from 0 to 2.4 kg/ m3day.

The microbial aggregation matrix within the compact granules is likely to serve as an effective protection barrier against high phenol concentrations. Due to the diffusion limitation, a substrate concentration gradient is developed at the surface of the granular matrix. This condition seems to be able to protect the microorganisms from toxicity effect by means of diluting the chemical compound below some threshold value and avoid substrate inhibition (Rittmann and McCarty, 2001 and Liu and Tay, 2004). Adav et al. (2007) reported that aerobic granules are capable of degrading phenol at 1.18 g phenol/g VSS/d. An addition of co-substrate such as glucose and ethanol is capable of treating phenol-containing wastewater (Wang et al., 2007 and Zhang et al., 2008).

44 2.7.5 Biosorption of Heavy Metals and Nuclear Waste

Aerobic granules seem to have the ability as a biosorbent towards some heavy metals that are often found in a wide variety and range of industrial wastewaters. Aerobic granules have shown capability in absorbing the heavy metals as revealed by other biomaterials such as fungus (Kumari and Abraham, 2007 and Patel and Suresh, 2007), marine algae (Daneshyar et al., 2007), waste activated sludge (Otero et al., 2003) and biosludge (Wang et al., 2006c). Granules have large surface area, high porosity and good settling properties that can be responsible for the performance as a good biosorbent.

Concentration gradient has become the driving force for the absorption of metals onto aerobic granules. The maximum biosorption capacities of individual Cu2+ and Zn2+ by aerobic granules are closely related to the initial concentrations of the metals in the reactor i.e. 246.1 mg/g and 180 mg/g respectively (Xu et al., 2006). Sun et al., (2008a) revealed that in the adsorption mechanisms, the functional groups such as alcoholic and carboxylate of the aerobic granules would be the active binding sites for the biosorption of Co2+ and Zn2+. The maximal adsorption capacity of the granules was 55.25 mg/g of Co2+ at pH 7 and 62.50 mg/g of Zn2+ at pH 5.

The ability as novel biomaterials for nuclear waste (soluble uranium) removal by the aerobic granular sludge has been demonstrated by Nancharajah et al. (2006). The effect of different pH levels (pH 1 to 8) and initial uranium concentrations (6 to 750 mg/L) are among the main focus of the study. In less than one hour, almost complete removal of uranium at concentrations ranging between 6-100 mg/L is reported. Rapid biosorption occurred in a pH range of 1 to 6 as compared to pH 7 and above. In the biosorption of uranium, an ion-exchange mechanism is observed to take place. Light metal ions such as Na2+, K2+, Ca2+ and Mg2+ are simultaneously expelled from the granules during the absorption of the uranium. The maximum biosorption capacity of uranium is reported to be at 218 2 mg/g dry granular biomass.

45 As a conclusion, granulation systems offer good removal performance of various types of pollutant. However, the behavior with respect to the removal performance, changes in the physical characteristics of the granules as well as the stability of the reactor systems varies in treating different types of pollutants. The biological activity and microbial diversity within the granules may also differ with the granules used in treating different types of wastewater. Even though there are many studies being reported on the use of anaerobic granular biomass in treating textile wastewater, the knowledge of the use of aerobic or facultative anaerobic granular biomass particularly with the application of intermittent anaerobic and aerobic reaction phase is still lacking. Therefore, this study is conducted with the objective of filling the insufficient knowledge with regard to the use of facultative anaerobic granular biomass in treating textile wastewater.

CHAPTER 3

DYE DEGRADATION PROCESS

3.1

Textile Industry

The textile industry, which is known as one of the main industrial trades, established its first textile processing factory way back in the 1500s (Neefus, 1982). The world production of natural and chemical fibres in 2003, have reached almost 63 million tonnes, 1.8 million tonnes or 2.4% more as compared to the production in 2002 which provided huge advantages for world economic values (Aizenshtein, 2004). In social terms, it gives benefit to more than 2.2 million workers through 114,000 textile-related companies with a turnover of about 198 billion Euros. In 2001, the European textile and clothing industries have contributed to about 3.4% of the EU manufacturing industrys revenue and granted 6.9% work opportunity to the citizens (IPPC, 2003).

Malaysia is also known for its textile and apparel, recognized around the world for quality and reliability. It has become one of the important industrial activities of this country. When the country started to embark on a path of exportoriented in the early 1970s, the growth on Malaysians textile and apparel industry shot up very high and accelerated with an export valued at RM 10.3 billion. This has

47 listed the textile industrial activities as the ninth largest contributor to total earnings from manufactured exports in 2007. The industry has provided more than 67,000 work opportunities through 637 licensed companies in textile production with investments of RM7.8 billion (MIDA, 2007).

Apart from running textile manufacturing as a large scale activity, quite a number of Malaysian fabric productions are conducted on small scales. One common practice from conventional cottage textile industries is that the textile effluents are mainly discharged directly into the drainage system without proper treatment. Even though textile manufacturing has added great value in terms of its economic and social aspects, it has also been identified as one of the significant environmental polluters. One of the greatest contributors to Malaysias GDP, textile industry activities have also been listed as the fourth industrial wastewater polluter discharging significant quantities of high level pollutants amounting up to 7.4% into streams. Fiber manufacturing and dyeing textile sectors are predominant for its contribution both to the economy and environmental emissions (Haroun and Azni, 2009).

The textile industry is also known for its longest and most complicated industrial chains in the manufacturing industry. It has diverse sectors in its production with respect to the raw material, processes and products equipment. The textile industry can be divided into different fragmented groups that produce and process textile-related products such as fiber, yarn or fabric for further processing into apparel, home furnishings and industrial goods. The most important stage that has been identified to contribute significant adverse impacts to environmental water pollution problems is the dyeing and finishing stage. This stage covers the bleaching, dyeing, printing and stiffening of textile products conducted in various processing steps. The purpose of the dyeing and finishing stage is to improve the serviceability and increase the durability of products to suit the demands of fashion and function (IPPC, 2000 and Savin and Butnaru, 2008). The finishing stage is also known as the wet processing. The term is given as wet due to the huge amount of water usage in most of the processes. In order to achieve the desired effect, a wide

48 range of chemicals, dyes and chemical auxiliaries are used. Textile processing

employs a variety of chemicals depending on the nature of the raw material and the desired product (Aslam et al., 2004). Any impurity from each of the processing stages will be discarded into wastewater treatment systems. The dyeing and finishing process of the textile industry has been recognized as the main contributor with respect to the amount of water usage and its quality (Savin and Butnaru, 2008).

3.2

Characteristics of Textile Wastewater

Dyes are used in many manufacturing activities as coloring agents to produce many types of goods such as textile, plastic, paper printing, leather, food and in specialized applications such as drugs, cosmetics and photochemical products (Zollinger, 1987). Among these, the textile industry is the largest consumer. Due to the high consumer demand, there are over 100,000 commercially obtainable dyes existing with more than 700,000 tonnes of dyes produced annually (McMullan et al., 2001 and Pearce et al., 2003). This scenario has resulted with the high generation of colored wastewater. The characteristics of textile wastewater for both its quantity and quality vary greatly depending on the type of raw materials, chemicals, techniques or specific process operations at the mill, equipment used and production design of the textile processes (Bisschops and Spanjers, 2003 and Dos Santos et al., 2006a). The prevailing management philosophy of a company also influences the amount of water usage.

Lacasse and Baumann (2006) reported that the textile industry gave adverse impact to the environment through its high pollutant discharge. In textile processing activities, about 10% of the chemicals in the pre-treatment and dyeing operation will remain, giving the desired design and color on the fabric. Meanwhile, the other 90% of chemicals will be discharged as textile effluent (IPPC, 2003). Due to the inefficiency of the treatment system, the textile industry has been recognized as one

49 of the main pollutant discharge. Apart from that, textile industrial activities involve large amount of water in its processes. Due to these factors, textile industrial processes have been listed as one of the top pollution contributor to the environment.

3.2.1 Quantity

One of the prominent profiles of the textile industry is its high water usage. It has been estimated that nearly one million metric tonnes of dyes is produced annually (Dos Santos et al., 2003). The average wastewater generation from a dyeing facility is estimated at between 3785-7570 million m3 per day. The dyeing and rising processes for disperse dyeing generate about 100 to 142 L of wastewater per kilogram of product. The textile industry presents its biggest impact on the environment through its primary water consumption that could reach up to 80-100 m3/ton of finishing textile (Savin and Butnaru, 2008).

Dye and pigments from printing and dyeing operations are the principal sources of color in textile effluent. Dyes and pigments are highly colored materials used in relatively small quantities (a few percent or less of the weight of the substrate) to impart color to textile materials for aesthetic or functional purposes. Desizing, scouring, bleaching, mercerizing and dyeing are the common cotton wet textile processing. Among these processes, the mercerizing and dyeing processes consume large volumes of water with a water usage of 232-308 L and 8-300 L for every kilogram of textile processed, respectively (Dos Santos et al. 2007). In typical dyeing and printing processes, 50 to 100% of the color is fixed on the fiber and the remainder is discarded in the form of spent dye baths or in the wastewater from subsequent textile-washing operations (EPA, 1997). The amount of dye lost into the wastewater depends upon the type of dyestuff used, methods and application route in the textile processing operation. It also depends on the intended color intensity that is required for each particular design (Willmott et al., 1998). During the dyeing

50 process, a range of 5-20% of acid dyes is lost in the effluent and in numerous cases these dyes are directly flushed into the receiving water body (Trovaslet et al., 2007). The release of dyes into the effluent may vary greatly leading to a wide range of total annual discharge between 30,000 and 150,000 tonnes (Faraco et al., 2009).

High content of salts in textile dyeing wastewater has been identified as a potential environmental problem. Many types of salt are either used as raw materials or produced as by-products of neutralization or other reactions in textile wet processes. Typical cotton batch dyeing operations use quantities of salt that range from 20 to 80% of the weight of goods dyed, with usual concentrations between 2,000 mg/L to 3,000 mg/L. Sodium chloride and sodium sulfate constitute the majority of the total salts used, while other salts such as magnesium chloride and potassium chloride are used as raw materials in lower concentrations (EPA, 1997).

3.2.2 Quality

Dye industrialized wastewater are normally characterized by high chemical and biological oxygen demand, suspended solids, high values of conductivity and turbidity and intense color owing to the presence of dye intermediates or residues and auxiliary chemicals added in many stages in textile processing (Mohan et al., 2007a and Miranda et al., 2009). Pollution from this manufacturing is very much related to the type and origin of the fiber involved. Textile processes with natural fibers generate higher pollution loads as compared to synthetic fibers. The usage of pesticide as preservation of natural fibers contributes to high COD concentrations in natural fibers textile processing wastewater. These pesticides are released into the wastewater during washing and scouring operations (Correia et al., 1994). Finishing processes generate wastewater containing natural and synthetic polymers and a range of other potentially toxic substances (Snowden-Swan, 1995).

51 Common characteristics of textile wastewater for cotton textile wet processing for different processing categories are shown in Table 3.1. The highest organic loading is generated from the scouring process with 8 g COD/L followed by bleaching and desizing processes. The desizing process produces the highest concentration of total solids which may come from impurity of the previous processes. The primary sources of biological oxygen demand (BOD) of the desizing process include waste chemicals or batch dumps, starch sizing agents, knitting oils, and degradable surfactants. Desizing, which is the process of removing size chemicals from textiles, is one of the industrys largest sources of wastewater pollutants in the U.S textile industry. More than 90% of the size used is disposed of in the effluent streams. The remaining 10% is recycled (EPA, 1997). Desizing processes often contribute up to 50% of BOD loading in wastewater from wet processing (Snowden-Swan, 1995). The dyeing process is a process where color is added to the fibres which normally require a large amount of water usage. Table 3.1 shows the dyeing process releasing the colored effluent with very high dye concentration of 1450-4750 of ADMI units (Bisschops and Spanjers, 2003; Dos Santos et al., 2006a; Dos Santos et al., 2007).

Table 3.2 summarizes the typical pollutant released by various associated textile manufacturing processes. Desizing, scouring and dyeing are among the processing steps that contribute to the most pollutant in textile waste stream. Source of metals such as copper, cadmium, chromium, nickel and zinc found in textile mill effluents include fiber, dyes, plumbing, and chemical impurities (IPPC, 2003). In some dyes, metals are the functional group which forms an integral part of the dye molecule. However, in most textile effluents, the metals present are simply from impurities generated during dye manufacturing.

52 Table 3.1: Characteristics of textile wastewater (Bisschops and Spanjers, 2003; Dos Santos et al., 2006a)
Process Desizing Scouring Bleaching Mercerising Dyeing Bleaching and Dyeing* COD (g/L) 4.6-5.9 8 6.7-13.5 1.6 1.1-4.6 0.2-5.5 BOD (g/L) 1.7-5.2 0.1-2.9 0.1-1.7 0.05-0.10 0.01-1.80 2.0-3.0 TS (g/L) 16.0-32.0 7.6-17.4 2.3-14.4 0.6-1.9 0.5-14.1 0.1-5.0 TDS (g/L) 4.8-19.5 4.3-4.6 0.05 pH 10--13 8.5-9.6 5.5-9.5 5-10 2-10 Color (ADMI) 694 153 1450-4750 280-2000

*Characterization of textile wastewater in Malaysia (Ahmed et al., 2005; Lau and Ismail, 2009; Ibrahim et al., 2009; Ibrahim et al. (in review))

3.3

Dye and Environmental Problems

In textile dyeing processes, dyes are lost into the effluent due to the incomplete exhaustion of dyes on the fibres. Approximately 2% of dyes are discharged directly into the aqueous effluent and 10 % are subsequently lost during the dyeing process of textile (Easton, 1995 and Pearce et al., 2003). The problem related to the dye lost is very much accelerated when the annual market for dye was reported to be more than 109 kg (Zollinger, 1987 and Dos Santos et al., 2007).

Reactive dyes are among the popular type of dyes used especially in textile dyeing processes of cellulose fibres and make up approximately 30 % of the total dye market (Kamilaki, 2000). Dyeing with reactive dyes contribute more problems due

53 to its low degree of fixation ability which causes as much as 50% of the dye lost into the wastewater stream. Losses of dye are due to the relatively low levels of dye-fiber fixation degree and to the presence of unreactive hydrolyzed dye in the dyebath. Dye hydrolysis takes place when the dye molecule reacts with water rather than with the hydroxyl groups of the cellulose. These problems become more complex with high water solubility and characteristics of the dyes involved (Pearce et al., 2003).

Table 3.2: Release of typical pollutants associated with various textile manufacturing processes (Crini, 2006 and Dos Santos et al., 2006a)
Steps in Textile Processing Main pollutants

Sizing

BOD, COD, metals, cleaning waste, size

Desizing

BOD from water-soluble sizes, lubricants, biocides, antistatic compounds, size agents, enzymes, starches, waxes, ammonia, Disinfectants and insecticides residues, NaOH, surfactants, soaps, fats, waxes, pectin, oils, sizes and antistatic agents, detergents, knitting lublicants, spin finishes, spent solvents Adsorbable organic halogens, sodium silicate or organic stabilizers, Hydrogen peroxide,high pH

Scouring/ washing

Bleaching

Mercerizing

NaOH and other salts, high pH Color, metals, salts, surfactants, sulphide, formaldehyde, toxics, organic processing aids, cationic materials, BOD, COD, sulfide, acidity/alkalinity, spent solvents BOD, COD, suspended solids, toxics, spent solvents

Dyeing

Finishing

The presence of very small amount of dyes in water even less than 1 mg/L for certain types of dyes is highly visible and undesirable (Robinson et al., 2001 and

54 Crini et al., 2007). The release of dyes into the environment has become a public concern since its presence gives adverse effects on aesthetic merit, water transparency and gas solubility in lakes, rivers and other water bodies (Banat et al., 1996). Since textile processing wastewaters typically contain dye concentration in the range of 10-200 mg/L (Pandey et al., 2007) these can be considered as highly colored and could impose aesthetic problems of the environment. Dyes that are released in the water body in the form of colored wastewater could lead to several adverse effects to the environment. Aquatic organisms are exposed to acute effects due to toxicity of the dyes. The intensity of light that penetrate into the water body will be reduced which will end up with reduction in the photosynthesis process by the plants in the aquatic ecosystem. Additionally, the dyes are chemically complex and photolytically stable which will make the dyes highly persistent in the environment for a long while. As the dye components are very difficult to be degraded, they persist in the environment. These conditions are even worse with the fact that many of the dyes are made from known carcinogens such as aromatic compounds and benzidines (Clarke and Anliker, 1980 and Brown and DeVito, 1993).

Based on the European criteria for the classification of dangerous substances, the acute toxicity effect of azo dyes is considered rather low with the LD50 values of 250-2000 mg/kg of body weight (Clarke and Anliker, 1980). However, the degradation products of the azo dyes include the aromatic amines and the impurities of the textile wastewater, are the compounds of concern due to their potential carcinogenicity (Novotny et al., 2006). Due to these circumstances, the release of dye-contained wastewater has become a source of public concern.

3.4

Treatment of Dyes

There are a number of approaches that have been used in treating textile industrial effluents. At present, the major techniques in textile wastewater treatment

55 mainly involve physical and/or chemical processes. However, such methods are often costly. Some treatments may remove color by just transferring one problem to another when those treatments produce accumulation of concentrated sludge which creates disposal problems (Pearce et al., 2003). Excessive use of chemicals in dye treatment creates secondary pollution problems to the environment.

Some of the physico-chemical techniques that have been applied for textile wastewater treatment are coagulation and flocculation (Harrelkas et al., 2009), electrokinetics coagulation (Kobya et al., 2003 and Alinsafi et al., 2005), precipitation (Solmaz et al., 2007), adsorption (Ong et al., 2008a and Sayed and Ashtoukhy, 2009), membrane filtration and nanofiltration (Miranda et al., 2009 and Unlu et al., 2009), ion exchange (Wu et al., 2008), ultrasonic mineralization (Maezawa et al., 2007) and electrolysis (De Jonge et al., 1996). Treatment using ozonation, Fentons reagent, electrochemical destruction and photocatalysis are some of the emerging techniques reported to have potential use for decolorization (Tang and Chen, 2004; Faouzi et al., 2006; Papadopoulos et al., 2007; Ay et al., 2009; Ma and Zhou, 2009). However, such technologies usually involve complicated procedures and are economically unfeasible (Chang and Lin, 2000). Table 3.3 shows some of the advantages and disadvantages of using physical and chemical technologies in color removal (Robinson et al, 2001 and Crini 2006).

The technique finally chosen for a particular textile wastewater treatment usually depends on types of dyes used in the textile processing, quantity and quality of the textile effluent, operational cost from energy consumptions, equipment, chemical requirement and as well as cost of handling the generated waste products. Environmental fate over the chosen treatment for textile wastewater is one of the significant factors that need to be taken into account. Treatment systems that can offer effective dye removal from large volumes of wastewater at low cost is a more preferable alternative in solving the textile wastewater problem and this can be achieved through biological and/or combination treatment processes.

Table 3.3: Advantages and disadvantages of the current methods of dye removal from industrial effluents (Robinson et al. 2001 and Crini , 2006)
Categories Physical treatment Technology Membranes filtration Adsorption on activated carbon Coagulation Ozonation Irradiation Ion exchange Photochemical Advantages Removes all types of dyes, produce a high quality treated effluent The most effective adsorbent, produce a high-quality treated effluent Simple, economically feasible Disadvantages High pressure, expensive, incapable of treating large volumes. Ineffective for disperse and vat dyes, require regeneration that increase in the expenses, loss of adsorbent, non-destructive process High sludge generation, handling and disposal problems

Chemical treatment

Applied in gaseous state: no alteration of volume Effective oxidation at lab scale Regeneration; no adsorbent loss No sludge production

Short half-life (20 min) Requires a lot of dissolved O2 Not effective for all dyes
Formation of byproduct

Electrochemical Breakdown compounds are non-hazardous destruction Fenton reagent Biological treatment Biomass

Very expensive
Sludge generation

Effective decolonization of both soluble and insoluble dyes

Biosorbents Peat Chitin and chitosan

Low operating cost, good efficiency and selectivity, no toxic Slow process, performance depends on some effect on microorganisms environmental conditions such as temperature, pH, salts Good adsorbent due to cellular structure Requires long retention time Low cost, abundant, renewable, biodegradable resources Resulted with pressure drop in sorption columns, cannot be used as insoluble sorbent under acidic conditions

56

57

Bioremediation using microbial biocatalysts to reduce the dyes present in the effluent offer potential advantages over physico-chemical processes. Such a system has become the main focus of recent research. However, many aspects have to be investigated in order to really gain advantages over the system especially when it comes to applying the treatment system in-situ. Each of the technique mentioned has its own limitations. Complete dye degradation seems to be difficult to achieve if the treatment only depends on a single treatment process. At present, a combination of different treatment techniques is being practiced in order to achieve complete mineralization of dyes.

3.4.1

Biodegradation of Dyes

Biodegradation of dyes means using a biological approach in decolorizing the dyes. It can be carried out using bacteria, algae or fungi. Advantages achieved by using biological approaches have been claimed by many researchers either by having partial or complete degradation of dyes (Kudlich et al., 1999; Chen et al., 2005; Frijters et al., 2006; Dos Santos et al., 2007; Mohan et al., 2007a; Ertugrul et al., 2008). Effective dye removal from large volumes of wastewater at a considerable low cost as compared to other techniques can be obtained through biological approaches. Furthermore, biodegradation does not contain complicated procedures (Pearce et al., 2003).

Microalgae which acted as the primary producers to the aquatic food chains were found to be competent as an ideal biosorbent for color removal from textile wastewater (Daneshvar et al., 2007). However, its application was limited due to low chemical and heat resistance (Ertugrul et al., 2008). Due to the advantages of using bacterial in dye degradation, extensive research have been conducted for the

58 past two decades involving the isolation and identification of bacteria capable of degrading various types of dyes (Rai et al., 2005).

The application of fungi is influenced by the bioadsorption capacity of the biomass. The absorbed dye compounds are degraded by the powerful extracellular ligninolytic enzymatic system (Dias et al., 2007). However, fungi are ineffective for the removal of reactive dyes. Reactive dyes are usually changed into a hydrolyzed form, losing its aptitude to bind to cellulose. In this condition, these dyes may no longer be successfully absorbed by the fungal biomass. The regeneration process by extraction with methanol is required to regain back the absorption capacity. However, the regeneration process may slightly reduce the percentage of absorption (Carliell et al., 1994).

Pretreatment process is required in order to increase the biomass absorption capacity. The pretreatment methods may include autoclaving, contacting with The chemicals, either organic chemicals (formaldehyde) or inorganic chemicals (natrium oxide, calcium chloride, hydrogen sulfide) (Fu and Viraraghavan, 2001). pretreatment process may increase the cost of the operation set-up and increase the operating time. The application of fungi in a large scale system has become a limitation since high growth of fungi may inhibit the growth of other useful microorganisms. Furthermore, fungi require low pH levels for optimum activity and need long hydraulic retention times for high dye removal (Chen et al., 2003). The main principle of dye removal using algae and fungi depends on the adsorption rather than the degradation process. As a result, the dyes still remain in the environment (Wang et al., 2009a). bacteria. Due to the drawbacks in using algae and fungi as the biodegradation agent, many studies have focused on the biodegradation process by

In recent reports, a number of studies have focused on the immobilized microorganisms able to decolorize textile wastewater (Kornaros and Lyberatos, 2006; Sirianuntapiboon et al., 2007; Somasiri et al., 2008; Sun et al., 2008b; Zhu et

59 al., 2008). The immobilization of dye-removing microorganisms provides important advantages including degradation at higher dye concentrations without lost of cell viability, protective environment for the dye degradation activities against changes in temperature, pH and effect of toxic compounds that co-exists in the wastewater (Ertugrul et al., 2008).

3.4.2

Bacterial Degradation of Dyes

Isolation of decolorizing bacteria started in the 1970s with cultured bacteria Bacillus subtilis able to degrade azo dyes (Horitsu et al., 1977), followed by isolation of Aeromonas hydrophila (Idaka et al., 1978) and Bacillus cereus (Wuhrmann et al, 1980). Nowadays, numerous capable dye degrader bacteria have been reported (Chen et al., 2003; Xu et al., 2007; Hsueh and Chen, 2007; Moosvi and Madamwar, 2007; Dave and Dave, 2009). A bacterial strain, Citrobacter sp. CK3, isolated from activated sludge from a textile paper mill was found capable of degrading Reactive Red 180 with 95% color removal within 36 hour incubation under anaerobic condition. It also exhibited high tolerance to dye concentration up to 1000 mg/L (Wang et al., 2009a). Some of the isolates are capable of degrading a broad spectrum of dyes which include azo, anthraquinone and triphenylmethane dyes such as Aeromonas hydrophila (Ren et al., 2006), Bacillus cereus strain DC11 (Deng et al., 2008) and Bacillus thuringiensis (Dave and Dave, 2009).

Most of the researches on color degradation mechanisms have been conducted and focused on the biotransformation of azo dyes, as, among the 10,000 dyes applied in textile processing industries, 60-70% are azo compounds (van der Zee, 2003a). Hence, most study in relation to dye degradation has been focused more on the azo dyes. Azo dyes are molecules with one or more azo (N=N) bridges linking substituted aromatic structures (Carliell et al., 1995). However, there have been several reports focusing on other types of dyes such as degradation of

60 anthraquinone dyes (Lee and Pavlostathis, 2004 and Trovaslet et al., 2007), phtalocyanine dyes (Nilsson et al., 2006) and triphenylmethane dyes (Shedbalkar et al., 2008).

3.4.3

Mechanisms of Biodegradation of Azo Dyes

Biodegradation

of

azo

dyes

can

occur

under

aerobic,

anaerobic

(methanogenic), and anoxic conditions by many different trophic groups of bacteria (Pandey et al., 2007). Biodegradation of azo dyes can occur as a direct degradation process through the presence of enzymes or as an indirect mechanism process. The indirect mechanism process involves the presence of other substances that could aid the degradation process. Many research studies have been concentrated under anaerobic degradation process due to the higher removal percentage of dye degradation as compared to aerobic condition.

3.4.3.1 Aerobic Dye Degradation Process

Aerobic biodegradation of azo dyes involve enzymatic reduction process. In this mechanism, enzymes play an important role in transferring reducing equivalents from the oxidation of organic substances or from the coenzyme to the azo dyes. Azo reductase is a specialized azo dye reducing enzyme found in some aerobic and facultative bacteria that are able to degrade simple molecular structure of the azo dye as sole carbon and energy sources (Zimmermann et al., 1984). Since the enzymes react specifically to the dye molecule, the reaction is known as specific enzymatic reaction (Blumel and Stolz, 2003 and Chen et al., 2004).

61 Pseudomonas aeroginosa can decolorize a few types of azo dyes under aerobic condition with the presence of external carbon sources (Nachiyar and Rajkumar, 2003). An obligate aerobic strain, Shingomonas 1CX, is able to grow on Acid Orange 7 (AO7) and used it as the source of energy, carbon and nitrogen (Coughlin et al, 2003). However, these bacterial strains could only cleave the N=N bond of a simple dye structure and utilize the amines as the source of carbon and energy for bacterial growth but not on complex azo dye structure. Xenophilus azovorans KF46 could utilize azo dye carboxy-orange I (Zimmermann et al., 1982) but this strain could not grow on more complex dye structure such as analogous sulfonated dyes, Acid Orange 20 and Acid Orange 7.

The azo dye, Acid red 151 (AR151) was successfully degraded under aerobic condition when this dye acted as the sole carbon source for microorganisms in a sequencing batch biofilter with a porous volcanic rock. The system showed a high percentage of color removal (99%) with initial concentration of 50 mg/L of AR151 (Bruiton et al., 2004). Arora et al. (2007) reported extensive degradation (95-98%) of monoazo disperse dye under shaking aerobic condition by bacterial strain Bacillus firmus isolated from local sewage.

Azo dye degradation has also been observed to occur under microaerophilic conditions (Sandhya et al., 2005; Xu et al., 2007; Franciscon et al., 2009; Khalid et al., 2008, Elisangela et al., 2009). Microaerophilic condition is where the percentage of oxygen is only 5% (Engelkirk et al., 1992). Several azo dyes were degraded in sequential microaerophilic-aerobic treatment condition by a facultative Klebsiella sp. Strain VN-31 with 94% of color removal (Franciscon et al., 2009). Another facultative Staphylococcus arlettae bacterium isolated from an activated sludge process of the textile industry, has successfully decolorized four different azo dyes under micraerophilic condition with percentage dye removal of more the 97% (Elisangela et al., 2009). However, since the aerobic biodegradation of azo dyes is restricted to the presence of azoreductase and only suitable for the degradation of simple molecule dye structures. This has led many researchers to conclude that azo

62 dyes are persistent under aerobic conditions (Pagga and Taegar, 1994 and Ong et al., 2008).

3.4.3.2 Anaerobic Dye Degradation Process

Dye degradation under methanogenic (anaerobic) condition could be participated by various types of bacteria trophic groups including acidogenic, acetogenic and methanogenic bacteria (Talarposhti et al., 2001; Bras et al., 2005; Ong et al., 2005a; Somasiri et al., 2008). Extensive studies on dye degradation have been carried out by many researchers using diverse groups of bacteria. Based on the molecular characterizations of microbial populations in anaerobic baffled reactors treating industrial textile wastes, sulfate reducing bacteria (SRB), -proteobacteria and Mehanosaeta species and Methanome donthylovorans hollandica of the methanogenic populations were among the prominent bacteria groups identified in the treatment of dye waste (Plumb et al., 2001). Dos Santos et al. (2006b) confirmed the ability of fermentative bacteria to use humic acid as electron acceptor in the reduction of azo dyes. The anaerobic treatment of wool dyeing effluents which contained predominantly methanogenic cultures have shown higher performance for color removal of more than 88% at the HRT of 24 hours (Bras et al., 2005). Ong et al. (2008b) had reported 100% degradation of 625 mg/L of initial concentration of Acid Orange 7 under limited oxygen supply (DO below 0.25 mg/L) without any addition of external carbon sources in granular activated carbon-biofilm configured sequencing batch reactor system. (1997) and many others. Study on the methanogenic consortia on decolorization has been studied earlier on by Carliell et al. (1996), Razo-Flores et al.

Based on many documented research reports, various types of azo dyes could be degraded by anaerobic bacteria. This gives an indication that azo dye reduction mechanisms are non-specific reactions (Moutaouakkil et al., 2003 and Hong et al.,

63 2007). Under this condition, in order for dye degradation to occur, organic carbon or energy source from simple substrates such as glucose, starch, acetate or ethanol or complex substrates such as whey and tapioca are required (Chinwetkitvanich et al., 2000, Isik and Sponza, 2005a, van der Zee and Villaverde, 2005). The rate of degradation defers depends on the addition of co-substrate and the dye structure itself (Pandey et al., 2007).

First-order kinetics with respect to dye concentrations has been reported by many researchers for the mechanisms of dye decolorisation (Isik and Sponza, 2004a; Ong et al., 2005b; Lourenco et al., 2006). According to van der Zee (2001a), the biological reduction of mono-azo dyes by anaerobic bioreactors followed the first order kinetics without any lag phase. Multiphase kinetics was observed for biological reduction of dyes containing azo linkages such as diazo and polyazo dyes. Meanwhile, Wijetunga et al., (2007) reported biodegradation of Acid Red 131 and Acid Yellow 79 under anaerobic condition using mixed anaerobic granular sludge followed by the first order and second order kinetics, respectively. Other researchers found dye decolorization occurring according to zero-order kinetics (Brown, 1981 and Dos Santos et al., 2004). Degradation of azo dyes (Acid Orange 7 and Reactive Red 2) by anaerobic granular sludge demonstrates zero order kinetics for biological dye reduction and second order kinetics for chemical dye reduction as a function of sulfide and dye concentrations (van der Zee, 2003b). The contradictory findings may be due to the different experimental conditions that imposed different rate-limiting step in the reduction of azo dye.

3.4.3.3 Anoxic Dye Degradation Process

Degradation of azo dyes could also occur under anoxic condition by maintaining the oxygen concentration levels in the range of 2.5-3.0 mg/L (Mohan et al., 2007b). Dye degradation under anoxic condition is considered as a non-specific

64 enzymatic reaction (Pearce et al., 2003 and Silveira et al., 2009). Azo dye decolorizations by mixed aerobic or facultative anaerobic microbes are reported to occur under anoxic conditions (Kapdan et al., 2000, Moosvi et al., 2005, Ren et al., 2006; Dafale et al., 2008). Aeromonas hydrophila strain, new isolated species found that could decolorize triphenylmethane, azo and anthraquinone dyes with more than 85% decolorization for azo and anthraquinone dyes within 36 hours under anoxic condition (Ren et al., 2006).

Decolorization of Remavol Black-B under anoxic condition was found to fit the first order kinetics with respect to dye concentration and electron donor (carbon source) as well as operational parameters, pH and temperature (Dafale et al., 2008). Significant degradation of azo dyes by a specific bacterial consortium was achieved in a two stage anoxic-oxic reactor system with 84% and 80% for color and COD removal in raw textile wastewater (Dafale et al., 2008). A novel bacterial strain isolated from soil samples contaminated with textile wastewater, identified as Pseudomonas sp. SUK.1, is capable of decolorizing Red BLI (50 mg/L) up to 99.28% within 1 hour under static anoxic condition at a pH range of between 6.5 to 7.0 and temperature at 30oC (Kalyani et al., 2008).

Dye degradation under anoxic conditions by facultative, anaerobic and fermentative bacteria seems to be affected by the type of substrate used as the external carbon sources (Pandey et al., 2007). The azo dye decolorization (Reactive Red 22) under anoxic condition was significantly decreased when glucose was used as the main carbon source for Pseudomonas luteola (Chang et al., 2001).

65 3.4.4 Mineralization of Aromatic Amines

The mineralization of aromatic amines can be considered as the second stage of dye degradation process after the cleavage of the azo bond. At this stage the amine compounds will be converted into harmless end products under aerobic condition. This degradation stage is very important since the persistent occurrence of these substances may impose significant adverse effects on the ecosystem.

Many of the aromatic amines released from the anaerobic degradation of azo dye are further mineralized in the aerobic conditions. Under aerobic treatment conditions, aromatic amines can be mineralized by non-specific enzymes through the hydroxylation and ring opening of the aromatic compounds by incorporating two oxygen atoms (Zissi and Lyberatos, 1996 and Pandey et al., 2007). The aerobic degradation of aromatic amines has been extensively studied such as the degradation of aniline (Konopka, 1993), chlorinated aromatic amines (Loidle et al., 1990), carboxylated aromatic amines (Russ et al., 1994), sulfonated aromatic amines (Sen and Demirer, 2003), benzene-based aromatic amines (Cinar et al., 2008) and nitroaniline (Khalid et al., 2009). Biodegradation of sulfanilic acid and 1-amino-2napthol has been successfully demonstrated by a bacterial culture in an aerobic rotating biological contactor (Coughlin et al., 2002). However, there are certain types of aromatic compounds that cannot be further degraded even in aerobic condition especially byproducts from the cleavage of reactive azo dye Reactive Black 5, Reactive Violet 5 and Direct Black 8 (Panswad and Luangdilok, 2000; Libra et al., 2004; Sponza and Isik, 2005).

The conversion of the aromatic amines is generally carried out by specialized aerobes. Under microaerophilic conditions where DO concentrations are in the range of 0.2 to 0.5 mg/l, certain aromatic amines including aniline, 1,4-diaminobenzene, 1amino-2-naphthol; catechol and 4-amonobenzoic acid can be completely degraded by Shewanella decolorationis S12 via the oxidative cleavage of the aromatic ring (Xu et al., 2007). Mixed bacterial culture identified as Acinetobacter sp., Citrobacter

66 freundii and Klebsiella oxytoca, showed complete degradation of 100 mg/L of nitroaniline within 72 hours under aerobic conditions (Khalid et al., 2009).

Aromatic amines are considered as stable biotransformation byproduct of azo dye degradation under anaerobic conditions due to the resistance for further degradation under anaerobic conditions (Stolz, 2001 and Sponza and Isik, 2005). However, amines with simple structure were reported able to be degraded under anaerobic condition such aniline and nitroaromatic compounds (De et al., 1994 and Razo-Flores et al., 1999). Complete degradation of aniline by methanogenic granular sludge was reported by Kato et al. (1993) and Tan et al. (1999). Amines that could not be further degraded will remain and contribute to the untreated COD level in the effluent.

The amine compounds can be autoxidized when exposed to air. This reaction will cause the colorless anaerobic dye degradation byproduct to become colored with difference color intensity. The increase of color during autoxidation of aromatic amines was confirmed by several researchers (Cruz and Buitron, 2001; Libra et al., 2004; Sponza and Isik, 2005). This reaction may reduce the overall percentage of color removal and further investigations are required to overcome this problem.

As discussed in the earlier sections, dye degradation mainly occurred under anaerobic or anoxic conditions while further degradation of the aromatic amines mainly takes place under aerobic conditions. Hence, many researchers are focusing more on having both anaerobic and aerobic conditions in textile wastewater treatment processes for complete and effective dye degradation either as integrated or sequential treatment systems.

67 3.5 Treatment System for Biodegradation of Azo Dyes

The requirement of azo dye cleavage prior to degradation through oxidative reaction of the aromatic amines allow the process of anaerobic and aerobic reaction phase as the logical prerequisite requirement for complete biodegradation for most colored substances through biological treatment (Knackmuss, 1996 and van der Zee and Villaverde, 2005). The applications of both anaerobic and aerobic conditions are indeed required for complete degradation of azo dyes (Melgoza et al., 2004). These conditions have become important features that need to be considered in designing the treatment system for textile wastewater.

The treatment condition that would be best for complete mineralization of the azo dye could be appropriate in two approaches. It uses two separate compartments or a reactor that separates the sludge in sequential anaerobic/aerobic treatment system (Khelifi et al., 2008) or integrated anaerobic/aerobic treatment in a single reactor system (Frijters et al., 2006 and Cinar et al., 2008). The study on the dye degradation process has been conducted by many researchers either in sequential treatment system such as a series of reactor systems or integrated treatment in a single reactor using sequential batch reactor. The study on dye biodegradation is also conducted under limited oxygen supply such as anoxic or microaerophilic condition, either in sequential or integrated reactor system. Different types of media have also been used as the degradation agents such as suspended biomass, biofilms and granules in different types of reactor systems to achieve the most effective treatment system for dye degradation of textile wastewater.

68 3.5.1 The Sequential Anaerobic/Aerobic Reactor System

In sequential anaerobic and aerobic reactor system, two separate reactors are used. The wastewater is first treated under anaerobic condition in an anaerobic reactor followed by an aerobic reactor for the aerobic treatment phase. The studies on the conversion of azo dyes using a sequential anaerobic and aerobic reactor system have been extensively studied by many researchers (Brown and Hamburger, 1987; Rajaguru et al., 2000; Kapdan et al. 2003; Libra et al. 2004; Mohanty et al., 2006). Materials such as charcoal and calcium alginate beads have been used in dye degradation process as the support materials for the immobilization of microorganisms in the reactor system. Activated sludge and anaerobic granular sludge are some of the common biomass used as the source of the microorganisms for biodegradation of textile wastewater. Table 3.4 summarizes some of the studies conducted on dye degradation using a sequential anaerobic and aerobic reactor system.

In this sequential treatment process, the main substrate source that represents the organic loading are consumed anaerobically and at the same time the cleavage of the azo dye takes place producing the aromatic amines as the byproduct. During the aerobic reaction phase, the amines are used as the additional carbon and energy sources for the microbes in the aerobic reactor. However, in the case of recalcitrant amines such as the sulphonated aromatic amines (Tan and Field, 2000 and Tan et al., 2005), these amines either remain in the reactor system or escape with the released effluents.

The first anaerobicaerobic full-scale treatment of textile wastewater was applied in the Netherlands in 1999 by a textile company known as Ten Cate Protect dealing with vat, disperse and reactive dyes. The company managed to remove 8095% of the dyes in the anaerobic reactor system. The removal of reactive dyes took place in the anaerobic treatment while the absorption of vat and disperse dye and also

69 the mineralization of the aromatic amines occurred in the aerobic reactor (Frijters et al., 2006).

Overall observation shows that high color removal is achieved under anaerobic condition while higher aromatic amines and COD removal can be accomplished under aerobic condition. A coupled system with anaerobic and aerobic reaction mode has confirmed to be a successful strategy in achieving complete biodegradation of azo dyes. Isik and Sponza (2006) suggested that having post aerobic treatment step would provide even more complete mineralization of textile wastewater. Increase in the retention time under anaerobic condition would increase the COD and color removal (Ong et al., 2005a). Most of the degradation of organic compound occurred at the early stage of aerobic reaction, so increase in the HRT of the aerobic system does not really effect the COD removal.

The addition of organic loading rate would improve the percentage of color removal (Ong et al., 2005b). This is because an increase in the organic loading rate means more of substrate is added into the treatment system. The presence of substrates such as glucose, sucrose, acetic acid acted as the electron donor. Increase of these substances multiplies the amount of electron donor transferred to the N=N bond and results with increase in color removal. Increase in organic loading rate showed a slight increase in COD removal due to the increase in the biomass production under aerobic condition. However, the COD removal deteriorates in the anaerobic treatment system when the organic loading rate increases. The reduction effect on COD removal is more obvious when there is increase in loading rate due to increase in the concentration of dyes. When comparing between static and shaking conditions, the static condition would exhibit more percentage color removal (Moosvi and Madamwar, 2007).

70 3.5.2 The Integrated Anaerobic/Aerobic Reactor System

Complete degradation and mineralization of dye-containing wastewater require both anaerobic and aerobic conditions. As discussed in the earlier section, many researches have been conducted with both anaerobic and aerobic treatment conditions for textile wastewater treatment. However, using two or more separate reactor systems may not be economical as it requires more area for reactor set-up. In order to achieve complete dye degradation for textile wastewater within a suitable working area, an economical budget integrated system has been introduced. In the integrated treatment system, the most important part is the development of different microniches occupied by different types of microorganisms. Control on the oxygen level is very important in order to develop different microniches in the reactor column. Type and concentration of co-substrate present in the wastewater may also influence the oxygen level within the reactor system during the degradation process (Tan et al., 2000). This means choosing the appropriate type and amount of substrate for use in the treatment system are also important aspects that need to be considered to achieve the desired removal and stability performance of the reactor system.

The integration of different microniches will be useful for the degradation of several types of pollutants in a single compartment or reactor system. Table 3.5 shows the summary of the integrated system of anaerobic and aerobic dye degradation process in a sequential batch reactor system. Based on the results of the integrated treatment system, most of the cleavage of the azo bond takes place under anaerobic condition while mineralization of the aromatic amines occurs mainly under aerobic condition. The results are comparable to the treatment systems that used separate reactor system.

Table 3.4: Sequential anaerobic-aerobic treatment system for dye degradation

Treatment system AnAHR-UASB Dye/Wastewater Azo dyes (Siriusgeib and Siriuslichtbraun). Simulated textile effluent; Reactive azo dye + modified starch Remazol Black-5 (100 mg/L)+ glucose Biomass/ Microorganisms Mesophilic anaerobic granular sludge and activated sludge Granules Removal Performance 56-90% (color); Methane production rate 22 mg COD/L/day UASB: 73% (COD); 84% (BOD); 64% (Color); Aerobic tank: 12% (COD); 11% (BOD); 11% (Color). Overall: 84% (COD); 96% (BOD); 75% (Color) UASB: 92-87% (COD); 50-76% (Methane Gas); CSTR: 28%, 42%, 90% (SRT: 1.7, 5.7 and 11d)(COD); 90-95% (Color) 85% (COD); 90% (Color) Reference Kalyuzhnyl and Sklyar (2000) O'Neill et al., (2000a)

UASB (HRT:24h) Aerobic tank (HRT:16 h) UASBCSTR

Anaerobic granule

Sponza and Isik (2002)

UASB (HRT: 0.5d) CSTR(HRT: 2d) RDR (HRT: 15h) UASB (HRT: 30h) CSTR (HRT: 4.5 d) UASB:CSTR

2,4 dinitrotoluene (DNT) (2-500 mg/L) + Molasses Reactive Black 5 (530 mg/L) + acetic acid Raw cotton textile wastewater + glucose Reactive Black 5 (RB 5) and Congo Red (CR)

Partially granulated anaerobic sludge; activated sludge Partially granulated anaerobic sludge; activated sludge Partially granulated anaerobic sludge; activated sludge

Sponza and Atalay (2003) Libra et al. (2004) Isik and Sponza, (2004a) Isik and Sponza, (2004b)

65% (Color); 85% (methane gas) UASB: 9-15% (COD); 46-55% (Color); Overall: 40-85% (COD); 39-81% (Color) RB5: 94.1-45.4% (COD); 79-73% (Color); CR: 92.3% (COD); 95.3-92.2% (Color) (HRT 3.5-0.5d)

71

Table 3.4: Sequential anaerobic-aerobic treatment system for dye degradation (Continued)
Treatment system UASB (HRT: 16.515 h) SBR (HRT: 2.5-2.3 h) UASB (HRT: 24 h) SBR (HRT:24 h) SBR1 (aerobic) SBR2 (anaerobic); HRT(24 h) Full scale: AnFBR (anaerobic) Aerobic basin Fermenter (anaerobic) Aerobic tank UAFB (HRT: 5-15 days)Aerobic tank UASB (HRT: 6-100 days)CSTR (HRT: 15-1days) Dye/Wastewater Direct Black 38 (6.1-213 g/m3h) + glucose Biomass/ Microorganisms Partially granulated anaerobic sludge; activated sludge Activated sludge Activated sludge Removal Performance UASB: 49% (COD); 80% (Color)(HRT: 15 d) CSTR: 67%(COD) (HRT:2.3d); Overall: 84% (COD); 52% (Color) (HRT: 2.9d). UASB: 25-45% (COD); >95% (Color); SBR: 90% (COD) 15% (COD-SBR1); 80% (COD-SBR2) Reference Sponza and Isik (2005)

Orange II (0-100 mg/L); STWW + sucrose Orange II (0-100 mg/L); STWW + sucrose Mixture of vat, disperse, reactive, anthraquinone and indigoids (40 mg/L) Reactive Black 5(100-3000 mg/L) Effluent from manufacturing textile and pharmaceuticals Mixed dyes (Reactive Black 5, Direct Red 28, Direct Black 38, Direct Brown 2 and Direct Yellow) (250 mg/L)

Ong et al. (2005a) Ong et al. (2005b) Frijters et al. (2006) Mohanty et al. (2006) Moosvi et al. (2007)

80-90% (COD); 80-95% (Color)

Activated sludge

>90% (COD); 46% (amines);HRT: 2 days

UAFB (charcoal support material); Aeration tank + P. aeroginosa ;5%, (v/v) UASB: Partially granulated anaerobic sludge; CSTR: activated sludge from dye industry

UAFB: 37-70% (COD); 7-58%(color); Aerobic Tank: 40-45% (COD); 35-40% (color) ; Overall: 94% (COD); 89% (color).

Overall removal: 97% (COD) and 91% Isik and (Color). CSTR: TAA (70-85%); HRT: 9-6 Sponza (2008) days.

72

Table 3.5: Integrated anaerobic-aerobic sequential treatment system for dye degradation
Treatment system SBR: Fill: <5min; React: Anaerobic (18/6 h); Aerobic (5 h); Settle: 55 min; Decant: 5 min. SBR: Fill: 15min:React: 18.5 h; 0.5 h (aerobic); Settle: 4 h; Decant: 0.25 h; Idle: 0.5 h. Dye/Wastewater Remazol Black B (10 mg/L) + Nutrient broth + Sodium acetate + glucose Remazol Black. STWW (+ starch,+ polyvinylalcohol (POVH),+ carboxymethyl cellulose) STWW (propionate : DB79 [1:50]); Dye (50% dye + 50% dispersing agent) Azo dye: Remozol Brilliant Violet 5 and Acid Orange; STWW + starch derivative Jane Sandolane MF-RL & Rouge Sandolane MF2BL dyes + antraquinone Biomass/ Microorganisms Sewage treatment plant Removal Performance 73-77% (Color-PAOs); 59-64% (Color-GAOs). 66% (18h HRT); 59% (6h HRT) of anaerobic contact time. 66% (TOC); 94% (Color) Reference Panswad et al., (2001a)

Anaerobic granules from UASB

Shaw et al. (2002)

SBR: VER: 75%. Intermittent agitation: 180 rpm

Activated sludge

65% (Color); 96% (amines); Overall removal: 96% (Color)

Melgoza et al. (2004)

SBR: Fill: 50 min; React: Anaerobic (10.5 h) / Aerobic (10 h) ; Settle: 1.5 h; Draw: 35 min; Idle: 10 min SBR: Fill: 48 h (static or mixed); Anaerobic: Aerobic (812h); Settle: 2 h; Draw: 48 min; Idle: 24 min

Activated sludge

80% (COD); 90-99% (Color) removal Albuquerque et al. and 80% of COD removal (2005)

Raw wastewater + aerobic sludge.

85% (soluble COD); 95% (BOD5)

Goncalves et al. (2005)

73

Table 3.5: Integrated anaerobic-aerobic sequential treatment system for dye degradation (Continued)
Treatment system SBR: Different anaerobic/aerobic residence time (2-19 h) Dye/Wastewater Vinylsulphonyl, monochlortriazine, Remazol Rot RR. Biomass/ Microorganisms Alcaligenes faecalis and Comamonas acidovorans Granular activated carbon; dye degrading microbes Facultative mixed bacterial culture Removal Performance 90% (Color-4-6 h of anaerobic cond. At 60 mg/L of dye conc.); >85% (COD); >90% (Color) at 500 mg/L dye conc. 88% (COD); 100% (Color) Reference Kapdan and Oztekin (2006)

SBCR: Fill: 3 h; React: 20 h; Draw: 0.45 h; Idle: 0.15 h.

Acid Orange 7; STWW + sucrose

Ong et al. (2008b)

SBR: Fill: 3 min; Anaerobic: Aerobic reaction 48 hrs(24:23.9); 12 hrs(12: 11.9); 12 hrs(6:5.9); Draw (3 min) SBR: HRT: 8 h; Working vol.: 2 L; VER: 50%,

Remazol Brilliant Violet 5R; STWW + glucose

Anaerobic: 75% (COD); 72%, 89% and 86% (color); Aerobic:64%, 92% and 89% (amines)

Cinar et al. (2008)

Malachite green (MG) as main carbon source

Aerobic digested sludge (+nutrient + micronutrient);

92.3% (Color)

Mondal and Ahmad (2009)

74

CHAPTER 4

DEVELOPMENT OF FACULTATIVE ANAEROBIC GRANULES

4.1

Introduction

In recent years, the ability of biodegradation process for treating different types of wastewater involving both anaerobic and aerobic processes has been widely reported in the literature (Jang et al., 2003; Arrojo et al., 2004; Su and Yu, 2005; Yilmaz et al., 2007; Wang et al., 2009b). Table 4.1 summarizes some of the studies conducted on anaerobic and aerobic/anoxic treatment process in SBR system treating different types of wastewater. The intermittent anaerobic and aerobic The treatment approach showed high percentage of COD and nutrient removal.

intermittent anaerobic and aerobic treatment process in wastewater treatment is important in obtaining complete degradation process particularly for treating recalcitrant compounds.

Since color removal and complete mineralization of the dyestuff could be achieved through the combination of both processes as discussed in Chapter 3, many studies have been conducted on the textile wastewater using the intermittent anaerobic and aerobic treatment approach (Kapdan et al., 2003; Goncalves et al., 2005; Albuquerque et al., 2005; Franciscon et al., 2009). Nevertheless, the 75

76 integrated process used so far, requires the use of two separate reactors to suit the different oxygen requirements of the degradation processes.

An attempt was therefore made to develop FAnGS which are capable to live and work under both anaerobic and aerobic conditions. While achieving both conditions fulfill the requirements to treat textile wastewater, the use of microbial granules enhances the treatment system as discussed in the previous chapter. Furthermore, the application of FAnGS under anaerobic and aerobic conditions requires the use of a single reactor.

In this study, the progress of the FAnGS development was monitored and their properties were determined. The effectiveness of the FAnGS in treating This synthetic wastewater during the development process was also assessed. chapter presents the results of the work conducted.

4.2

Materials

All of the chemicals/reagents used in this experimental work are listed in Table 4.2 while the analytical equipments are listed in Table 4.3. Distilled water generated from a water distiller was used throughout the experiment. temperature for daily use. Trace elements and mixed dye were prepared as stock solutions and were kept at room Synthetic textile dyeing wastewater (STDW) was prepared three times a week for influent supply to the reactor system. In order to avoid contamination, the mixture of the synthetic wastewater, trace elements and dye were carried out in a laminar flow.

76

77

Table 4.1 Sequential batch reactor system with intermittent anaerobic/aerobic/anoxic reaction phase treating variety types of wastewater
Reference Biomass Wastewater Reaction phase/HRT Performance/Remark

Jang et al. (2003) Lin et al. (2003) Meyer et al.(2003) Zhu and Wilderer (2003) Arrojo et al. (2004)

Activated sludge of MWTP Sludge from MWTP Sludge enriched with GAO Activated sludge Sludge from industrial wastewater

SWW (Carbon source: glucose) SWW (Carbon source: acetate) SWW (Carbon source: acetate) SWW (Carbon source: glucose & pepton) Industrial WW and SWW (Carbon source: acetate) Dairy WW

HRT: 6 hours; Feed: 15 min; Aerobic: Anoxic: 4.75 hours (2:1); Settling: 45 min; Decant: 15 min HRT: 6 hours; Feed: 5 min; Anaerobic: 120 min; Aerobic: 226; Settling: 5 min; Decant: 4 min. HRT: 8 hours; Feed: 5 min; Anaerobic 1.5 hours; Aerobic: 2 hours; Settling: 25 min; Decant: 5 min HRT: 6 hours; Feed: 15 min; Anaerobic: 1 hour 40 min; Aerobic: 3.5 hours; Settling: 20 min; Decant: 15 min HRT: 3 hours; Feed: 3 min; Anoxic/Aerobic: 171 min; Settling: 1 min; Decant: 30 min; Idle: 3 min HRT: 8 hours; Feed: 5 min; Anaerobic: 0-60 min; Aerobic: 335-345 min; Settling: 5 min; Decant: 4 min; Idle: 5 min. HRT: 4 hours; Feed: 5 min; Aerobic: 220 min; Settling: 5 min; Decant: 10 min HRT: 7.2-6.2 hours; Fill: 120 min (anaerobic); Aerobic : 220 min; Settle: 2-60 min; Decant/Idle: 15 min. HRT: 6 hours; Feed: 5 min; Aerobic: 230; Anaerobic: 119 min;Settling: 2 min; Decant: 4 min.

Removal: Nitrification (97%); COD (95%)

P-accumulating organisms were enriched in the granule as the substrate P/COD ratio was increased. Dense and highly active aggregates of microbes can lead to mass transport limitation Removal: COD (78-94%)

Removal: Nitrification (70%); COD (85-95%)

Schwarzenbeck Sludge et al. (2005) biomass Su and Yu (2005) Cassidy and Belia (2006) Qin and Liu (2006) Activated sludge Flocculating sludge Activated sludge

Removal:90% (CODtotal); 80% Ntotal; 67% Ptotal

Soy-bean WW Abattoir WW SWW (Carbon source: ethanol)

Removal: COD (98-99%- after 7 days) Removal: COD and Phosphates (>98%); Nitrogen (>97%) Removal: COD (95-97); Nitrogen (99-100%)

77

78

Table 4.1 Sequential batch reactor system with intermittent anaerobic/aerobic/anoxic reaction phase treating variety types of wastewater (Continued)
Reference Zhang et al. (2006) Biomass Activated sludge of MWTP Activated sludge of MWTP Aerobic granule Activated sludge of MWTP Aerobic granules Flocculating sludge Mixed sludge with anaerobic granule Wastewater Raw swine manure WW Reaction phase/HRT HRT: 3.3 days; Cycle time: 8 hours; Anaerobic: 1hour 15 min; Anoxic/aerobic: 2 hour 45 min; Anaerobic: 1 hour 30 min; Anoxic/aerobic: 2 hours; Settling: 30 min HRT: 8 hours; Cycle time: 4 hours; Feed: 4 min; Anoxic (no stirring): 30 min; Aerobic: 200-210 min: Settle: 1-11 min; Decant: 5 min HRT: 6.7-13.3 hours; Feed: 18 min; Anaerobic (50-60 min); Aerobic (160-400 min); Post Aerobic (80 min) HRT: 6H; Aerobic: 4.75 hours; Anaerobic: 1.25 hours; Feed: 0.25 hours; Settle: 0.75 hours; Decant: 0.25 hours HRT: 13.3 hours; 8 hour cycle; Feed: 18 min; Anaerobic/anoxic: 60 min; Aerobic: 315 min; mixed anoxic: 80 min; Settle: 2 min; Decant: 5 min HRT: 8 hrs; Feed: 60 min; Anaerobic: 60-0 min; Aerobic: 334-405 min; Settling: 15-4 min; Decant: 5 min; Idle: 5 min HRT: 6 hours: Feed: 5 min; Anaerobic: 80 min; Aerobic: 260 min; Settling/Decant/Idle: 5 min Performance/Remark Removal: Total Nitrogen (97.5%); Total Phosphorus (95%); COD (96%); BOD5 (100%); Turbidity (95%) Removal: COD (95%); 2,4-dicholophenol effluent (94%) Removal: Soluble Nitrogen (93%); Soluble COD (85%); Soluble Phosphorus (89%) Removal: COD (95-98%); NH4+-N (47-99%) depending on the NH4+-N loading rate Total removal: Soluble COD (85%); Ammonia (99%); Phosphates (98%) Nitrogen removal rate: 4.5-9.0 kgCOD/m3d.

Wang et al. (2007) Yilmaz et al. (2007) Kim et al. (2008) Lemaira et al. (2008a) Wichern et al. (2008) This Study (2009)

SWW (Carbon source: glucose & 2,4-dichlorophenol) Abattoir WW/SWW (0-100% ratio) SWW (Carbon source: glucose & acetate) SWW; different ratio of SWW and abattoir WW Dairy WW

Synthetic textile dyeing WW

Removal: COD (93%); Ammonia (95%); Color: (62%)

*MWTP-municipal wastewater treatment plant; WW-wastewater; SWW-synthetic wastewater; GAO-glycogen accumulating organisms.

78

79 Table 4.2: List of reagents used in the experiment

Chemical Solutions/Reagents Synthetic wastewater Trace element Mixed dyes Sludge sewage Anaerobic granules Dye degrader microbes COD reagent Nessler reagent Concentrated H2SO4 Nitric Acid Gold sputter Crystal violet Iodine Ethyl alcohol Safranin Nitrogen gas Oil emulsion Sodium hydroxide, NaOH Hydrogen cloride, HCl pH adjustment Anaerobic condition (Section 4.2.4.5 (b)) Observation under 100x microscope magnification Gram Staining (Section 4.2.4.5(a)) Applications Wastewater model compounds (Section 4.2.1)

Granular precursor (Section 4.2.2) COD measurement (Section 4.2.4.4(b)) Ammonia measurement (Section 4.2.4.4.(c)) Digestion of mineral (Section 4.3.3) Sample coating (FESEM)

4.2.1 Wastewater Composition

Synthetic wastewater with the following composition was used: NH4Cl 0.16 g/L, KH2PO4 0.23 g/L, K2HPO4 0.58 g/L, CaCl22H20 0.07 g/L, MgSO47H2O 0.09 g/L, EDTA 0.02 g/L and trace solution 1 ml/L. The carbon sources used in this experiment were glucose (0.5 g/L), ethanol (0.125 g/L) and sodium acetate (0.5 g/L). The trace elements used were based on the composition recommended by Smolders et al. (1995). The composition of the trace element was H3BO3 (0.15 g/L),

80 FeCl34H2O (1.5 g/L), ZnCl2 (0.12 g/L), MnCl24H2O (0.12 g/L), CuCl22H2O (0.03 g/L), NaMoO42H2O (0.06 g/L), CoCl26H2O (0.15 g/L), and KI 0.03 g/L. Mixed azo dyes consisted of Sumifix Black EXA, Sumifix Navy Blue EXF and Synozol Red K-4B with total concentrations of 50 mg/L was used in this study. The mixture gave an initial COD of 1270 mg/L; 1020 ADMI (102 Pt-Co) and average ammonia concentration of 38 mg/L. The pH of the synthetic wastewater was adjusted to 7.0 0.5 before feeding.

Table 4.3: List of equipment used in the experiment Equipments IFAnGSBioRec Filter paper Filter apparatus Incubator Furnace Glass column Orbital shaker Spectrophotometer COD reflux Flame Atomic Absorption Spectrophotometer Stereo microscope Digital image management and analyzer Scanning electronic microscope Coating system DO meter & Data acquisition software Water bath Autoclave Air pump Water distiller Laminar flow Sieve Manufacturer/Product UTM/Fabricated Wartmann/125 mm diameter Sartorius/DOA-P504-BN ELBA/EMO-1706 Interscience Sdn. Bhd./Carbolite UTM/Fabricated Protech/Orbital Shaker Model 720 HACH/DR/4000U HACH/DRB 200 Perkin Elmer/Analyst 400 Leica Mycrosystems Wetzlar GmbH/Leica DMLS ARC PAX-CAM/PAX-ITv6 Carl Zeiss/Zeiss Supra 35 VPFESEM Biorad/Polaron Division SEM Coating System ISTEK/PH/ISE/DO Meter Model 125 PD Memmert / Julabo PT30511 Hirayama/Hiclave HV-50 RESUN/LP-100 air-pump Apex/Water Distiller ERLA/CFM Series Air Cabinet Laminar Flow WYKEHAM FARRANCE SLOUGH ENGLAND/British Standard Test Sieve (0.1-2.0 mm) 80

81 As discussed in Chapter 3, the most commonly used dyes in the textile processing are the azo dye. Hence, a mixed azo dye was applied in this study in order to provide a closer resemblance to the real textile wastewater that usually consist of several mixtures of dyes. Furthermore, synthetic textile wastewater was used in this study in order to ensure the consistency of the wastewater quality with respect to the compositions and concentrations. High variation in the wastewater compositions may result in difficulty in analyzing and interpreting the results of the study.

4.2.2 Granules Precursor

The development of the FAnGS involved the mixture of sludge sewage treatment plant (Taman Sutera, Indah Water Konsortium Treatment Plant System) and textile wastewater treatment plant (Ramatex Industry Sdn. Bhd., Sri Gading Industrial Park). Sludge from sewage treatment plant was used since the sludge contains very high concentration of microbial populations that may be useful for granule development. The sludge from textile wastewater was used since the microorganisms that are present in the textile sludge may have already acclimatized to the textile wastewater and would be easier to grow in the synthetic textile wastewater used in the study. The acclimatized microbes in the textile sludge may also have high capability in dye degradation process. This is important for the development of granules that is going to be used for treating textile wastewater. An equal volume of sludge from a municipal sewage treatment plant and a textile mill wastewater treatment plant were mixed. The sludge inoculums were sieved with a mesh of 1.0 mm to remove large debris and inert impurities. Figure 4.1 shows the location of the sewage treatment plant (Indah Water Konsortium Treatment Plant System, Taman Sutera) and textile wastewater treatment plant (Ramatex Industry Sdn. Bhd., Sri Gading Industrial Park) where the sludge samples were taken.

82

Figure 4.1

Location of textile industry; Ramatex Industry Sdn. Bhd., Sri Gading

Industrial Park, Batu Pahat and sewage treatment plant; Indah Water Konsortium Treatment Plant System, Taman Sutera, Skudai.

82

83 Anaerobic granules collected from a UASB treating paper mill industrial effluent (Denmark) were used as the seed sludge. For every 1 L of sludge mixture, about 100 mL of anaerobic granules of sizes less than 1 mm diameter were used as seed for the granulation process. The MLSS of the anaerobic granules were 3.3 g/L. Dye degrader microbes were also added into the sludge mixture. These microbes were obtained from previous research that has successfully isolated the microbes from textile wastewater treatment plants (Nawahwi, 2009 and Ibrahim et al., 2009). The sludge mixtures were acclimatized with synthetic textile dyeing wastewater for two months prior to the experimental start-up.

4.2.3 Reactor Set-up

The schematic representation of the IFAnGSBioRec set-up is given in Figure 4.2. The design of the reactor used in this study was based on several studies conducted by previous researchers such as Wang et al. (2004) and Zheng et al. (2005) with several modifications. A water jacketed column reactor was used in the study. The column was designed for a working volume of 4 L with internal diameter of 8 cm and a total height of 100 cm. The water jacketed column was designed to provide temperature controlled conditions by allowing the circulation of hot water from a water heating circulation system (Julabo PT30511) to the water jacketed column of the reactor system. The temperature of the heating system was set at 30oC. The wastewater was fed into the reactor from the bottom of the reactor. Air was supplied into the reactor by a fine air bubble diffuser also located at the bottom of the reactor column. The decanting of the wastewater took place via an outlet sampling port located at 40 cm height from the bottom of the reactor. The reactor system was equipped with dissolved oxygen meter and pH meter (Istek , Korean Model 125 PD) for the continuous monitoring of the DO and pH level throughout the experiment. The 4-L laboratory scale of IFAnGSBioRec used in this study is shown in Figure 4.3. Figure 4.4 shows the preparation carried out in the development of FAnGS for textile wastewater treatment.

84

4 DO probe o o o o o o Effluent o o Sampling point o o o 3 8

pH probe

o o o o o o o o o o o o

Influent 9 2

1. Influent tank 6. Mass-flow controller 8. Timer controller

2-5. Peristaltic pumps 7. Air pump 9. Effluent tank

Figure 4.2 Schematic layout of the IFAnGSBioRec system (Wang et al. (2004) and Zheng et al. (2005)

84

85

Figure 4.3 The IFAnGSBioRec system used in this study

86 4.3 Analytical Methods

The FAnGS was analyzed for biological, physical and chemical characteristics. The biological characteristic of the FAnGS was investigated in terms of morphological, structure as well as cellular observation of the microbes within the FAnGS. The microbial activities of the granules were investigated with respect to their specific oxygen uptake rate (SOUR) and specific methanogenic activity (SMA). The physical characteristics of the FGS tested include settling velocity, granular strength and sludge volume index (SVI). analyzed includes their mineral content. retention time. The chemical aspect of the FAnGS Profiles of the reactor system were

evaluated on the biomass concentration retained in the reactor as well as the sludge Figure 4.5 shows the experimental analysis conducted for characterization of the FAnGS.

4.3.1

Biological Characteristics

4.3.1.1 Morphological and Structural Observation

The morphological and structural observations of the granules were carried out by using a stereo microscope equipped with digital image management and analyzer (PAX-ITv6, ARC PAX-CAM). The microbial compositions of the granules were observed qualitatively with a scanning electronic microscope (FESEM-Zeiss Supra 35 VPFESEM). The granules were left dried at room temperature prior to gold sputter coating (Bio Rad Polaron Division SEM Coating System) with coating current of 20 mM for 45 s.

86

Figure 4.4 Preparation frame work for granule development

87

88

Figure 4.5 Characterizations of FAnGS

88

89 The cellular observation of the microorganisms present within the FAnGS was carried out using gram staining. The gram staining procedures were carried out by preparing a smear onto a glass slide. The slide was heated in order to fix the smear onto the slide. The fixed smear was covered with one or two drops of crystal violet for one minute. The stained smear was poured off and carefully washed with distilled water. Then, iodine was added and left for 1 minute before once again washed off with distilled water. The preparation was then washed with ethyl alcohol of 95%. The slide was counterstained with safranin for 1 to 2 minutes prior to rinsing with distilled water. A drop of oil was placed on the slide and was examined under 100x magnification of stereo microscope (Leica DMLS). Gram-positive cells appear violet and gram negative cells appear red when observed under the light microscope.

4.3.1.2 Microbial Activity

The microbial activity of the FAnGS was conducted by measuring the oxygen utilization rate (OUR), specific oxygen utilization rate (SOUR) and specific methanogenic activity (SMA). The OUR and SOUR measurements were performed by following Standard Methods (APHA, 2005). The OUR value was measured during both the first and second stage of the aerobic reaction phase. The profile of dissolved oxygen (DO) concentration in the reactor was measured continuously online using a DO electrode (Istek, Model 125 PD). The data were electronically recorded using data acquisition software (Istek, Model 125 PD).

The OUR measurement was conducted as soon as the aeration phase started. Sample for the reactor system was taken from the sampling port and used to fill the 300 mL BOD bottle. Then, the DO probe was immediately inserted into the BOD bottle. The sample in the BOD bottle was mixed using a magnetic stirrer at 20 rpm. The initial DO was measured as DOa. The time taken for the DO to reduce to 2

90 mg/L was measured as T. DOb was measured at the end of each OUR measurement (i.e. when the DO level in the BOD bottle nearly reaching to 2 mg/L). Then the sample in the BOD bottle was returned back into the reactor. The DO measurement is repeated with 10 minutes time interval with new samples from the reactor until the aeration phase completed. For the SOUR measurement, the biomass concentrations collected in the BOD bottle were quantified and measured as M. The measurement for OUR and SOUR were conducted at room temperature. The calculations for OUR and SOUR are given in the equations below:

where OUR = SOUR = DOa DOb T M = = = = Oxygen uptake rate (mg/L.h) Specific oxygen uptake rate (mL CH4/g VSS.h) Initial dissolved oxygen (mg/L) End dissolved oxygen (mg/L) Time (min) Granular biomass (mg/L)

The SMA analysis was conducted according to Erguder and Demirer (2008) with several modifications where a 500 mL BOD bottle seeded with FAnGS with final concentrations of 1-2 g VSS/L and basal medium (250 mL effective volume). The bottle was purged with N2 gas mixture for 5 minutes to obtain an anaerobic condition. The bottle was then sealed with a rubber septum. Acetate acid (HAc) was fed into the serum bottle at the concentration of 3000 mg/L. The experiments were conducted at room temperature (28 2oC). The production of methane gas (CH4) was determined daily for 5-7 days by using liquid displacement methods containing concentrated KOH stock solution (20 g/L) (Erguder and Demirer, 2005a). After

91 each gas measurement, the bottle was manually shaken. At the end of the SMA assay, the VSS content in the bottle was measured. The SMA was calculated as the maximum CH4 produced per gram of VSS per hour (mL CH4 g/VSS.h) (Zitomer and Shrout 1998).

4.3.2

Physical Characteristics

4.3.2.1 Settling Velocity

The settling velocity was determined by recording the average time taken for the individual granule to settle at a certain height in a glass column filled with tap water (Linlin et al., 2005).

4.3.2.2 Sludge Volume Index

The SVI value could be calculated by measuring the bedvolume of the sludge biomass in the reactor divided with the dry weight of the biomass in the reactor. The bedvolume can be obtained by measuring the bed height of the sludge biomass that settled in the reactor 5 minutes after the aeration phase stopped. The bedvolume is obtained by multiplying the bedheight with the surface area of the bedcolumn (Beun et al., 1999).

92 4.3.2.3 Granular Strength

Determination of the granules strength was based on Ghangrekar et al. (1996). Shear force on the granules was introduced through agitation using an orbital shaker (Protech Orbital Shaker Model 720) at 200 rpm for 5 minutes. At certain degree of the shear force, parts of the granules that were not strongly attached within the granules will detach. The ruptured granules were separated by allowing the fractions to settle for 1 minute in a 150 ml measuring cylinder. The dry weight of the settled granules (SG) and the residual granules in the supernatant (RG) were measured. The ratio of the solids in the supernatant (RG) to the total weight of the granular sludge (SG+RG) used for granular strength measurement was expressed as the integrity coefficient (IC) in percent. This percentage indirectly represents the strength of the granules. The higher the IC value the lesser the strength of the granules and vice versa. The calculation for the IC value is given in the equation below.

where IC RG SG = = = Integrity coefficient Residual granules (mg) Settled granules (mg)

4.3.2.4 Biomass Concentration

The biomass concentration in term of mixed liquor suspended solid (MLSS) and mixed liquor volatile suspended solid (MLVSS) were measured according to

93 Standard Methods (APHA, 2005). Ten (10) mL of samples were filtered using 45m filter paper (Wartmann) using a filter apparatus (DOA-P504-BN). The filter papers with samples were then weighed (Ma) before heated at 150oC for one hour. After the samples were allowed to cool in the desiccators, the filter papers were weighed again (Mb). Then the filter papers were heated at 550oC for 15 min and were weighed (Mc) after the filter papers were allowed to cool in the desiccators. The MLSS and MLVSS were measured using Eq. 4.4 and Eq. 4.5, respectively.

where, MLSS MLVSS Ma Mb Mc V = = = = = = Mixed liquor suspended solid (mg/L) Mixed liquor volatile suspended solid (mg/L) Weight of filter paper with sample before heating at 150oC (mg) Weight of filter paper with sample after heating at 150oC (mg) Weight of filter paper with sample after heating at 550oC (mg) Sample volume (mL)

4.3.2.5 Sludge Retention Time

Sludge retention time (SRT) is determined as follows (Beun et al., 1999).

94

where, Xr VT Qe Xe = = = = Mixed liquor volatile suspended solid in reactor (mg/L) Total working volume in reactor (L) Effluent flowrate (L/d) Mixed liquor volatile suspended solid in effluent (waste sludge) (mg/L)

4.3.3

Chemical Characteristics

The granules were analyzed chemically for their mineral content which includes Ca2+, Mg2+, Na+, K+, Fe2+, Ni2+ and Co2+. The procedures for acid digestion of the granular sample were based on Ghangrekar et al. (2005) with some modification. The granular sample was evaporated to dryness in an incubator (EMO-1706) at 105oC. About 5 g of the dry sludge were dissolved in a minimum quantity of concentrated sulfuric acid giving a brownish solution. Then, concentrated nitric acid was added into the solution until it turned colorless. The solution was diluted with distilled water to a total volume of 25 mL for mineral and metal determination. The mineral contents were determined by using a Perkin Elmer Analyst 400 Flame Atomic Absorption Spectrophotometer (FLAA).

95 4.3.4 Removal Performance

Synthetic wastewater samples of the influent and effluent from the IFAnGSBioRec were used for the quantification of removal performance of the Chemical Oxygen Demand (COD), color and ammonia. centrifuged prior to measurement. All samples were This step is carried out to prevent any

interference that may caused by the presence of suspended particles in the samples.

4.3.4.1 Color

A quantitative estimation of the color intensity was carried out by calorimetric approach. Color was analyzed using a HACH Spectrophotometer (DR/4000U) according to Procedure No.1660. Using distilled water as blank, the method gives color value in terms of American Dye Manufacturing Index (ADMI)

4.3.4.2 Chemical Oxygen Demand

Chemical oxygen demand was quantified using a HACH Spectrophotometer (DR/4000U) according to Procedure No. 2720. Each sample was added to the COD reagent (High Range Digestion for COD, Cat. 21259-15) and was digested at 150oC for 2 hours in COD reactor (Model DRB 200). After the digestion was completed, the sample was allowed to cool at room temperature before the COD levels were measured using the spectrophotometer.

96 4.3.4.3 Ammonia

The determination of ammonia was according to the Nessler Method (APHA 2005). One (1) mL of sample was diluted using 25 mL deionized water. Then three drops of mineral stabilizer was added into the samples and mixed. This was followed by adding another three drops of dispersing agent and mixed. Lastly, 1 mL of Nessler reagent was added into the sample and mixed again before the sample was left to react for 1 minute. The sample was then measured using a HACH Spectrophotometer (DR4000/U) according to Method No 2400.

4.4

Experimental Procedures

During the start up period, 2 L of mixed sludge and 2 L of synthetic textile wastewater were added into the reactor system making the final volume of 4 L with a total sludge concentration after inoculation of 5.5 g/L. The system was supplied with external carbon sources consisting of glucose, sodium acetate and ethanol which gave a substrate loading rate of 2.54 kg COD/m3d. The calculation for the OLR is given in Appendix A. The hydraulic retention time (HRT) of the reactor was 6 hours and was divided into several phases.

The reactor was operated in successive cycles of 6 hours, each one with an intermittent anaerobic and aerobic reaction phase. All of the operation of peristaltic pumps, circulation of influent, air diffuser and decanting process were controlled by means of a timer. The reaction phase was started with an anaerobic phase for 40 min, followed by an aerobic reaction phase for 130 min. The reaction phase was repeated with another 40 min of anaerobic phase and 130 min for a second aerobic reaction phase. During the anaerobic reaction phase, the wastewater in the reactor system was allowed to circulate. The wastewater from the upper level of the reactor

97 system was pumped out of the reactor column and returned back through the valve located at the bottom of the reactor. The circulation process was carried out by using a peristaltic pump (Cole-Parmer System Model; 6-600 rpm). The wastewater was circulated at a rate of 18 L/h. anaerobic phase ended. The circulation system was stopped when the The circulation process is required to achieve a

homogeneous distribution of substrate as well as a uniform distribution of the granular biomass and restricts the concentration gradient. Each of the cycle comprised of 5 min filling, 340 min reaction, 5 min settling, 5 min decanting and 5 min idle.

Samples of the synthetic wastewater from the IFAnGSBioRec were taken twice a week. Fifteen (15) mL of influent sample as the initial value was taken from the influent tank before the new cycle operation started, while another 15 mL of the effluent sample was taken from the effluent tank after the effluent was released during the decanting phase as the final values. The samples were filled in a separate 15 mL centrifuge tube. Samples were centrifuged for 5 min at 4000 rpm at 4oC in order to pellet down all of the suspended particles from the samples. Demand (COD), color and ammonia removal. The supernatant was used to measure the removal performance of the Chemical Oxygen

Ten (10) mL of sample was taken into 15 mL of centrifuge tube from the top portion of the reactor about 10 minutes after the filling stage ended. The sample was measured as the initial concentration of the suspended solids. Another 10 mL of sample was taken from the effluent after the decanting stage. When conducting sampling for the measurement of the suspended solids, the effluent from the reactor was collected in a 5L beaker. The effluent in the beaker was mixed before the sample taken with the purpose to get a correct representative value of the suspended particles in the effluent. These samples were used for the measurement of the suspended particles in the influent and in the effluent. Lastly, another 10 mL of sample volume was taken during the aeration phase. In order to get a representative value, the samples were taken at two sampling points i.e. the upper and lower

98 sampling ports. Both samples were then mixed in a beaker and analyzed for the MLSS and MLVSS.

The bedheight of the biomass in the reactor was also measured twice a week for the calculation of the SVI. The bedheight was measured immediately after the settling time ended and before the wastewater was drained out during the decanting time. The measurement of the OUR and the SMA were conducted a few days before the experiment ended which is after the FGS has reached the maturation stage. The granular sample was taken almost once in two weeks for the measurements of the physical properties of the FAnGS.

Table 4.4 shows the successive phase for one complete cycle of the IFAnGSBioRec. The dissolved oxygen (DO) concentrations remained low during the anaerobic condition (0.2 mg/L) and reached saturation concentrations during the aerobic phase. The superficial air velocity during the aerobic phase was 1.6 cm/s. The calculation of the superficial air velocity is given in Appendix A. The pH during the reaction process varied in the range of 6.0 to 7.8 and the temperature of the experiment was at 30oC. The reactor system was operated for a period of 66 days. Two litres of the wastewater remained in the reactor after the decanting stage yielding a volumetric exchange rate (VER) of 50%. At this settling time (5 min), only particles with settling velocity larger than 4.8 m/h remained in the column. Any particles having smaller settling velocity would be washed out in the effluent.

99 Table 4.4: One complete cycle of the IFAnGSBioRec Successive Phase Filling One complete cycle (6 hours) 5 min Anaerobic React 1st phase 2nd phase Settling Decant Idle Total cycle length 40 40 5 min 5 min 5 min 360 min Aerobic 130 130

4.5

Results and Discussion

4.5.1

Morphology of Facultative Anaerobic Granular Sludge

A week after inoculation in the reactor, visual and microscopic observations of granules formation were made. placed under vigorous shaking. At this stage, the developed granules were Within a week, the anaerobic seed granules composed more of loosely clumped sludge which could easily break up into pieces if underwent morphological changes from spherical in shape and black in color with average diameter of 1 mm into smaller grey granules due to exposure to the aeration as mentioned earlier. On day 30, two different types of granules were clearly observed in the reactor as shown in Figure 4.6.

100

Figure 4.6

The morphological development of facultative anaerobic granular

sludge (scale bar at steady-state equals to 1mm). Pictures were taken using a stereo microscope with magnification of 6.3X. (a) Granules developed from the activated sludge. (b) Granules developed from anaerobic granules patches.

101 Figure 4.6a shows mainly irregular-shaped with yellow colored granules (Type A) that are solely developed from the activated sludge. In Figure 4.6b, the anaerobic granules that have fragmented into smaller pieces have formed different sizes of granules (Type B) that contained pieces of anaerobic granules. The outer layer of the latter were yellow in color indicating the domination of aerobic or facultative microorganisms while the darker spots within the granules indicate the presence of anaerobic fragments originated from the anaerobic granules. Beun et al. (1999). The formation of Type A granules could be elucidated by the mechanisms explained by The development was initiated from the mycelial pellets that were retained in the reactor due to high settling velocity. These mycelial pellets eventually become the support matrix for the bacteria growth. Bacteria that were able to attach to this matrix were retained and suppressed the growth of filamentous microorganisms and became the dominant species in the reactor.

The formation of Type B granules has been discussed by Linlin et al. (2005). These granules were formed through a series of physical and morphological changes. The anaerobic granules initially disintegrated into smaller size flocs and debris when exposed to aeration forces in the SBR column. Some of the granules and debris that were too small were washed out with the effluent while the heavier ones were retained in the column and acted as nuclei for the formation of new granules. Having these types of granules that consisted of the combination of aerobic and anaerobic portions within the granules could increase the possibility of degradation process that requires both aerobic and anaerobic conditions for complete degradation particularly for textile wastewater. Figure 4.7 shows the obvious morphological differences between sludge particles during the initial stage of the experiment and matured FAnGS at the final stage (day 66) of the experiment. The average sludge particles are 0.02 0.01 mm (Figure 4.7a), while the FAnGS developed with the average particle diameter size of 2.3 1.0 mm with maximum size reaching up to 4 mm (Figure 4.7b).

102

Figure 4.7 Pictures of sludge particles during the initial stage of the experiment (a) and matured FAnGS granules at the 66 days of the experiment (b). Pictures were taken using a stereo microscope with magnification of 6.3X (scale bar equals to 1 mm)

103 The microstructure of the FAnGS that was examined using SEM is shown in Figure 4.8. The SEM observation of the mature granules shows the domination of non-filamentous coccoid bacteria that is tightly linked and embedded to one another and form a rounded shape on the surface of the granule and covered with extracellular polysaccharides substances (EPS) (Figure 4.8a). The absence of filamentous bacteria in the developed granules may be due to the experimental conditions that did not favor their growth such as high concentrations of DO during the aerobic phase (i.e. 7.0 0.5 mg/L) and considerably high organic loading rates (2.4 kg COD/ m3d) (Chudoba, 1985; Eckenfelder, 2000; Zheng et al., 2006). Figure 4.8b shows the presence of cavities between the clumped bacteria. These cavities are anticipated to be responsible in allowing a smooth mass transfer of substrates or metabolite products in and out of the granules (Tay et al., 2003 and Toh et al., 2003).

4.5.2

Cellular Characterization of Facultative Anaerobic Granular Sludge

The seeding sludge under microscopic observation showed a typical morphological structure of conventional activated sludge that contained the filamentous bacteria as the typical dominant species. The gram staining of the seeding sludge in Figure 4.9a shows the presence of filamentous bacteria as the main dominating species and mostly resulted with negative gram staining. The mature FAnGS reveal more of fat-rod and coccal-types bacterial morphotypes. The microbial presence resulted with both positive and negative gram stains (Figure 4.9b). This shows that there are changes in the dominancy of microorganisms in the development of the FAnGS.

104

Cavities

Figure 4.8

FESEM microstructure observations on mature facultative anaerobic

granular sludge under the magnification of 10,000K. (a) Coccoid bacteria tightly linked to one another. (b) Cavities that appear between bacteria clumped inside the granules

105

Figure 4.9 The changes on the microbial population during the process development of the FAnGS observed by gram staining procedures under microscopic magnification of 1000K (a) The sludge being dominated by the filamentous organisms. (b) Changes in the domination species within the FAnGS

106 4.5.3 Microbial Activity

A typical DO concentration profile for one complete cycle and the OUR profile during both of the aerobic reaction phases are shown in Figure 4.10. OUR gives an indication of the performance of biological activity of microorganisms in the reactor in terms of oxygen utilization. Higher OUR values designate of high biological activity and vice versa.

PI and PIII show the first and second stages of anaerobic reaction phase, respectively. At these anaerobic stages, most of the dye degradation process occurred where amines as the byproduct, were released (Sponza and Isik, 2005), while PII and PIV represent the first and second stage of aerobic reaction phase, respectively. Most of the co-substrates provided to the reactor system were consumed within few minutes of the first aerobic reaction phase (PII) and is known as the feast period. During the feast period, the DO concentrations in the reactor were low (about 4 mg/L). The high utilization of DO during the feast period was also indicated by the high OUR which was 281 mg/L.h. The amines, which were produced during the anaerobic reaction phase (PI), were mineralized under this aerobic condition (PII) as they cannot be further degraded under anaerobic phases (Stolz, 2001 and Sponza and Isik, 2005).

When all the carbon sources (substrate and amines) in the wastewater have being utilized, an endogenous respiration process took place, referred as the famine period. The DO concentration immediately increased to around 7.0 mg/L which was closed to the DO saturation level. The OUR also reduced to 14 mg/L.h indicating low utilization of DO. The transition from the feast to famine phase was clearly observed with the drastic increase of the dissolved oxygen and the extreme drop of the OUR within few minutes of the aerobic reaction phase (PII).

107

PI

PII

PIII

PIV

Figure 4.10

The profile of dissolved oxygen and oxygen uptake rate in one

complete cycle of the IFAnGSBioRec system () Dissolve oxygen, () Oxygen uptake rate (PI and PIII-Anaerobic phase; PII and PIV- Aerobic phase)

Since there was no addition of substrate during the second aerobic reaction phase (PIV), the consumption of DO during this phase was also low. This is shown by high DO levels reaching saturation values of 7.6 mg/L. A sharp increase in the OUR was also observed at the beginning of this phase. Apparently, the residual dyes which were not degraded in Stage PI and PII were transformed into smaller molecules (e.g. amines) during the second stage of the anaerobic phase (PIII). These smaller molecules were further mineralized in Stage IV which resulted in a sharp increase in the OUR. As the concentration of these molecules were reduced, the OUR also became lower until it reached a minimum of 11 mg/L.h. Table 4.5 shows the OUR value during both of the aerobic reaction phases.

108

Table 4.5: The OUR levels during the aerobic reaction phase of one complete cycle OUR (mg/L.h) Aerobic reaction phase Begin react End react

1 stage (PII) 281 39 14 2

st

2nd stage (PIV) 167 51 11 2

The SOUR of the FAnGS was determined before the termination of the experiment. The SOUR was 51.1 6.8 mg DO/g VSS.h. This value was slightly lower than those of the aerobic granules reported by Tay et al. (2001a) which ranged from 55.9- 69.4 mg DO/g VSS.h and higher than the coupled granules reported by Erguder and Demirer (2005a) (6-47 mg DO/g VSS.h). The SMA of the FAnGS is lower (10.3 mL CH4/g VSS.h) than the one reported by Erguder and Demirer (2005a) (14-42 mL CH4/g VSS.h). However, despite the low SMA emission, it provides the evidence of the existence of methanogens within the FAnGS.

4.5.4

Size of the Facultative Anaerobic Granular Sludge

The shear force imposed in the development of granules in this experiment, in terms of superficial upflow air velocity (i.e. 1.6 cm/s), resulted in the development of FAnGS with average diameter of 2.25 mm. This is in the range normally observed for anaerobic and anoxic granules. According to Peng et al. (1999), the diameter of the developed aerobic granule is in the range of 0.3 to 0.5 mm which is much smaller as compared to anaerobic and anoxic granules that could develop up to 2 to 3 mm. The strong shearing force produced by mixing and aeration during the aerobic phase prevents the development of bigger aerobic granules. However, reduction in famine period may also lead to the formation of bigger aerobic granular sizes (Liu and Tay, 2006). Other factors and conditions of the experimental set-up could also resulted to much bigger sizes (eg. 5 mm) as reported by Liu and Tay (2004).

109 4.5.5 Settling Velocity of the Facultative Anaerobic Granular Sludge

The average settling velocity of the sludge and anaerobic granular sludge used as the seeding in this experiment were 9.9 0.7 m/h and 42 8 m/h respectively. The average settling velocity of the anaerobic granular seed is in accordance with those reported by Schmidt and Ahring (1996) which was in the range of 18-100 m/h. The settling velocity of the FAnGS increased from 17.8 2.6 m/h to 83.6 2.6 m/h at the end of experiment. The average settling velocity of the mature FAnGS at the end of the experiment reached almost 80 7.6 m/h. The settling velocity obtained from this study was almost three times greater than the settling velocity of the aerobic granules reported by Zheng et al. (2005).

The increase in settling velocity has given significant impact on the biomass concentration in the reactor. The relationship between the concentration of the MLSS and settling velocity of the granules is shown in Figure 4.11. Despite the short settling time (5 min in this experiment), the high settling velocity possessed by the developed FAnGS enabled the granules to escape from being flushed out during the decanting phase. Such conditions have caused more FAnGS to retain in the reactor and resulted in the increase of biomass concentration.

The SVI value has also improved from 276.6 mL/g at the initial stage of the experiment to 69 mL/g at the end of the experiment indicating the good settling properties of the granules which is favorable in wastewater treatment plant operation. Figure 4.12 demonstrates the SVI profile along with settling velocity. As the SVI value improved, the granular settling properties increased from 50 m/h to about 80 m/h.

The SVI value achieved in this experiment is in agreement with the result reported by McSwain et al. (2004) with SVI values of 115 36 ml/g (settling time 10 min) and 47 6 ml/g (settling time 2 min). The higher settling velocity and lower

110 SVI value of the mature FAnGS as compared to previous reports by other researchers indicate that the formation of granules seeded with anaerobic granules would develop better settling properties of the granules.

Figure 4.11 concentration

The relationship between the biomass concentrations retained in the

reactor with the settling velocity of the FAnGS () Settling velocity; () Biomass

4.5.6

Granular Strength of the Facultative Anaerobic Granular Sludge

The granular strength of the granules was measured based on the integrity coefficient (IC) as mentioned earlier. The smaller the value of IC, the higher the strength and ability of the granules to clump themselves from being broken due to shear force of the aeration. Figure 4.13 shows the profile of IC of the developed FAnGS as a function of time. The IC reduced as the granules developed. With an initial value of 30, the IC was reduced to about 9 at the termination of the

111 experiment. A sharp reduction of IC was observed after 40 days of the experimental run. According to Ghangrekar et al. (2005), granules with integrity coefficient of less than 20 were considered high strength granules. The reduction in IC value indicates the increase in the strength of the bond that holds the microorganisms together within the developed granules.

Figure 4.12

The relationship between the SVI values and settling velocity of the

FAnGS () SVI, () Settling velocity

During the early stage of the granule development, the microbes within the granules were loosely bounded to each other. At this stage, the granules may consist of more cavities which make the granules less dense, as manifested by low settling velocity. As more microbes are linked together, the granules increase in size. Under certain selective pressures (i.e. short settling time, hydrodynamic shear force, starvation of the microbial cell) within the reactor, microbes may produce more extrapolysaccarides (EPS) (Lin et al., 2003 and Qin et al., 2004a). As reported by Zhang et al. (2007b) and Adav and Lee, (2008a), the EPS contribute greatly to the strength and the stability of aerobic granules. When more EPS are being produced

112 by the microbial cells, they form a cross-linked network and further strengthen the structural integrity of the granules. The cavities within the granules will be filled with the EPS as it is a major component of the biogranule matrix material. This caused the granules to become denser and stronger as shown by their high settling velocity and low IC value at the end of the experiment.

Figure 4.13 The profile of integrity coefficient representing the granular strength of the FAnGS

The physical characteristics of the seed sludge and the matured FAnGS are summarized in Table 4.6. The developed FAnGS possess the biomass characteristics that are desirable in the biological wastewater treatment system.

113 Table 4.6: Characteristics of seed sludge and FAnGS Characteristics SVI (mL/g) Average diameter (mm) Average settling velocity (m/h) IC MLSS (g/L) MLVSS (g/L) Seed Sludge 276.6 0.02 0.01 9.9 0.7 92 6 2.9 0.8 1.9 0.5 FAnGS 69 2.3 1.0 80 8 9.4 0.5 7.3 0.9 5.6 0.8

4.5.7

Biomass Concentration and Sludge Retention Time

The profile of the biomass concentration (i.e. MLSS) after seeding with the anaerobic granules is shown in Figure 4.14. rapid decrease in the biomass concentration. During the first few days of the The MLSS reduced from initial experiment, almost half of the sludge was washed out from the reactor causing a concentrations of 5.5 g/L to 2.9 g/L mainly due to the short settling time used in the cycle (i.e 5 min). During this initial stage, the anaerobic granules were also observed to disintegrate into smaller fragmented granules and small debris resulted from shear force caused by the aeration during the aerobic stage. These small fragments have poor settling ability and were washed out from the reactor. This caused an increase in the suspended solids concentration in the effluent. However, as the experiment continued, the concentration of the biomass increased and finally reached 7.3 g MLSS/L when the experiment was discontinued on the 66th day. The profile of MLVSS follows the same trend of MLSS, ranging from 1.9 g/L to 5.6 g/L.

114

Figure 4.14 The profile of biomass concentration in the SBR. () MLSS, () MLVSS

The mean cell residence time (SRT) also increased from 1.4 days at the initial stage to 8.3 days on the 66th day, indicating the accumulation of the biomass in the reactor. As less biomass was washed out during the decanting period, the increase in SRT is another manifestation of good settling characteristics resulting from the high settling velocity. Nonetheless, the benefit of high SRT will depend on the goal of the treatment process (Tchobanoglous et al., 2004). The SRT is affected by the settling velocity. The profiles of the settling velocity and the SRT as a function of time are given in Figure 4.15.

4.5.8

Mineral and Metal Content

The concentration of mineral and metal contents in sludge, newly developed and matured FGS were determined in mg/g of dry sludge and presented in Table 4.7. The trend of the concentration of the metal differs according to specific metals.

115

Figure 4.15

The settling velocity profile in relation to mean cell residence time

(SRT). () SVI, () SRT

The concentration of Na+ and K+ shows not much difference in the newly developed and matured granules as compared to the content in the sludge. However, there is an obvious increment on the concentration of Ca2+ and Mg2+ within the matured granule. The concentration of Fe2+ is slightly reduced in newly-developed and matured FAnGS as compared to the concentration in the sludge. As for the concentration of Ni2+ and Co2+, there is not much difference when compared between the newly-developed and matured granules. The concentration for both of these metals is lower as compared to the concentration in the sludge.

Stable concentrations of Na+ and slight decrease of K+ concentrations in the sludge and the matured granules may indicate that these monovalent cations are not involved in the granulation process. However, it was reported that at high concentrations, Na+ and K+ may cause adverse effect on the granules formation. It could cause reduction in sludge concentration, settling velocity of the sludge, granular strength and treatment efficiency (Ghangrekar et al., 2005). The

116 monovalent cation, Na+ could become the reason for detrimental impact on the flocculation system (Sobeck and Higgins, 2002).

Table 4.7: Comparison of mineral content at different stages during the development of FAnGS Mineral contents (mg/g of dry sludge) Mineral / Metal Ca2+ Mg2+ Na+ K+ Fe2+ Ni2+ Co2+ Sludge 1.53 0.02 0.13 0.01 0.22 0.06 1.31 0.06 2.32 0.02 0.60 0.02 0.149 0.003 Newly developed Matured FAnGS FAnGS (1 week) (10 weeks) 2.01 0.58 0.322 0.003 0.25 0.49 1.15 0.03 1.90 0.04 0.22 0.01 1.021 0.002 4.65 0.04 1.75 0.08 0.24 0.05 0.93 0.05 1.98 0.08 0.23 0.01 0.02 0.01

The developed FAnGS in this study showed higher accumulation of Ca2+ and Mg2+ as compared to the newly-developed and the seed sludge. This may indicate the involvement of these elements in the granulation process. Based on the divalent cation bridging theory, the presence of Ca2+ and Mg2+ promotes equivalent floc properties (Soberck and Higgins, 2002). The presence of Ca2+ was reported to intensify the granular strength and enhance the granulation process (Grotenhuis et al., 1991b; Jiang et al., 2003; Ghangrekar et al., 2005). These divalent cations are postulated to be able to stimulate granulation by neutralizing the negative charges on the bacterial cell surfaces that were developed due to the strong van der vaal attraction forces (Grotenhuis et al., 1991a and Tay et al., 2000). The Ca2+ acts as a cation bridge that interconnects between the EPS molecules and bacterial surfaces. The connections form as stiff polymeric gel-like matrix that could augment the

117 granulation development (Costerton et al., 1987; Sutherland, 2001; Jiang et al. 2003).

The presence of Ca2+ in the form of calcium carbonate and calcium phosphate produced an inert support for the bacteria to grow in the granulation process (Yu et al., 2001). Calcium uptake around 60 mg/g of Ca2+ in the granule has been reported to be the ideal concentration for good granule characterization with high strength and good settling property (Ghangrekar et al., 2005). Anything higher than this concentration was not recommended since it could cause increase in the ash content in the sludge due to chemical precipitation. Too much of Ca2+ could also give a detrimental effect on the performance and stability of the reactor systems and decrease in specific activity of the sludge system (Yu et al., 2001). Ca2+ at concentrations higher than 780 mg/L could caused 60-90% reduction in COD removal and serious cementation in the sludge bed through the high precipitation of Ca2+ (Langerak et al., 1998).

The Ni2+ and Co2+ uptake into the granules might be influenced by the metal requirement of the variety of microorganisms present in the reactor system (Ghangrekar et al., 2005). However, at higher concentration, these metals could inhibit some of the biological mechanisms of organisms (Bae et al., 2000).

4.5.9

Removal Performance

The performance of the FAnGS (after acclimatization stage) based on the removal of COD, color and ammonia is given in Figure 4.16 to 4.18. Figure 4.16 and 4.17 shows that, at the initial stage of the operation, the percentage removal for COD and ammonia were 71% and 67%, respectively. The removal efficiency has increased to 94% for COD and 95% for ammonia at the end of the experiment. The

118 increase in removal efficiency indicates the occurrence of high biological activity in the reactor system. During the first month, the removal efficiency for COD and ammonia was fluctuating. However, the removal became more stable for the remaining period.

Figure 4.16

Profile of COD removal during FAnGS development in

IFAnGSBioRec system. () Influent COD, () Effluent COD, () COD removal

The removal efficiency for color as shown in Figure 4.18 was fluctuating almost throughout the experiment. The percentage of color removal was about 25% during the start up and increased to 62% at the end of the experiment. The average color removal was 55%. This low percentage of the color removal may be due to insufficient HRT. As dye substances are recalcitrant and difficult to be degraded, more time is required by the organisms to degrade the dyes. The inconsistent percentage for color removal may be also contributed by the unstable condition of the aromatic amines, the byproduct of dye degradation which easily oxidized when exposed to oxygen during the aerobic phase. As will be discussed in Chapter 6, the removal of color becomes better at higher HRT.

119

Figure 4.17

Profile of Ammonia removal during FAnGS development in () Influent ammonia, () Effluent ammonia, ()

IFAnGSBioRec system. Ammonia removal

Figure 4.18

Profile of color removal during FAnGS development in

IFAnGSBioRec system. (100 ADMI 1 Platimun-Cobalt). () Influent color, () Effluent color, () Color removal

120 Figure 4.19 shows the percentage of removal of COD, ammonia and color in a complete 340-minute reaction mode of the SBR system recorded on the 66th days of experiment. The profile and the percentage of removal for COD and ammonia were almost the same while the removal of color was much lower. After 340 minutes of intermittent anaerobic and aerobic modes, about 93%, 95% and 62% of COD, ammonia, and color respectively were removed.

PI

PII

PIII

PIV

Figure 4.19

The removal for COD, ammonia and color in one complete cycle of

the SBR system () Color, () COD, () Ammonia

During the first anaerobic phase (PI) (0-40 min), approximately 15% and 4% of COD and ammonia respectively, were removed. In the first aerobic phase (PII) (40-170 min), about 69% of the COD was removed while 80% of the ammonia was oxidized. In the second anaerobic phase (PIII) (170-210 min), only about 5% of COD and ammonia were removed while the remaining (about 4% for COD and 6% for ammonia removal) were removed in the second aerobic phase (PIV) (210 to 340 min). As for color, about 46% and 16% were removed during the anaerobic and aerobic phases respectively.

121 The degradation and decolorization of dye during the anaerobic condition has been widely reported in the literatures (van der Zee et al., 2001a and Dos Santos et al., 2007). The electrons from the electron donor are transferred to the N=N bond of the azo dye causing the cleavage of the bond forming aromatic amines in anaerobic condition. The amines are then degraded under aerobic condition reducing the COD value of the wastewater. In addition to the degradation mechanism, dye removal may also occur via adsorption onto the biomass. The removal of ammonia which mainly takes place during the first aerobic stage is expected to be caused by the nitrification process.

Based on the removal performance of the system, it has been proven that the developed FAnGS is capable of performing the degradation process during the anaerobic and aerobic phases. This indicates the presence of facultative and anaerobic microorganisms in the FAnGS. According to Li and Liu (2005), when the granules grew to a size larger than 0.5 mm, the diffusion of oxygen into the inner part of the granules became a limitation. This may give an indication of the presence of anaerobic microorganisms within the centre of the FAnGS since the average size of FAnGS developed in this study was 2.3 1.0 mm. Aerobic microorganisms may be present at the outer layer of the granules which easily access the oxygen molecule and mainly responsible for the COD removal. live under both anaerobic and aerobic conditions. Meanwhile, the facultative microorganisms may be present in any part of the FAnGS due to their capability to

4.6

Conclusions

Several conclusions could be drawn from the study and they are as follows: i. Stable FAnGS could be cultivated in the SBR system with the application of intermittent anaerobic and aerobic reaction modes during the reaction

122 phase. The matured granules showed the domination of non-filamentous bacteria that were tightly linked and embedded to one another and covered with EPS. The morphology of the developed granules is affected by the seed used in the development process.

ii. After 66 days of operation, matured FAnGS has reached an average diameter of 2.3 1.0 mm with settling velocity of 80 8 m/h. The SVI value of the biomass has decreased from 276.6 mL/g to 69 mL/g at the end of 66 days, also indicating the excellent settling properties of the granules. The SRT increased from 1.4 days during the initial stage to about 9 days at the end of the experiment demonstrating the accumulation of biomass in the reactor system. The FAnGS is also structurally strong as shown by a low IC value of 9.4 0.5. By comparing the settling property of the FAnGSs with the granules developed by other researchers, the cultivation of granules seeded with anaerobic granules resulted in better granules.

iii. The development of FAnGS is positively-correlated with the accumulation of divalent cationic Ca2+ and Mg2+ in the granules suggesting the role played by the cations in the granulation process.

iv. The developed FAnGS was able to remove COD and ammonia in the wastewater up to more than 93%. Although the average removal of color was only about 55%, the results indicate the viability of the granulation system in treating textile wastewater under intermittent anaerobic and aerobic phase strategy.

v. OUR/SOUR and SMA analysis proved the presence of anaerobic and aerobic microorganisms activities in the FAnGS and capable of performing the degradation process both in anaerobic and aerobic conditions.

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CHAPTER 5

EFFECT OF AGGREGATION AND SURFACE HYDROPHOBICITY BY SELECTED MICROBES FROM FACULTATIVE ANAEROBIC GRANULAR SLUDGE

5.1

Introduction

Granulation is a complex process. Development of compact aerobic granules is initialized by the formation of small aggregates (Adav et al., 2008a) that does not rely on the need for carriers or artificial surfaces for cell attachment (Liu and Tay, 2002). The mechanisms involved in granulation are subjected to a multiple-step process that involves many aspects of physicochemical and microbiological features. These steps may be affected by many factors including types and concentrations of substrate in the influent, nature of the seed sludge, availability of essential nutrients, presence of extracellular polymeric substrates (EPS), composition of the media that may contain different concentrations of divalent cations, pH and temperature of the experiments and also the operational set-up of the reactor system (Dignac et al., 1998; Linlin et al., 2005; Liu and Tay, 2007c; Yi et al., 2008; Wang et al., 2009b). The interaction of microorganisms during the initial stage of the granulation process may play a significant role in the assurance of successful development of the granulation process. Investigation on factors that may affect the mechanisms during

124

the initial stage of the granulation process should be considered as a crucial aspect to be explored.

Aggregation ability and the surface hydrophobicity (SHb) of bacteria are two independent traits that can be used as an indirect method for evaluating the adhesion ability of bacteria (Marin et al., 1997; Ibrahim et al., 2005; Rahman et al., 2008). Since the adhesion ability is postulated to be involved in the granulation process, study on autoaggregation or coaggregation (CAg) and SHb of the granules and/or among the bacteria found within the granules have become one of the main focuses in the foundation for a better understanding on the granulation mechanisms. Few attempts have been made by researchers concentrating on the effect of aggregation and SHb (Dabert et al., 2005; Wang et al., 2005a; Ivanov et al., 2006). However, the knowledge on this aspect particularly on the development of dye degrading granule is very limited. Not much research has been carried out to study the factors affecting aggregation and SHb among the microbes involved in the granulation process.

This study was conducted to investigate the response on CAg and SHb of selected mixed bacteria which have been isolated from FAnGS. Deeper investigation at microscopic level was carried out to determine the effect of substrate concentration, pH and temperature on the CAg and the SHb of the microbes isolated from the FAnGS developed in the IFAnGSBioRec system described in Chapter 4.

5.2

Materials

Some of the chemicals/reagents and equipments used in this study are listed in Section 4.2 and are described in Section 4.3. In addition, another list of chemicals

125

and reagents and analytical equipment specified used in this experiment of this chapter are given in Tables 5.1 and 5.2, respectively.

Table 5.1: List of reagents used in the experiment Chemical/Reagent Nutrient agar Potassium phosphate, K2HPO4 Xylene (C6H4(CH3)2) Nutrient broth Promega DNA extraction kits Promega DNA purification kits Ethanol Agarose gel TAE buffer Bromophenol blue SDS Loading dye (Appendix C-2) Glycerol Gene Ruler Ladder Mix Marker Reverse and Forward primer PCR reaction solution Promega WizardSV gel PCR product purification (Appendix C-4) PCR clean-up system PCR amplification (Appendix C-3) Gel eletrophoresis (Appendix C-2) Buffering system for electrophoresis (Appendix C-2) DNA extraction and purification (Appendix C-1) Applications Spread plate (Section 5.3.1) Washing buffer (Section 5.3.6) Surface hydrophobicity assay (Section 5.3.6) Bacteria culture (Section 5.3.7)

126

Table 5.2: List of equipment used in the experiment Equipment Microcentrifuge Turbidity Meter Optical Density Meter Vortex 0.2 m Filter Syringe filter Microwave Eletrophoresis Apparatus Power pack UV Transilluminator Thermocycler Manufacturer/Product Sartorius/Sigma 1-14 HANNA Instrument/ H 93703 Microprocessor BUCK Scientific/100 VIS Spectrophometer IKA/MS 1 Minishaker Sartorius (M) Sdn. Bhd./Minisart-Ny25 Sartorius (M) Sdn. Bhd./Minisart-Ny25 ELBA/EMO-1706 BIO-RAD Laboratores/MiniSub-Cell BIO-RAD Laboratores/PowerPac Basic Vilber Lourmat/ TFX-35 Vilber Lourmat Perkin Elmer/GeneAmp PCR System 9700

5.3

Analytical Methods

5.3.1 Chemical Oxygen Demand and Color Removal

In this study, the removal efficiency of COD and color of each of the isolated bacteria from the FGS were conducted by batch experiment. The COD and color removal were measured according to the analytical methods described in Section

127

4.3.4. The specific COD and color degradation rate were obtained by dividing the percentage of COD or color removal with the biomass concentration for each of the individual bacteria and time taken for the removal to take place. methods as described in Section 4.3.2.4. The biomass concentrations for each of the bacteria were measured according to the analytical

5.4

Experimental Procedures

The experiments in this study were conducted in two stages. The first stage involved the isolation of microorganisms from the FAnGS and screening of microorganisms based on their ability to degrade COD and dyes. Figure 5.1 shows the experimental work conducted on the characterization of the microbes isolated from the FAnGS. The degradation of dyes was measured as the percentage of color removal. In the second stage the effect of substrate concentration, pH and temperature on CAg and SHb of selected bacteria as a mixed culture were investigated using factorial and response surface methods. The summary of the experimental work for this second stage is shown in Figure 5.2.

5.4.1 Isolation Procedure of Bacteria Strain

The synthetic textile dyeing wastewater (STDW) was prepared as explained in Section 4.2.1. For the bacteria isolation, the synthetic dyeing wastewater which was used as the growth medium was sterilized by autoclaving the media for 20 min at 121oC. The mixed dyes, glucose and minerals were filtered through 0.2 m membrane filter for sterilization purposes. Then, these materials were added

128

separately into the autoclaved medium. Using aseptic techniques, a small amount of mature granules were added to synthetic dyeing medium (15 mL) and mixed in a sterilized beaker in order to disintegrate the granules. The supernatant was serially diluted with medium (101 to 108 fold dilutions) before inoculation on to nutrient agar (NA). Eight dilution bottles filled with 9 mL of sterilized distilled water were prepared for sample dilution process. The samples were serially diluted by transferring 1 mL of samples from the lower serial dilution (10-1) to the next serial dilution bottle (10-2) until the eighth serial dilution bottle (10-8). Finally, about 1 mL of each dilution was spread onto nutrient agar using a glass spreader. The isolation of microorganisms was carried out by the spread plate method (Madigan et al., 2000). The plate was inverted and incubated at room temperature and were monitored over several weeks. Pure bacterial cultures were obtained by repeatedly subculturing onto new nutrient agar plates until single pure colonies were obtained. The single bacterial colonies were investigated for morphological and cellular characteristics.

5.4.2 Morphological Characterization

The pure culture bacteria isolated from FAnGS were characterized based on the colony and cellular morphology of single colonies obtained. The colony morphology on the nutrient agar was characterized for their size, shape, color, margin and elevation. The colonies were examined using a stereo microscope (Leica Zoom 2000) and the Pax-it Image Analyzing System. The analytical method of gram-staining for the cellular morphology examination was according to Section 4.3.1.1.

Figure 5.1 Characterization of microbes isolated from the FAnGS granules

129

130

Figure 5.2 Experimental work for the investigation on the effect of substrate concentration, pH and temperature on the percentage of coaggregation and surface hydrophobicity of the mixed culture

131

5.4.3 Identification of Microorganisms Isolated from Facultative Anaerobic Granular Sludge

Bacteria isolated from the FAnGS were further investigated for identification of the microorganisms. The identification of the microorganisms involved several stages beginning with the DNA extraction by using the DNA extraction kits (Promega) in order to obtain the genomic DNA of the microbes. The successful isolations of the genomic DNA were identified by running the gel electrophoresis. In order to increase the magnitude of the isolated DNA, the genomics DNA were then amplified by using the polymerase chain reaction (PCR) amplification process. In the PCR process, two universal primers were used to amplify the 16S rRNA gene of the selected bacteria. The amplified DNA was purified using PCR purification kits before the sequencing of the genomic DNA was performed. The genomic DNAs were sent to Vivantis Sdn. Bhd. for the sequencing purposes. Through the result of the sequencing process, the identifications of the bacteria were obtained through the BLASTn search which was carried out at the National Center of Biotechnology Information (NCBI). The detailed procedure of microbial identification by using 16S rDNA sequence analysis is provided in Appendix B.

5.4.4 Specific Growth and Screening for Dye-Degrading Bacteria

The cells used in the specific growth observation were at the exponential growth in mixed dye (MD) medium. Each bacterium was inoculated in separate bottles with 10% v/v of pure culture into the sterilized synthetic textile dyeing wastewater. The experiments were performed in duplicate in 1 L schott bottle. Each bottle contained 1L of MD medium with an initial mixed dye concentration of 100 mg/L. A mixture of substrate consisted of glucose; acetate and ethanol were added at the concentration of 1500 mg/L. Individual strains were introduced into the

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medium and were cap immediately and incubated at room temperature.

The

decolorizations of the mixed dye were measured at regular intervals during incubation. The cell growth was monitored by optical density (OD) measurement with an Optical Density Meter (100 VIS Spectrophometer) at wavelength of 600 nm. The specific growth rate and dye degradation rate were calculated by performing a linear regression on initial curves of cell growth and dye decolorization, respectively against time.

5.4.5 Autoaggregation Assay

Samples which were used for the growth and dye degradation rate were used for CAg and hydrophobicity assay. The synthetic textile dyeing wastewater in each of the schott bottle was allowed to reach complete decolorization (more than 90%) before autoaggregation assay was carried out. The aggregation assay was conducted based on the procedure presented by Rahman et al. (2008) with several modifications. Each of the samples was aerated using an air diffuser which was connected to an air pump at a rate of 5 L/min. The aeration was stopped 15 min after the growth of the bacteria reached the stationary phase. Fifteen (15) mL of samples from each bottle was taken for the autoaggregation assay.

The initial turbidity of each sample was measured using a turbidity meter (H 93703 Microprocessor) as the initial reading. Then, the samples were centrifuged at a slow centrifugation speed of 650 g for 2 min as described by Malik et al. (2003) before the turbidity measurements were taken again. The CAg ability was expressed as coaggregation percentage and calculated by using the equation below:

133

(5.1) where, CAg% = CA0 CAi =

=

Percentage of coaggregation The absorbance of cultured media at 0 h The absorbance of cultured media after centrifugation

From the CAg%, the mixed culture could be classified into three groups: high coaggregation (HCAg: >70% CAg), medium coaggregation (MCAg: 20-70% CAg) and low coaggregation (LCAg:<20% CAg) cultures. Lee, 2008b). A high aggregation index denotes a strong tendency of the cells to agglomerate into an aggregate (Adav and

5.4.6 Surface Hydrophobicity Assay

The surface hydrophobicity (SHb) of the bacterial strains either in the form of single or as mixed cultures were based on the microbial adhesion to hydrocarbon assay. SHb of the mixed bacteria was determined according to the methods described by Zavaglia et al. (2002) and Canzi et al. (2005). Fifteen (15) mL of samples were taken from the sample bottles used for growth and dye degradation rate tests in the previous experiments. The bacterial cells were harvested by centrifugation at 14,000xg for 5 min. The samples were washed twice with 50 mM K2HPO4 (pH 7.0) and then resuspended in the same buffer to obtain an absorbance of about 0.5 at 660nm. Five (5) mL of bacterial suspension was mixed with 1 mL of xylene (C6H4(CH3)2) by vortexing for 120s and then allowed to stand for 1 hour at room temperature. The absorbances of the bacterial suspension in the aqueous phase after mixing (Ai) were compared to the absorbance taken at the initial stage of the experiment (A0). Changes in absorbance due to the bacterial adhesion to the hydrocarbons were measured as 660 nm by using an Optical Density Meter (OD)

134

(Jenwai-6300 Spectrophometer). equation:

The surface hydrophobicity was expressed as

surface hydrophobicity percentage (SHb%) and calculated by using the following

(5.2)

where, SHb% = A0 Ai = = Percentage of surface hydrophobicity The absorbance of sample before mixing with xylene The absorbance of sample after extraction with xylene.

5.4.7 Effect

of

Substrate

Concentration,

pH

and

Temperature

on

Coaggregation and Surface Hydrophobicity

Based on the results of the previous analysis which includes dye degradation tests, autoaggregation and SHb assay, six out of twelve isolated bacteria were selected for further study. These selected bacteria were labels as bacteria BS1FAnGS, BS6FAnGS, BS7FAnGS, BS10FAnGS, BS11FAnGS and BS12FAnGS.

These bacteria were used as a mixed culture to determine the effect of substrate concentration, pH and temperature on the CAg of and SHb of the mixed culture. Each of the selected single bacteria cell were cultured separately in nutrient broth until the measurement of the OD was near to 1. Then, the cell cultures were harvested and re-suspended in saline water. The individual bacteria were then mixed together and about 25 mL (10%v/v) of sample culture were inoculated in a separate 500 mL schott bottle containing STWW with the concentration of dye at 10 ppm.

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The substrates containing glucose, acetate and ethanol were used as the mixed external carbon sources. The final pH of the culture media, the temperature for sample incubation and the concentration of substrate used in the experiment were varied depending on the experimental design. The synthetic textile dyeing wastewater which has been inoculated with the selected mixed culture was allowed to decolorize before the CAg and SHb assay were carried out. After the synthetic textile dyeing wastewater was decolorized more than 90%, the sample were aerated by supplying air at a flow rate of 5 L/min. The samples were aerated continuously for 5 hours. Ten (10) mL samples were taken hourly throughout the aeration phase and used for the CAg and SHb assay.

5.4.8 2-Level Factorial Experimental Design

The effects of substrate concentration, pH and temperature on CAg and SHb of the selected mixed culture in the synthetic textile dyeing wastewater were investigated using a 2-level factorial experimental design. The range values of the factors considered in the experiments are listed in Table 5.3. For a three variables factorial design, a complete matrix would have been 23 which are equal to 8 experimental runs. Since the experiments were conducted in duplicate, a total of 16 experiments were carried out. Table 5.4 shows the factorial design of the study. Each of the independent variable was investigated at a high (+1) and a low (-1) level. The experimental design and the analysis were carried out using MINITAB statistical software.

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Table 5.3: The variables and their range of high and low values used in the factorial experiment
Variables A: Substrate B: pH C: Temperature
o

Unit mg/L

Low Value 500 5

High Value 3000 9 40

20

Table 5.4:

Two-level fractional factorial design with three variables (in coded

levels) conducted in duplicate (not in randomized order)

Factor 1 Run No. A: Substrate CASE01 CASE02 CASE03 CASE04 CASE05 CASE06 CASE07 CASE08 CASE09 CASE10 CASE11 CASE12 CASE13 CASE14 CASE15 CASE16 -1 -1 +1 +1 -1 -1 +1 +1 -1 -1 +1 +1 -1 -1 +1 +1 B: pH -1 -1 -1 -1 +1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 +1 C: Temperature -1 -1 -1 -1 -1 -1 -1 -1 +1 +1 +1 +1 +1 +1 +1 +1 Factor 2 Factor 3

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5.4.9 Response Surface Methodology (Central Composite Design)

The Response Surface Method (RSM), namely Central Composite Design (CCD) was used to investigate the possibility of non-linear effect of the selected variables. In addition to the factorial trials, the CCD was run with five replicate at the central point together with + and points. This was employed to fit the secondorder polynomial models and to obtain an experimental error of the experiment. The range and levels of experimental variables investigated in this study are the same used in the factorial design process as presented in Table 5.3. For CCD, a total of twenty experimental runs were carried out. Table 5.5 shows the design matrix for substrate, pH and temperature as the variables in coded units for the CCD experimental run.

Based on the CCD design matrix, the experiments were divided into three parts; a 23 factorial design point run (CASE01 to CASE08), the star point run (CASE09 to CASE14) and the centre point runs (CASE15 to CASE20). In this study, the responses were the percentage of CAg and SHb of the mixed microorganisms selected from the FAnGS. The main and synergistic effects of the factors were determined based on the factorial points and centre point runs. The nonlinear response behavior was analyzed using the star points and centre points runs. The centre point runs were repeated six times in order to achieve a better estimation of the experimental error (pure error). The experimental runs were conducted in randomized order. The main reason of randomization is to reduce bias of unexpected element during experimental runs.

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Table 5.5: Two-level of CCD experimental run in coded units

Factor 1 Run No. A: Substrate CASE01 CASE02 CASE03 CASE04 CASE05 CASE06 CASE07 CASE08 CASE09 CASE 10 CASE 11 CASE 12 CASE 13 CASE 14 CASE 15 CASE 16 CASE 17 CASE 18 CASE 19 CASE 20 -1 1 -1 1 -1 1 -1 1 -1.682 1.682 0 0 0 0 0 0 0 0 0 0 B: pH -1 -1 1 1 -1 -1 1 1 0 0 -1.682 1.682 0 0 0 0 0 0 0 0 C: Temperature -1 -1 -1 -1 1 1 1 1 0 0 0 0 -1.682 1.682 0 0 0 0 0 0 Factor 2 Factor 3

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5.5

Results and Discussion

5.5.1 Morphological and Cellular Characterization of Bacteria Isolation from Facultative Anaerobic Granular Sludge

A total of twelve microorganisms have been successfully isolated as pure culture bacteria from the FAnGS developed in synthetic textile wastewater in the IFAnGSBioRec system. All of the 12 bacteria were characterized in terms of form, shape, edges and colony surface for their colony characteristics. Gram staining procedure was carried out for each bacterium in order to characterize the cellular morphology of the isolated bacteria. The pure bacteria isolates were named accordingly as BS1FAnGS to BS12FAnGS. Almost all of the isolated pure culture showed the presence of EPS indicated by slimy and gleaming emergence. Table 5.6 shows the results of the cellular and colony morphology of the isolated pure culture from FAnGS. C-3. The examples that illustrate the colony and cell morphology used to describe the characteristics of the isolated bacteria are given in Appendices C-2 and

5.5.2 Screening for Dye Degrader and Autoaggregator from Bacteria Strain Isolated from Facultative Anaerobic Granular Sludge

All of the 12 pure bacteria cultures isolated from FAnGS were screened for the ability of dye degradation and autoaggregation. The specific COD and dye degradation rates were also determined to assist the selection of the isolated bacteria. Table 5.7 shows the bacterial growth rate, percentage of color and COD removal, specific COD and dye degradation rate, percentage of aggregation and hydrophobicity of the individual bacteria. The percentage of COD removal was

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measured during the decolorization of the mixed dye medium where most of the decolorization took place after seven days of incubation period. Not much of the COD was removed during the decolorization that occurred under anaerobic condition. Most of the individual bacteria isolate showed a slight increase in the COD during the anaerobic phase. COD reduction was higher during the aerobic reaction phase.

Table 5.6: Morphological and cellular characterization of the twelve isolated bacteria from FAnGS
Bacteria strain BS1 FAnGS BS2 FAnGS BS3 FAnGS BS4 FAnGS BS5 FAnGS BS6 FAnGS Gram staining + + + Cellular morphology Palisades Streptobacilli Coccobacilli Long rod streptobacilli Palisades Rod Rod Streptobacilli Palisades Long rod streptobacilli Coccobacilli Coccobacilli Form Circular Punctiform Punctiform Irregular Circular Irregular Irregular Punctiform Circular Irregular Irregular Circular Colony morphology Edge Entire Entire Entire Undulate Entire Undulate Undulate Entire Entire Undulate Undulate Entire Surface Smooth Smooth Smooth Dry, powdery Dry Dry Dry, powdery Smooth Smooth Dry, powdery Dry, powdery Smooth Elevation Convex Convex Convex Raised Flat Flat Raised Convex Convex Raised Raised Convex

BS7 FAnGS BS8 FAnGS BS9FAnGS BS10FAnGS BS11 FAnGS BS12 FAnGS

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Based on the ability to degrade dye and the competency of the individual bacteria to form aggregates, the six bacteria i.e. BS1FAnGS, BS6FAnGS, BS7FAnGS, BS10FAnGS, BS11FAnGS and BS12FAnGS are selected for further analysis. These six selected bacteria show high capability to degrade the dye with the percentage of more than 85% and a moderate range of COD removal with the percentage of 1556%. Based on the percentage of autoaggregation and SHb, all the selected bacteria are of high CAg and high SHb with more than 70% for both CAg and SHb. The identity of the selected bacteria was investigated to classify the genus and species of the bacteria through 16S rDNA sequence analysis.

5.5.3 Analysis of the Isolates from Facultative Anaerobic Granular Sludge

Molecular techniques of bacteria identification involved several steps which include bacteria isolation, PCR amplification process either by using specific or universal primer, characterization and finally determination of taxonomic and phylogenetic class of bacteria isolates via 16S rDNA sequence analysis (Margarita et al., 2001). PCR is a powerful tool with its ability to exponentially amplify specific nucleic acid sequences in a short period of time. The amplification of deoxyribonucleic acid (DNA) via PCR is achieved through multiple cycles of in vitro DNA replication (Kuslich et al., 2008). The result of nucleic acid sequence analysis will complement and confirmed the conventional bacteria identification assay. In this study, the corresponding 16S ribosomal DNA (rDNA) sequence analysis was applied rather than using the ribosomal RNA (rRNA) since the former approach would grant stable and more informative analysis as compared to the latter. Furthermore, the analysis of 16S ribosomal DNA (rDNA) would be able to provide information of bacteria identification up to the genus and species level via its sequence polymorphisms (Fox et al., 1993).

Table 5.7: Characteristics and performance of the isolated bacterial from the FAnGS
Bacterial growth rate Bacteria strain Anaerobic phase 0.026 0.024 0.027 0.027 0.030 0.044 0.030 0.077 0.027 0.035 0.032 0.030 Aerobic phase 0.229 0.233 0.173 0.147 0.179 0.072 0.195 0.141 0.206 0.143 0.174 0.159 Max biomass (mg /L) 2.28 2.05 1.73 1.42 1.74 1.48 2.28 1.40 1.60 2.07 2.19 2.05 Color removal (ADMI) (%) 86.5 83.0 77.9 82.6 82.7 91.5 87.1 82.1 79.3 86.7 88.0 82.7 Specific dye degradation rate (mg/g/h) 0.287 0.278 0.040 0.221 0.045 0.229 0.285 0.150 0.136 0.150 0.425 0.196 COD removal (%) 47.3 10.3 10.2 40.6 27.3 15.2 37.6 12.1 40.0 54.5 33.3 40.9 Specific COD degradation rate (mg/g/h) 1.80 4.40 0.29 2.06 0.77 0.73 1.52 0.32 1.94 2.29 1.32 1.69 Surface Hydrophobicity (%) 82.8 1.6 39.6 1.8 61.4 2.5 35.5 1.3 90.2 2.8 97.3 1.4 87.4 1.1 12.5 2.7 21.5 3.2 85.7 1.8 90.2 4.2 88.4 3.3

Autoaggregation (%)

BS1FGS BS2FGS BS3FGS BS4FGS BS5FGS BS6FGS BS7FGS BS8FGS BS9FGS BS10FGS BS11FGS BS12FGS

97.3 1.4 40.2 0.9 20.4 1.5 35.7 1.1 18.5 1.8 77.4 1.2 77.9 1.5 31.4 1.1 12.5 1.4 85.5 1.4 82.8 1.8 61.4 1.5

142

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As discussed earlier, six bacteria have been selected for further analysis for their taxonomic and phylogenetic status. The bacteria were extracted for their ribosomal DNA and were amplified through the PCR amplification process by using universal primers with forward and reverse primer. In this study, all the DNA of the selected bacteria was successfully extracted individually. These were shown by the clear formed band on the agarose gel electrophoresis. The amplified 16S rDNA sequences were purified using protein purification kits (Promega PCR Clean-up System). The purification kits were used to remove impurities that may interfere the recovery of clear DNA band. Figure 5.3 shows the qualitative analysis of the PCR products. The observation of visible and clear bands from Lane II to VII indicates the successful isolation of high concentration and purity of the genomic DNA. Based on the comparison with the DNA marker Ladder, the obtained genomic DNA extraction was more than 10kbp indicating that the DNA samples are pure enough to be used as a template in the PCR amplification process for further analysis.

The amplification of the PCR products after purification by using Vivantis GF-1 Gel DNA Recovery Kit is shown in Figure 5.2. Lane II to VII shown in Lane I Figure 5.4 represents the genomic DNA PCR product of bacteria strain BS1FAnGS, BS6FAnGS, BS7FAnGS, BS10FAnGS, BS11FAnGS and BS12FAnGS, respectively. represent the DNA ladder marker. The PCR product of the selected bacteria strain were successfully amplified and purified based on the comparison with the DNA ladder. The results indicated that all PCR products could be observed at approximately 1.5 kbp.

The amplified 16S rDNA of the PCR products were sent to Vivantis Technologies Sdn. Bhd. for the sequencing process. The 16s rDNA sequencing results were then compared with the Genbank database at the National Center for Biotechnology Information (NCBI) using the nucleotide-Basic Local Alignmentt Search Tool (BLASTn) program for the identification of genus and species of the isolated bacteria (Altschul et al., 1990). Through the BLASTn analysis, the alignment scores based on forward and reverse sequence of partial 16S rDNA of the

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selected bacteria strains were determined.

BLASTn is a nucleotide-matching

program with improved search speed and firm statistical establishment to support the database searching. A reliable justification of a homology is obtained through high percentage of DNA nucleotide similarity, an E-value of less than one and a high score of more than 80 bits (Altschul et al., 1990 and Bergeron, 2002).

10 kbp

Figure 5.3 Agarose gel electrophoresis of DNA extraction Lane I: DNA ladder marker Lane II: Genomic DNA extraction of BS1FAnGS Lane III: Genomic DNA extraction of BS6FAnGS Lane IV: Genomic DNA extraction of BS7FAnGS Lane V: Genomic DNA extraction of BS10FAnGS Lane VI: Genomic DNA extraction of BS11FAnGS Lane VII: Genomic DNA extraction of BS12FAnGS

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I II III IV V VI VII VIII

1.5 kbp

Figure 5.4 Agarose gel electrophoresis of purified PCR amplification product Lane I: DNA ladder marker Lane II: Amplified 16S rDNA of BS1FAnGS Lane III: Amplified 16S rDNA of BS6FAnGS Lane IV: Amplified 16S rDNA of BS7FAnGS Lane V: Amplified 16S rDNA of BS10FAnGS Lane VI: Amplified 16S rDNA of BS11FAnGS Lane VII: Amplified 16S rDNA of BS12FAnGS

The sequencing result analysis reveals that BS1FAnGS could be identified as Pseudomonas veronii. A full length of sequencing containing 1394 nucleotides base pair was obtained from the NBCI showing 100% similarity with strain Pseudomonas veronii UFZ-B547. The results showed that the E value was zero with a very high total score of 2555 indicating a reliable justification of a homology. Based on the alignment scores of sequence generated from the forward primer, 338 sequence nucleotides were obtained. From this sequence, it shows that BS6FAnGS has 94% similarity with Bacillus cereus strain S10 with a total score of 525 and E-value of 5e145 generated from forward primer.

Meanwhile, based on the 595 nucleotides

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sequence result generated from the reverse primer has 98% similarity to Bacillus cereus with a total score of 1066 and E-value of zero. Based on the forward and reverse sequence analysis that show high percentage of similarity, high total score and less than one of E-value, BS6FAnGS is identified as Bacillus cereus.

Strains BS7FAnGS, BS10FAnGS and BS12FAnGS were identified as Pseudomonas sp. However, based on the alignment score of the sequence generated by the forward and reverse primers were not from the same species since there were differences in the arrangement of nucleotides bases. Based on the obtained sequences, these three strains can be grouped under the same type of Pseudomonas genus. The analysis sequence for strain BS7FAnGS showed the forward and reverse sequences with 383 and 620 nucleotides based pair, respectively. Based on the 383 nucleotides forward sequence, strain BS7FAnGS demonstrate 98% similarity, total score of 686 and zero Evalue with Pseudomonas citronellolis strain NK 2.C2-1. Meanwhile, the reverse primer generated 620 nucleotides sequence that showed 99% similarity with Pseudomonas sp.J9(2007). The total score was high (1110) and the E-value was zero. Based on the obtained result, therefore strain BS7FAnGS was identified as Pseudomonas sp. Strain BS10FAnGS was identified as Pseudomonas sp. based on the 99% similarity and zero E-value of the forward (538 nucleotides) and reverse (787 nucleotides) sequences with high total score of 966 and 1443, respectively. The closest relative to the strain identified from the forward nucleotides sequences was Pseudomonas trivials strain BIHB 745. Meanwhile, the Pseudomonas sp. mandelli was identified as the closest relative to strain BS10FAnGS based on the reverse nucleotides sequence. BS12FAnGS was also identified as Pseudomonas species based on the full length sequences with 1403 based pair nucleotides. The generated full sequences exhibit 99% similarity to strain Pseudomonas species with a very high total score of 2536 and zero E-value.

BS11FAnGS is identified as Enterobacter sp. This was also based on the full length sequencing result analysis that show 99% similarity, high total score of 2603 and zero E-value with Enterobacter sp. VET-7, Enterobacter sp. L3R3-1 and

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Enterobacter asburiae strain J2S4. The detailed results on the DNA sequencing analysis and the BLASTn analysis for selected bacteria strain are provided in Appendix D. Table 5.8 shows the result of the alignment scores of sequences generated as the percentage of similarity and the confirmed identification of the bacteria strains BS1FAnGS, BS6FAnGS, BS7FAnGS, BS10FAnGS, BS11FAnGS and BS12FAnGS. The detailed characterizations of the identified bacteria strain describing the physical and chemical distinctiveness is given in Table 5.9 (John et al., 1994).

5.5.4

Effect of Substrate, pH and Temperature on Coaggregation and Surface Hydrophobicity

In this study, aeration was applied to generate shear force effect rather than using physical shaking as commonly reported in previous aggregation research papers (Rahman et al., 2008; Nishiyama et al., 2007; Adav and Lee, 2009). The reason of using aeration is to allow a close resemblance of a real condition that took place in the granulation process. As discussed in Chapter Four, the aeration was used to introduce the shear effect onto the microorganisms in the reactor to allow the initialization of the granulation process.

In this chapter, focus is made on investigating the effect of substrate, pH and temperature (terms as variables) on CAg and SHb (terms as responses) of the selected mixed culture from FAnGS in synthetic textile dyeing wastewater. MINITAB statistical software is used as the analytical tool for factorial design analysis. The effect of these variables was presented by the response produced by a change in the level of the factors investigated. When the effect of one variable is affecting the responses of other variables, there is an interaction effect between the variables studied.

Table 5.8: Taxonomic and phylogenetic characteristic of the isolates from FAnGS
No. of bases used to establish identity 1394 Forward seq. 338 BS6FGS Reverse seq. 595 Forward seq. 383 BS7FGS Reverse seq. 620 Forward seq. 538 BS10FGS Reverse seq. 787 BS11FGS BS12FGS 1429 1403 99 99 99 0 0 98 99 0 1443 2603 2536 98 98 0 1110 966 16S rRNA gene sequence identity (%) 99 94 5.00E-146 1066 686 Bacillus cereus partial Pseudomonas citronellolis strain NK 2.C2-1 Pseudomonas sp.J9(2007) Psedumonas trivialis strain BIHB 745 Pseudomonas sp. mandelii Enterobacter sp. VET-7 Pseudomonas veronii strain INA06 Enterobacter sp. Pseudomonas sp. Taxonomic affliation(s) Pseudomonas veronii

Isolates

E value

Total score

Closest relative Pseudomonas veronii 6S rRNA Bacillus cereus strain S10

BS1FGS

2555 525

Bacillus cereus

Pseudomonas sp.

Pseudomonas sp.

148

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Table 5.9: Characteristics of identified selected bacteria strains from FAnGS

Bacteria strain Characteristics

Bacillus cereus gram positive or facultative anaerobic spore forming rod (BS1FAnGS) well grown in anaerobic condition; usually found in soil, air and water frequently found in pasteurized milk, causing spoilage because of the production lipases and proteases grew and produced a protease using wool as sole of carbon and nitrogen associated with rice based food poisoning denitrifying bacteria; associated with the formation of "clumping" in secondary clarifiers and forming in anaerobic digester. identified to have capability in degrading the azo dye compounds (Khehra et al., 2005; Pourbabaee et al., 2005; Deng et al., 2008) Pseudomonas gram positive veronii (BS6FAnGS) non-spore forming facultative anaerobic capable in degrading ketones (Onaca et al., 2007) Pseudomonass sp. gram negative with non-spore forming (BS7FAnGS, obligated aerobic; facultative anaerobic BS10FAnGS, floc forming bacteria that could initiate floc formation in the activated sludge process BS12FAnGS) used as bioaugmentation in sanitary sewer system and biological treatment plan have the capability to degrade phenol and phenolic compounds easily degrade sulfur containing compounds that are associated with malodor production capable as denitrifying species many species from genus Psuedomonas were identified capable in degrading dye compounds (El-Naggar et al., 2004; Khehra et al., 2005; Barragan et al., 2007) widely distributed in nature some species are pathogenic for humans, animals or plants Enterobacter sp. gram negative with non-spore forming (BS11FAnGS) aerobic, facultative anaerobic, optimal temperature 30-37oC capable as denitrifying species and involve in fermentation process acted as phosphorus accumulating organisms (PAO) in enhance biological phosphorus removal system many species from genus Enterobacter sp. capable in degrading dye compounds (Moutaouakkil et al., 2003; Barragan et al., 2007) commonly distributed in fresh water, soil sewage, plants vegetables, animal and human faces

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The results from the factorial design are presented in the form of an ANOVA table and table that provides information on the estimated effects with coefficients. The ANOVA table gives a summary of the significance of the main and interaction effects by observation on the P-value. Table of the estimated effects with coefficients shows the P-value associated with each individual model term. In this factorial design, the responses obtained were statistically evaluated with the confidence levels of above 90% (P-value less than 0.1).

The experimental results for factorial design analysis are given in Table 5.10. The summary table of the ANOVA that shows the main, two and three ways interaction effect is given in Table 5.11. Detailed discussions for both responses of this study are based on the results obtained after five hours exposure to shear force through the aeration process. With respect to the experimental conditions exploited in the study, the P-value (at 0.1 level of significant) indicates that all factors, i.e. substrate, pH and temperature show significant effect on CAg. The significant interaction effects are only observed between pH and temperature (pH Temperature) while the three way interaction (Substrate pH Temperature) is not significant. As for the responses of SHb, all model terms (main, 2-way and 3-way interaction) except for the 2-way interaction between substrate and temperature (Substrate Temperature), are all significant. Figure 5.5 shows the Pareto chart generated by MINITAB for CAg and SHb of the mixed culture. The vertical disconnected red line represents the significance level determined by the statistical software. The horizontal column bar that does not reach the disconnected red line would imply the insignificance of the model terms. The detailed results of the analysis constructed by the software are given in Appendices E1 to E6.

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Table 5.10: Experimental results for 2-level factorial design analysis Run No. CASE01 CASE02 CASE03 CASE04 CASE05 CASE06 CASE07 CASE08 CASE09 CASE10 CASE 11 CASE 12 CASE 13 CASE 14 CASE 15 CASE 16 Coaggregation (%) 49.6 44.7 67.2 64.1 46.8 44.1 65.6 62.9 65.2 61.9 84.5 85.4 38.8 38.7 57.4 57.6 Surface Hydrophobicity (%) 19.0 21.7 33.8 29.4 37.3 41.2 39.6 36.9 32.1 31.5 53.9 52.0 20.4 18.2 8.6 6.8

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Table 5.11: The P-values of the estimated main and interaction effects of variables substrates, pH and temperature on to the percentage of coaggregation and surface hydrophobicity after six hours aeration phase

Effects

Coaggregation (%)

Significanta

Surface Hydrophobicity (%)

Significanta

The Main Substrate pH Temperature The 2-way interaction Substrate pH Substrate Temperature pH Temperature The 3-way interaction Substrate Temperature
a

< 0.0001 < 0.0001 0.0004

Yes Yes Yes

0.001 < 0.0001 0.0019

Yes Yes Yes

0.5607 0.4827 < 0.0001

No No Yes

< 0.0001 0.8624 < 0.0001

Yes No Yes

pH

0.4679

No

0.0008

Yes

significant at = 0.1

5.5.4.1 Factorial Analysis: The Main Effect of Substrate on Coaggregation

With respect to the main effect on the percentage of CAg, the substrate shows a significant effect with P-value of less than 0.0001. The substrate gave a positive effect with estimated main effect of 19.36. This means that increase in the

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concentration of substrate will cause an increase in the CAg process.

Such

phenomenon may due to the increase in the cell growth of the mixed culture when the substrate is increased. The presence of more cell biomass increases the collision among the cells and may cause more CAg to take place. Liu and Tay (2002) reported that the collision between particles is one of the key factors that influences the formation and stabilization of biofilms, anaerobic and aerobic granules.

The cell surfaces and their characteristics change with the alteration of the surrounding environmental condition (van Loosdrecht et al. 1989). van Loosdrecht et al. (1987) reported that increase in the substrate flux which cause increase in the bacterial growth is capable in changing the cell SHb. Hence, in order to accelerate the granulation start-up process, it was suggested that high OLR should be applied during the granulation process. Moy et al. (2002) reported that there was no negative effect on the granulation process when the OLR was increased as high as 15 kg/m3day.

The effect of collision among the microorganisms which was due to high biomass production at high substrate concentration was prominent during the initial stage of the aeration phase where the amount of substrate was still high. During the first hour of aeration phase, the percentage of CAg was about 57%, with the estimated significant effect of +7.775 and the P-value of 0.005 (Detailed results for one to four hours of CAg assay are given in the Appendices E1 to E4). As the aeration phase was prolonged up to six hours, the effect of substrate has increased (estimated effect +19.36) with the percentage of CAg at high substrate concentrations increased to 68%. The difference may due to the production of EPS during the starvation the phase after long aeration times. Microorganisms will produce more EPS when undergoing starvation conditions when most of the substrate in the medium is being utilized (Wang et al., 2006a). The formation of EPS that covers the cell surface from physicochemical point of view could be regarded as polyelectrolyte adsorbed onto a colloidal particle. The presence of EPS on the cell surface could alter the characteristics of the physicochemical properties of the cell

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surface which includes the surface charge, surface hydrophobicity and others. Varon and Choder (2000) reported that there is physical change of the bacterial surface during starvation condition. Bacteria were observed to produce connecting fibrils that emerged on the surface of the cell. The connecting fibrils acted as a form of microbial communication which is believed to be involved in the initial stage of cell aggregation. The changes in the bacterial surface properties are important aspects with regard to the flocculation, adhesion and granulation process (Veiga et al., 1997 and Liu et al., 2004d).

Under the condition where cells are poor with EPS, the cell surface will be dominated by electrostatic interactions resulting with the repulsion among the cells and cause separation between cells. Cells with poor EPS will follow the DerjaguinLandau-Verwey-Overbeek (DLVO) theory which indicated that increase in surface charge will lead to increase in the repulsion electrostatic interactions between approaching surfaces, resulting with a weakening of the bonding within cell. This condition may occur in the flocculation process that is poor with EPS. On the other hand, cells which are rich with EPS would be dominated with the polymeric interaction that resulted with enhancement of cell adhesion (Tsuneda et al., 2003b). Furthermore, the interaction effect between polymers is much greater as compared to the repulsion effect due to the increase of the surface charge. The amino of exoprotein (PN) which is one of the components of the EPS that are able to neutralize the surface charge. With the presence of EPS, the effect of surface charge will be too weak to inhibit sludge flocculation. Since increase in substrate concentration is associated with increase in cell biomass, the effect of substrate concentration could indirectly affect the aggregation of cells through the changes on the production of EPS particularly during the starvation phase.

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Figure 5.5 The pareto chart of the percentage of (a) coaggregation and (b) surface hydrophobicity after six hours of aeration phase (A: substrate; B: pH; C: temperature; : 0.1)

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5.5.4.2 Factorial Analysis: The Main Effect of pH on Coaggregation

The effect of pH is also significant on the percentage of CAg with the Pvalue of less than 0.0001. The pH gave an opposite effect onto the CAg process as compared to the effect of substrate. It was observed that the pH increase caused a decrease in CAg. The pH variables gave a -13.84 of estimated main effect onto the CAg process.

Under neutral conditions, microorganisms would be negatively-charged due to the ionization of carboxyl, sulphate and phosphate that acted as functional groups in the cells surface (Sutherland, 1982 and Wiley et al., 2008). According to the DLVO theory, when two surfaces possess the same charges, there will be a repulsive force between the two surfaces resulting with prohibition of cell aggregation. This repulsive force is known as Gibbs Free Energy. In acidic conditions, there will be an excess of ions H+ which will cause neutralization of the cell surface charge. Such condition will reduce the free Gibbs energy. Reduction in the electrical repulsion in turn favors cell-to-cell approach and would initiate the formation of cell aggregation (Derjaugin and Landau, 1941). When the pH is higher, the excess of OH- would enhance the surface charges of the bacteria cell and increased the free Gibbs energy and caused the cell to be driven even further apart.

Nonetheless, aggregation was also observed to occur under neutral pH conditions. 2008b). This may suggest that beside the electrostatic repulsion, other mechanisms such as SHb may also be involved in cell aggregation (Adav and Lee, There is evidence that cell hydrophobicity is inversely correlated to the quantity of the surface charge of the microorganisms (Liao et al., 2001). An increase of cell hydrophobicity reflects reduced negative charges on the bacterial surface.

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5.5.4.3 Factorial Analysis: The Main Effect of Temperature on Coaggregation

Temperature was also found to have a mainly positive significant effect on the CAg (P-value less than 0.001). However, the estimated main effect of temperature was only +5.56 suggesting that the effect can be considered weaker as compared to the other two variables. Increase in temperature within the range of this experimental condition would increase the microbial activities which include metabolisms and mobility of the microorganisms. In other words, increase in temperature may increase the specific growth rate of the microorganisms. According to Liu et al. (2004a), the microbial aggregation process is faster at higher specific growth rates of the microorganisms. Increase in cell growth rate will lead to increase in cell biomass and the percentage of collisions between the cells resulting in increase in the probability of cell aggregation.

Increase in temperature will also result in decrease of the free Gibbs energy. As mentioned earlier, reduction of the Gibbs free energy will favor the aggregation of cell. The same observation was reported by Ibrahim et al. (2005) who said that as the temperature of the incubation increased, the ability of the Bifidobacteria to perform aggregation also increased. Figure 5.6 showed the main effect of variables substrate, pH and temperature on the CAg process.

5.5.4.4 Factorial Analysis: The Interaction Effect on Coaggregation

As shown in Figure 5.7, among the three variables, the interaction effect was only observed to be significant between variable pH and temperature with a P-value less than 0.0001. At low temperatures, the percentage of CAg was almost the same at low and high values of pH (pH 5.81 and 8.19) which was about 53%. However,

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when the temperature increased, the percentage of CAg in acidic conditions increased up to more than 70% while the percentage of CAg in alkaline conditions reduced to less than 50%. The main effects of acidic and high temperature conditions have caused the percentage of CAg to increase to 66% and 62%, respectively. The interaction between pH and temperature has increased CAg up to about 75%.

Figure 5.6 Main effects plot on the coaggregation

When the pH of the experimental condition was alkaline, the increase in the temperature has reduced the percentage of CAg. As discussed earlier, when the temperature increases, the free Gibbs energy between two same surfaces charge particles decreases and eventually would enhance CAg. However, in alkaline conditions, there are possibly high concentrations of OH- ions which cause the repulsive force between cells to become even greater and could have overshadowed the effect of high temperatures. Increase in temperature would also cause the movement of the particles to increase based on the theory of Brownian movement (Tchobanoglous et al. 2004) causing the cell particles driven even more apart. This could be the possible reason of why during high temperature and high pH conditions, the CAg among the cell reduced.

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Figure 5.7 Interaction effects plot on the coaggregation process ( Centre point)

The relationship between substrate and pH and between substrate and temperature shows no significant interaction effect since the lines are almost parallel to each other. The 3-way interaction effect between variable substrate, pH and temperature were insignificant with a P-value of 0.4679.

5.5.4.5 Factorial Analysis: The Main Effect of Substrate on Surface Hydrophobicity

Surface hydrophobicity represents one of the physicochemical characteristics of cellular surface of the microorganisms. Hydrophobicity of sludge is believed to play a crucial role and acted as the triggering force towards aggregation (Mahoney et al., 1987; Liu et al., 2004b, Wang et al., 2005a). According to Liu et al. (2004b), the

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surface cell hydrophobicity could be induced by the conditions of the cultures and capable of initiating the cell-to-cell aggregation. It is believed that the hydrophobicity of the cell is one of the most important attraction forces in the microbial aggregation and high cell hydrophobicity seems to be a prerequisite for biogranulation to take place.

Previous studies indicate that changes in cell SHb is affected by many factors that cause stress to the culture condition such as starvation, growth rate, growth substrate, pH and temperature (Liu et al., 2004b). However, different types of bacteria, as single or as mixed culture may response differently especially when the bacteria is found in different types of solution or wastewater. According to Liu et al., (2004b), the knowledge regarding the role of cell hydrophobicity in the biogranulation process is far from complete. Under stressful culture conditions, bacterial cells would change its cell hydrophobicity (Bossier et al., 1996 and Mattarelli et al., 1999). The bacteria would become more hydrophobic and lead to the strengthening of the cell to cell interaction of a microbial structure. It is a form of protective mechanisms of the cells against unfavorable environmental conditions.

The result of the 2-level factorial design experimental run shows that substrate has significant effect on SHb with the P-value of 0.001 and estimated effect of +4.95. Figure 5.8 shows the main effect of the variables on the SHb of the mixed culture used in this study. The substrate has caused positive effect on the SHb where as the increase in substrate has caused increase in SHb. Increase in the substrate concentration means more food supplied to the microorganisms which will increase the bacterial growth. When more bacteria are present, more EPS will be produced when faced with the starvation stage. Increase in substrate may also induce the bacterial growth rate which would increase the production of the EPS. Increase in the EPS would cause increase in the SHb and the bacteria would become more hydrophobic which may facilitate adhesion or aggregation process (Kjelleberg et al., 1987; Liu et al., 2003c; Jiang et al., 2004b). The effect of substrate has the same pattern to the percentage of CAg and SHb with the presence of EPS playing a

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prominent role for both responses. The EPS is known to be capable in mediating both the cohesion and adhesion of cells and play a fundamental role in sustaining the structural integrity in the development of biofilm, anaerobic granules and aerobic granules (Tsuneda et al., 2001 and Flemming et al., 2007).

Figure 5.8: Main effects plot of variables for the percentage of SHb

5.5.4.6 Factorial Analysis: The Main Effect of pH on Surface Hydrophobicity

The pH caused a highly significant effect on SHb with P-value less than 0.0001 and an estimated effect of -8.05. Increase in pH from pH 5.81 to pH 8.19 has caused a decrease in the percentage of SHb from 34% to 27.5%. During acidic condition, the presence of H+ ions in the media solution would cause neutralization of the surface charge of the bacterial cell and reduced the electrostatic force. Since the cell hydrophobicity is inversely correlated to the quantity of the surface charge of microorganisms (Liao et al. 2001), the percentage of SHb increased and would enhance the aggregation of the cells as the surface charge of the microorganisms is reduced. When the pH of the media solution increases (alkaline), there will be more

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of OH- presence in the solution.

This will enhance the repulsive force of the

bacterial surface charge and reduced the chances of the bacteria cell forming aggregates subsequently reducing the percentage of SHb.

Difference in pH of the media can also be associated with the type of growth substrate used in the growth media. It was reported that the formation of anaerobic granules in the UASB was highly affected by the substrate composition of the media solution (Liu et al., 2003e). This is because different types of growth substrate may cause acidic or alkaline conditions when being hydrolyzed. The concentration of H+ and OH- will have an effect on the surface tension of the liquid media and eventually affecting the SHb of the bacteria cell.

The effect of pH on the SHb may basically depend on the net surface charge of the exoprotein of the bacteria cell which eventually depends on the type of protein of the bacteria cell wall. However, further investigations are required for a better understanding on the effect of pH on the SHb particularly on the association of different proteins of the bacteria cell wall.

5.5.4.7 Factorial Analysis: The Main Effect of Temperature on Surface Hydrophobicity

The P-value of 0.002 indicates that the temperature of the experimental conditions has a highly significant effect on the SHb. The estimated effect of -4.43 showed that temperature has caused a negative effect on the SHb.
o o

In this

experiment, when the temperature was increased from about 24 C to 36 C, there was a significant reduction of the percentage of SHb from 32.5% to 28%.

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As temperature increases, the liquid surface tension decreases (Moraes et al., 2008), allowing the adhesion of hydrophilic cells and resulted with reduction in the percentage of cell SHb. The same observation was reported in the study carried out by Blanco et al. (1997), on 42 strains of Candida albicans. The majority of the strains become hydrophobic at lower temperature (20oC) as more cell-to-cell aggregation takes place. When the temperature was increased to 37oC, the strains may undergo changes in their surface property and become hydrophilic with less aggregation taking place. In the development of anaerobic granules in the UASB reactor system, the bacteria cells become more hydrophilic and grow in rather loose association when the liquid surface tension in the UASB is lesser than 50 mN/m. When the liquid surface tension is larger than 56 mN/m, the adhesion of more hydrophobic bacteria cells will take place and form bigger aggromeration (Thaveesri et al., 1995 and Grootaerd et al., 1997).

The effect of temperature has given an opposite result for the percentage of aggregation and SHb eventhough it was claimed that the SHb is the triggering force for cell aggregation (Liu et al., 2004b). Rahman et al. (2007) has categorized bacteria (Bifidobacteria) into high, medium and low autoaggregator groups. Among the high autoaggregator, increase in temperature has caused a decrease in the percentage of autoaggregator. As for the medium and low autoaggregators, increase in temperature has caused an increase in the percentage of autoaggregator. In addition to temperature, other factors such as pH of the media, types of protein bound on cell surface of the different bacteria strains may give a different effect on the ability to autoaggregate. Further investigation is required in order to investigate the effect of temperature onto the autoaggregation and SHb among different bacteria strains under different experimental conditions.

Furthermore, different types of bacteria may response differently when exposed to different environmental conditions with regard to the ability to adhere either among cells or on to a solid surface. Different types of protein may also be secreted or produced at different temperature conditions resulting with a different

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degree of the cell hydrophobicity (Maclagan and Old, 1980). Mattarelli et al. (1999) reported the production of lipoteichoic acid and other mechanisms have caused high hydrophobicity of cells incubated at low temperature (25oC) as compared to 37oC incubation temperature condition. This study involved the use of a mixed selected bacteria culture which individually may give different responses towards the temperature changes. The overall effect may be affected by the most dominant species in the group and may differ from the expected outcome.

5.5.4.8 Factorial Analysis: The Interaction Effect on Surface Hydrophobicity

The interaction effect between variables is given in Figure 5.9.

The

interaction effect between substrate with pH and pH with temperature were highly significant with the P-value of less than 0.0001. The values of the estimated effects show a strong relationship for both the above significant interactions. The interaction effect between substrate and temperature is insignificant with the P-value of 0.862. The insignificant interaction is shown by the parallel line representing the SHb responses of SHb during high and low levels of substrate and temperature variables. The plot in Figure 5.9 describes the interaction between substrate and pH, showing at high concentrations of substrate, change in pH from a lower value to a higher value has caused the SHb to reduce. When the concentration of substrate is low, the increase in pH has caused a slight increase in the SHb of the mixed microorganisms.

The phenomenon observed is believed to be caused by the media solution used in preparing the synthetic textile dyeing wastewater which consists of ethanol, glucose and sodium acetate. During the aeration phase, when the degradation of substrate took place, hydrolysis of acetate released more of OH- that could cause the media to become more alkaline (Voet and Voet, 2004). At high pH conditions,

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where there will be more of OH- in the solution, hydrolysis of acetate will further increase the concentration of OH- and make the media solution more alkaline. Increase in the OH- will increase the surface tension due to the repulsive electrostatic force. This condition will make the cell to be driven apart and reduce the changes to form aggregation and that probably could elucidate on why the percentage of aggregation reduce even more when the substrate increase in the alkaline condition (high pH value). In this condition, it is believed that the electrostatic force is much stronger as compared to the polymeric interaction eventhough during high substrate concentrations.

Figure 5.9: Interaction effect plots for the percentage of SHb ( Centre point)

At low pH, hydrolysis of acetate that produces more OH- will neutralize the existing H+. In this situation, the electrostatic force is reduced and when the substrate concentration is increased, the production of EPS may contribute to cells hydrophobicity and make the polymeric interaction force become predominant and able to promote cell adhesion. This explanation is based on the report of Tsuneda et al. (2003b) who claim that if the amount of EPS is relatively small, the cell adhesion will be inhibited by the electrostatic interaction (referring to the alkaline condition of

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this experiment) and if the EPS is relatively large, cell adhesion will be relatively enhanced by the polymeric interaction.

For the interaction effect of pH and temperature, the plot in Figure 5.9 shows that when the pH of the media (synthetic textile dyeing wastewater) was acidic, increase in temperature caused the SHb to increase from about 25% up to more than 40%. However, when the pH of the media was alkaline, increase in temperature has caused reduction on SHb from almost 40% to less than 10% of SHb. Increase in temperature has enhanced the effect of pH either in acidic or basic conditions. In the acidic media where the ion H+ is abundant, most of the negative surface charges of the bacteria cell are neutralized. This will reduce the free Gibbs energy and reduce the repulsive effect between the cells. Increase in temperature will cause an increase in the collision between the cells (increase in the Brownian movement) which will encourage the interaction between cells and enhances the aggregation process. In the alkaline media condition, abundance of ion OH- may increase the repulsive effect of surface charge. Increase in temperature which increased the cell movement may cause the cell with high free Gibbs energy (due to increase in the surface charges) to be driven further apart from one another and reduce the changes to form aggregates.

A significant 3-way interaction effect of substrate, pH and temperature is also within the experimental condition with the P-value of 0.001. The curvature effect of the significant interaction was further investigated by using the response surface experimental design and will be discussed in the next section.

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5.5.5 Response Surface Analysis

The results of the CCD analysis are shown in Table 5.12. The analysis was carried out using full quadratic terms including linear, square and interaction with the aid of Design-Expert 7P statistical software. The summarized results of the analysis of variance (ANOVA) for the percentage of CAg and SHb are shown in Table 5.13. The detailed results consisting of estimated regression coefficient and ANOVA table are given in Appendices E7 to E9.

For CAg, based on the P-value, all the linear terms show significant values. The model exhibits significant non-linearity for substrate and pH with the P-values of 0.0042 and 0.0427, respectively. However, temperature shows non-curvature effect with the P-value of 0.2397. The interaction between pH and temperature are found to be highly significant with the P-value of 0.02 while the other two interactions are not significant (P-values more than 0.2). The R-squared value of the model is acceptable (86.63%) but the P-value for the Lack of Fit Test (LOFT) is significant (0.0029). This implies that the analytical understanding of the model is not statistically accurate. It may indicate that the process appears to be too complex to model.

In order to improve the model, another attempt was carried out by omitting two interaction terms (Substrate pH, Substrate Temperature) and one square term (Temperature Temperature). All the omitted terms were selected based on the insignificant result obtained from the full quadratic terms analysis. The results of this analysis with omitted terms are given in Appendix E8. Omitting the selected insignificant terms lowered the P-value of all the included terms (Reduced quadratic terms) compared to the previous analysis (Full quadratic terms). The R-squared term reduced from 86.63% to 84.06% and the LOFT increased from 0.0029 to 0.048%. The drop of the R-squared value points out that the omitted terms only cost 2.5% in goodness of fit and this is acceptable. Eventhough the LOFT P-value has

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improved by more than sixteen times higher compared to the analysis of the full quadratic terms, the value still implies the inadequacy of the fitted model.

Table 5.12: Experimental results for CCD analysis

Run CASE01 CASE02 CASE03 CASE04 CASE05 CASE06 CASE07 CASE08 CASE09 CASE10 CASE11 CASE12 CASE13 CASE14 CASE15 CASE16 CASE17 CASE18 CASE19 CASE20 Coaggregation (%) 49.6 67.2 46.8 65.6 65.2 84.5 38.8 57.4 68.8 73.0 54.2 5.50 29.3 58.3 59.4 56.4 55.1 60.3 57.5 60.2 Surface Hydrophobicity (%) 19.0 33.8 37.3 39.6 32.1 53.9 20.4 8.6 50.9 70.7 62.4 7.8 71.2 83.9 75.2 77.2 72.7 74.0 76.2 72.2

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Table 5.13: Summary of the P-value of the response surface modeling analysis Coaggregation (%) Term Full Quadratic Terms Linear + Square + pH Temperature The P-valuea Substrate pH Temperature Substrate*pH Substrate*Temperature pH*Temperature Substrate*Substrate pH*pH Temperature*Temperature R-squared value Lack of Fit (LOFT)
1997)

Surface Hydrophobicity (SHb) (%)

0.0067 0.0022 0.0369 0.6329 0.8147 0.02 0.0042 0.0427 0.2397 86.63% 0.0029

0.0037 0.0009 0.0262 0.0133 0.0016 0.0352 84.06% 0.048

0.3582 0.075 0.9194 0.3596 0.8854 0.1221 0.0423 0.0014 0.3393 75.89% <0.0001

0.01 0.04: Highly significant; 0.05 0.1: significant; 0.1 0.2: less significant; < 0.2: insignificant (Vecchio,

The response surface analysis of SHb shows that among the investigated variables, only pH shows a significant effect with the P-value of 0.0750. The model also shows significant non-linearity for both substrate and pH with the P-value of 0.0423 and 0.0014. Temperature shows the same response for the square term as in the CAg assay with insignificant effect with P-value of 0.3393. The R-squared value of this model eventhough slightly lower as compared to the value for responses of coaggregation is still considered within acceptable range with a percentage of 75.88%. However, the P-value for the Lack of Fit Test (LOFT) is significant (less than 0.0001) for this model indicating the inaccuracy of the statistical model. Nonetheless the best statistical models that can be used to represent the responses

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(CAg and SHb) within the range of the experimental conditions in this study as accomplished from the CCD design analysis are given in Table 5.14.

Table 5.14: Mathematical models in terms of actual factors

Responses Statistical Model = -30449.2 - 4.02A + 7114.5B + 843.1C 108.1BC + 1.410-3A2 329.1B2

(Coagggregation)2 (%)

Surface Hydrophobicity (%)

= -1122.2 + 0.12A + 239.7B + 18.1C 6.510-3AB 2.0110-4AC 1.4BC 1.910-5A2 13.8B2 0.1C2

A: Substrate (mg/L); B: pH C: Temperature (oC)

The predicted versus actual plots for the responses of CAg and SHb are shown in Figure 5.10. The plots reveal that the actual values are distributed relatively near to the straight line in both cases. The predicted and actual values obtained from this experiment are considered to be statistically acceptable indicated by the high value of the R-squared calculated from the analysis of variance of this study conditions.

The response surface and contour plots which illustrate the relationship between the percentage of CAg and independent variables of pH and temperature is shown in Figure 5.11. The figure shows the surface plots that represent the effect of varying pH and temperature at fixed substrate concentrations (1750 mg/L). It can be seen that increasing the temperature value at low pH levels, has caused an increase in the CAg process with a high percentage of 69.5%. However, at high pH levels, the percentage of CAg was slightly decreased with increase in the temperature. Having a condition at high temperature levels and low pH conditions would be the best condition for achieving the maximum percentage of CAg.

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7144.08

5365.49

Predicted

3586.91

1808.33

29.75

(a)
29.75 1808.33 3586.91 5365.49 7144.08

Actual
83.85

64.13

Predicted

44.41

24.69

4.97

(b)
4.97 24.69 44.41 64.13 83.85

Actual

Figure 5.10: Predicted versus actual data for (a) coaggregation and (b) surface hydrophobicity

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(a)

(b) Figure 5.11: (a) Contour and (b) 3D response surface plots representing relationship between pH, temperature and percentage of coaggregation

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Figures 5.12 to 5.13 show the relationship between SHb and independent variables of substrate, pH and temperature, respectively. The contour plots of the RSM are drawn as a function of two variables at a time, holding other remaining factors or variables at a fixed level.

The surface plots of the interactions between variables for the percentage of SHb as the observed response was found to be a symmetrical mound shape shown in Figures 5.12 and 5.13. Meanwhile, the contour plot in Figure 5.14 appears to be of elliptical shape. As shown in Figure 5.12 where the variable temperature was held at 30oC, at any substrate concentration level, the percentage of SHb was low both at high and low levels of pH (8.19 and 5.81). However, the response increased as the pH level approached the neutral pH condition (pH 7). The percentage of SHb was the highest (77.34%) when the pH and substrate level were at 6.7 and 1967.9 mg/L, respectively.

Figure 5.13 illustrates the percentage of SHb as a response in a contour and response surface plots for the interaction between varying concentrations of substrate and temperature. The interaction was observed at constant pH level (pH 7) as the hold value. The symmetrical mound shape plot shows that at any temperature level, the percentage of SHb increases as the substrate concentration increased from 1006.75 mg/L to1750 mg/L. However, as the concentration of substrate increases from 1750 to 2493.25 mg/L, the response started to reduce. The highest percentage of response is 75.91% obtained at substrate concentration of 1909.63 mg/L and temperature of 30.05oC. The point that shows the highest percentage of SHb was positioned at the middle of the symmetrical mound shape plots.

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(a)

(b) Figure 5.12: (a) Contour and (b) 3D response surface plots representing relationship between the concentration of substrate, pH and percentage of surface hydrophobicity

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(a)

(b) Figure 5.13: (a) Contour and (b) 3D response surface plots representing relationship between the concentration of substrate, temperature and percentage of surface hydrophobicity

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Having substrate variables at constant concentration of 1750 mg/L, Figure 5.14 demonstrates the contour and response surface plots of surface hydrophobicity at varying pH and temperature values. The percentage of response reduces from 71.34 to 50.17% when the temperature reduced from 35.95 to 24.05oC at a pH level of 5.81. However, at a high pH level (8.19) the response increases from 32.96 to 52.02% when the temperature of the experiment reduced from 35.95 to 24.05oC. The graph also shows that at a high temperature (35.95oC), the percentage of response was doubled when pH reduced from 8.19 to 5.81 at a temperature of 35.95oC. As the temperature of the experiment was set at 24.05oC, there was not much change on the percentage of response as the pH level reduced in the same manner. When the temperature and pH levels were at 32.68 and 6.6, respectively, the predicted percentage of SHb shows the highest value of 77.14. The response illustrated in Figures 5.12 to 5.14 shows that the maximum predicted percentage of SHb is indicated by the surface confined in the smallest curve of the contour diagram.

5.6

Conclusions

i.

The FAnGS is consisted of different types of bacteria where among the twelve bacteria successful isolates demonstrated different morphological and cellular characteristics.

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(a)

(b) Figure 5.14: (a) Contour and (b) 3D response surface plots representing relationship between pH, temperature and percentage of surface hydrophobicity

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ii.

Among the twelve bacteria isolated from FAnGS, all of the isolates show the same growth pattern under anaerobic (slow growth) and aerobic condition (fast growth). However, they show different capability in degrading the COD and dye compound with different degradation rate. The twelve isolated bacteria also demonstrate different SHb and ability in performing aggregation when imposed under high shear force. Most of the isolated bacteria that exhibit high percentage of SHb also show high aptitude to form aggregate.

iii.

With the aid of molecular technology via PCR application, the six selected bacteria that show considerable high capacity in degrading dye and COD and at the same time also able to perform aggregation and high SHb, have been identified as Bacillus cereus, Pseudomonas veronii, three species of Pseudomonas genus and Enterobacter sp. Based on the literature, all of the six selected isolates are capable to grow both under anaerobic and aerobic conditions. This ability may become as one of the reasons for the survival of these bacteria to grow within the FAnGS.

iv.

Within the experimental condition of this study, all the selected variables investigated imposed a significant linear effect on the CAg process. The substrate concentration and temperature show a positive effect on CAg as the concentration of substrate and the degree of the temperature increased, while pH imposed negative effect on CAg when the pH level change from acidic to alkaline condition. However, the interaction effect onto CAg was only significant between pH and temperature. Substrate concentration and pH showed significant non-linear effect on CAg, while the effect of temperature was not significant. The three way interaction between the three variables was not significant.

v.

All of the three variables, substrate, pH and temperature demonstrate a significant effect on SHb. Variable substrate shows an increase in percentage

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of SHb as the concentration of substrate increased. However, the percentage of SHb reduced as the degree of temperature reduced and the pH change from acidic to alkaline condition. The interactive effects were observed to occur between pH and substrate and between pH and temperature. All of the variables exhibit significant three way interaction effect. With respect to the non-linearity effect of variables, pH and substrate indicated significant effect but the result for temperature was opposite.

CHAPTER 6

THE EFFECT OF HYDRAULIC RETENTION TIME ON FACULTATIVE ANAEROBIC GRANULAR SLUDGE

6.1

Introduction

The applications of granulation techniques for dye degradation have been reported by many researchers. Most of the studies were focused on using anaerobic granules under anaerobic condition since major decolorization process occurs under this condition (Bras et al., 2005; Isik and Sponza, 2005b; Somasiri et al., 2008). The UASB reactor system containing anaerobic granules was capable in treating raw textile wastewater with 90% and 92% of COD and color removal, respectively with 24 hours of HRT (Somasiri et al., 2008). Color removal higher than 88% was reported by Bras et al. (2005) treating mixed monoazo and diazo in a methanogenic laboratory-scale UASB system at 24 hours HRT.

There are also several reports on the application of aerobic condition for color removal. Most of the studies use activated sludge, suspended cells or biofilms as the biomass compositions (Vives et al., 2003; Buitron et al., 2004; Sandhya et al., 2005; Sirianuntapiboon and Srisornsak, 2007; Sirianuntapiboon and Sansak, 2008). Almost 100% of color removal was reported for the degradation of Direct Blue and Direct

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Red in a series of granular activated carbon system and sequential batch reactor system operated under aerobic condition at HRT of 7.5 days (Sirianuntapiboon and Sansak, 2008). Buiton et al. (2004) reported an average of 80% color removal was observed for the degradation of Acid Red 151 in a sequential biofilter packed with porous volcanic rock-pozolane under aerobic condition.

Since complete mineralization of dye containing wastewater requires both anaerobic and aerobic conditions, several attempts have been conducted to investigate the removal efficiency under both operating conditions using anaerobic granules with a series of anaerobic and aerobic reactor systems (Sponza and Atalay, 2003; Isik and Sponza, 2004a; Isik and Sponza, 2004b). However, the application of granular system in an integrated textile wastewater treatment system has not been much reported (Shaw et al., 2002).

Table 6.1 shows previous research studies on dye degradation process in integrated reactor systems using different forms of biomass under different reaction phase conditions. The table shows that the overall color removal percentage is very much affected by the HRT particularly during the anaerobic phase. Color removal increased as the retention time of anaerobic phase increased (Panswad et al., 2001a; Buitron et al., 2004; Goncalves et al., 2005). However, different types of biomass either in the form of suspended cells, activated sludge or granules, used in the treatment system for treating different types of dyes (single or mixed) may result in different removal efficiencies.

Successful cultivation of FAnGS has been achieved using synthetic textile dyeing wastewater in an integrated reactor system under intermittent anaerobic and aerobic reaction phases as reported in Chapter 4. biomass for treating the textile wastewater. The FAnGS consisting of anaerobic, aerobic and facultative microorganisms may become the most suitable However, knowledge on the performance of color removal using FAnGS under different HRT is lacking. The changes in terms of the granular properties, performance of COD and color removal

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as well as the performance of the reactor system towards the effect of different HRT are the main focus of this chapter. Biokinetic parameters such as biomass growth rate (), endogenous decay rate (kd), observed biomass yield (Yobs) and theoretical biomass yield (Y) were also investigated in relation to the changes of HRT of the anaerobic and aerobic reaction phases.

6.2

Materials

All of the chemical or reagents and equipments used in this study were given in Section 4.2. In addition, an Orion 2 Star pH-Benchtop meter (SN-016655) was used to measure the redox potential of the wastewater while conducting the experiment. However, in this experiment the concentration of the carbon sources (glucose, acetate and ethanol) was increased giving an initial OLR of 2.5 kg COD/m3day.

6.3

Analytical Methods

The effect of HRT was investigated with respect to the changes in the microbial activity, physical characteristics and removal performance of the granular biomass in the IFGSBioRec. Figure 6.1 shows the experimental analysis conducted for this study.

Table 6.1: Dye degradation process using integrated reactor system

Reactor system SBR Biomass Activated sludge Sludge Dye Raw wastewater containing disperse, sulfur & reactive dyes Reactive Black 5 & Reactive Blue 5,19, 198 Reactive azo dye Procion Red H-E7B Remazol Black B Remazol Black B Remazol Brilliant Violet 5R & Remazol Black B Remazol Black reactive dye Acid Orange 7 Reaction phase/HRT Conventional SBR (Aerobic, 10 hrs); Anoxic+ anaerobic (2 hrs) / Aerobic (8 hrs) Anoxic/Anaerobic (18 hrs)/ Aerobic (5hrs) Anaerobic (24 hrs ) /Aerobic(16 hrs) Performance Color reduction was not so good due to less contact hour during anaerobic condition Color removal: at 20 mg/L of dyes (6366%); at 100 mg/L of dyes (32-58%) 63.9% (anaerobic stage) and 11.1% (aerobic stage) Reference Pansuwan et al. (1999) Luangdilok and Panswad (2000) ONeil et al. (2000b) Panswad et al. (2001a) Panswad et al. (2001b) Lourenco et al. (2001) Shaw et al. (2002) Coughlin et al. (2002)

SBR

SBR SBR SBR SBR

Activated sludge Activated sludge Activated sludge Activated sludge Anaerobic granules Aerobic biofilm

Anoxic+Anaerobic/ Aerobic: Color removal: 26.9-60.5% (anaerobic); (0/11; 2/9; 4/7; and 8/3 hrs) 1.9-16.7% (aerobic) Anaerobic (18/6 hrs) /Aerobic (5hrs) Anaerobic (9-11 hrs) /Aerobic (8-12 hrs) Anaerobic (18.5 hrs) /Aerobic (30 min) HRT: 1.5 hrs 73-77% (enriched with PAOs); 59-64% (enriched with GAOs) of color removal 90% removal for RBV; 75% removal for RBB 94% (color removal); 66% (TOC removal) 90% of color removal

SBR RDBR

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Table 6.1: Dye degradation process using integrated reactor system (Continued)
Reactor system SBR Biomass Activated carbon Porous volcanic rock Aerobic sludge Bio-sludge Dye Reactive dyes Reaction phase/HRT Anoxic (14;17.5 hrs)/ Oxic (6; 2.5 hrs) HRT: 4-24 hrs Anaerobic (12;8 hrs)/ Aerobic (8;12;12 hrs) Aerobic (19 hrs) /Anaerobic (3 hrs) /Anoxic (0.5 hrs); HRT : 3 d Anoxic (30 min)/ Aerobic (23 hrs)/Anoxic (30 min) Anoxic (8 hrs) / Aerobic (16 hrs) Anaerobic (20 hrs) Performance >80% (COD removal); 18-25% (color removal). 99% color removal (14-16% contributed by porous material ) 85% (COD removal); 95% (BOD5 removal) Color; COD; BOD5; TKN removal: STIWW (98.5%; 96.9%; 98.6%; 93.4%); RWW (75%; 71; 96.7;63%) 100% (color removal); 92% (COD removal) 12-85% (color removal ); 95% (COD removal ) 100% (color removal); 88% (COD removal) Highest color removal (86.5%), Highest COD removal (94.1%) Reference Pasukphun and Vinitnantharat (2003) Buitron et al. (2004) Goncalves et al. (2005) Sirianuntapiboon et al. (2006) Mohan et al. (2007b) Smith et al. (2007) Ong et al. (2008b)

SBBR SBR SBR

Azo dyes Acid Red 151 Azo dyes Vat dye

SBR Reaction vessel SBR

Activated sludge Sludge Granular activated carbon Facultative anaerobic granule

Acid Black Azo dye Reactive azo dyes Acid Orange 7

SBR

Mixed azo dyes

Anaerobic/Aerobic HRT=3:3;6:6;12:12; 18:6; 6:18 hrs

This study (2009)

184

Figure 6.1 Experimental analyses on the effect of HRT on granular biomass in treating synthetic textile dyeing wastewater

185

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6.3.1 Microbial Activity

The microbial activities as the observation of the biological characteristics were measured based on the OUR of the granular biomass used in this experiment. The procedure for OUR measurement is as described in Section 4.3.1.2.

6.3.2 Physical Characteristics

The physical changes were investigated in terms of the concentration of the MLSS and MLVSS, SRT, settling velocity and the sizes of the granular biomass. The settling velocity of the granules was measured as described in Section 4.3.2.1. The measurement of MLSS and MLVSS concentrations were according to Standard Methods (APHA, 2005) as given in Section 4.3.2.4. The granular sizes were measured by sieving 250 mL of suspended granular biomass using different sizes of sieve mesh. The amount of the sieved granules were divided with the total granular biomass and measured as a percentage of volume fractions.

6.3.3 Removal Performances

The removal performances of the reactor system with respect to color and COD removal were analyzed according to the description in Section 4.3.4.1 and Section 4.3.4.2, respectively. The redox potential levels were measured throughout the experiment using an Orion 2 Star pH-Benchtop meter (SN-016655).

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6.4

Experimental Procedures

The FAnGS which was developed in the IFAnGSBioRec as discussed in Chapter 4 was used as the granular biomass in this study. The size of FAnGS selected for this experiment was in the range of 0.3-2.5 mm. The FAnGS was inoculated into the bioreactor at a ratio of 1:4 of the working volume of the reactor system. One (1) L of acclimated mixed sludge as described in Section 4.2.2 was also added into the reactor system. During the start-up of the experiment, 2 L of synthetic textile dyeing wastewater was filled into the reactor. This has made the concentration of MLSS and MLVSS during the start up of the experiment as 23.2 g/L and 18.4 g/L respectively. During the first two month of the start-up, 10% (v/v) of selected dye degrader microbes were added twice a week into the reactor.

The operation steps of one complete cycle of the IFAnGSBioRec are shown in Table 6.2. The HRT were varied between 6 to 24 hours in order to study the effect of HRT towards the removal of COD and color by the FAnGS in the continuous operation of IFAnGSBioRec. During the anaerobic react phase, circulation of wastewater was carried out using peristaltic pump (Cole-Parmer System Model, 6600 rpm), pumping out the wastewater from the upper part of the reactor system and entering back into the system at the bottom of the reactor. The circulation was conducted at a flow rate of 18 L/h. Throughout the aerobic react phase, air pump was used to provide the oxygen to the system. The air was supplied at a superficial air velocity of 2.5 cm/s. The reaction phases were operated intermittently starting with anaerobic and followed by aerobic. The reaction phase was then continued with a second anaerobic phase followed by a second aerobic phase. Then, the biomass was allowed to settle during the settling phase. The detailed descriptions on the reaction phase variation are discussed in Section 6.5.2.

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Table 6.2: Operation steps during single cycle operation Sequence phase Fill Reaction Anaerobic Aerobic Settle Decant Idle Varies* Varies* 5 min 5 min 5 min Off On Off Off Off On Off Off Off Off Phase period 15 min Air supply Off Recirculation Off

6.5

Results and Discussion

6.5.1 Microbial Activity

The microbial activity was measured based on the oxygen uptake rate of a complete one cycle operation. The OUR of a complete cycle was measured several times before each of the stages ended. The OUR measurement of each stage of the experiment showed that most of the external substrate was consumed more or less within the first 30 minutes of each aerobic reaction phase. Figures 6.2 to 6.3 show the profiles of the OUR throughout the experiment from Stage I to Stage VI. The OUR profile (Figure 6.2) shows that the initial measurement of the OUR reduced as the HRT increased (Stage I to Stage III). This is due to the reduction in the OLR as the HRT increased. Less oxygen is required as the concentration of the organic loading reduced. After a sharp increase of OUR at the beginning of each cycle in all stages, the OUR measurement was constantly low until the end of the cycle. The low measurement of OUR gives an indication that most of the external substrates have been consumed. This also means that the microorganisms in the reactor system were under starvation phase.

At this phase, no further degradation was observed

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eventhough the HRT was extended.

During the starvation phase, endogenous

respiration will take place, except at the beginning of the second phase of aerobic reaction where there was a short increase in the OUR. In this case, the short increase on the OUR measurement was due to the mineralization of amines, the byproduct of dye degradation during the second anaerobic reaction phase. As the duration of anaerobic reaction phase increased, the short pulse increased as shown in Figure 6.3 (a and b) of Stage IV and V, respectively. Stage IV and Stage V were operated with the same HRT and organic loading but different in the anaerobic and aerobic reaction phase ratio.

6.5.2 Physical Profile of the Reactor System

The details of the experimental conditions of the reactor system are shown in Table 6.3. The HRT of the experiment was increased from 6 hours in Stage I to 24 hours in Stage III. The increase in the HRT resulted with a reduction of the OLR supplemented into the reactor system from 2.5 to 0.6 kg COD/m3day. The HRT for Stage III to VI was kept constant i.e. 24 hours, but the duration of anaerobic and aerobic reaction phases was varied. From Stage III onwards, the OLR was increased to 0.8 kg COD/m3day by increasing the concentration of the carbon sources in the synthetic textile dyeing wastewater. The temperature of the treatment system was kept constant at 30.0 2.0oC while the pH throughout the experiment was between 6.3 and 8.0.

Table 6.4 shows the oxidation reduction potential (ORP) values measured during the second phase of the anaerobic and aerobic reactions during the experiments. The ORP profile of all the stage corresponded very well with the dissolved oxygen. The ORPs were recorded with more negative values when the anaerobic reaction phase increased while during the aerobic phase the ORP varies between +98 to +177 mV.

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(c)

Figure 6.2

OUR profile of (a) Stage I (Aerobic phase 2.84 hours), (b) Stage II

(Aerobic phase 5.84 hours) and (c) Stage III (Aerobic phase 11.84 hours)

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(b)

(c)

Figure 6.3 OUR profile of (a) Stage IV (Aerobic phase 11.84 hours), (b) Stage V (Aerobic phase 5.84 hours), (c) Stage VI (Aerobic phase 17.84 hours)

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Table 6.3: Details of experimental condition of the IFAnGSBioRec

Phase (hours) Stage Days covered 1st Anaerobic I II III IV V VI 49 43 51 43 46 46 1.42 2.92 5.92 5.92 8.92 2.92 Aerobic 1.42 2.92 5.92 5.92 2.92 8.92 2nd Anaerobic 1.42 2.92 5.92 5.92 8.92 2.92 Aerobic 1.42 2.92 5.92 5.92 2.92 8.92 6 12 24 24 24 24 2.5 1.3 0.6 0.8 0.8 0.8 HRT (hrs) OLR (kg COD/ m3day)

, where X = COD concentration of the influent (mg/L); Vadd= Volume of influent added in each cycle operation (mL); Vtotal = Total working volume of the experiment (mL); T = Hydraulic retention time (hour).

Table 6.4: Oxidation Reduction Potential Stage I II III IV V VI Anaerobic Reaction Phase -124 27 -219 33 -358 29 -355 51 -407 21 -225 28 Aerobic Reaction Phase 125 19 129 24 174 34 151 17 112 21 177 15

The biomass profile at steady state with stepwise increment of HRT (Stage I to III) and variation of reaction phases (Stage IV to VI) are shown in Table 6.5. As shown in Table 6.5, it is apparent that the biomass concentration (MLSS) in the reactor decreased and the VSS in the effluent was also reduced with the increase in the HRT (Stage I to III). The reduction of the biomass concentration in the reactor

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may be due to the lower value of OLR applied in the reactor system as the HRT increased.

Table 6.5: Biomass concentrations at different stages of the experiment

Reaction Phase
Anaerobic (hours) Aerobic (hours) MLSS (g/L) MLVSS (g/L) VSS/SS Effluent (VSS g/L) SRT (day)

Stage I
2.8 2.8 35.3 1.6 31.9 1.8 0.90 0.34 0.16 27.6 13.4

II
5.8 5.8 28.7 0.6 24.5 2.2 0.85 0.31 0.11 42.4 10.2

III
11.8 11.8 25.2 1.8 18.5 2.2 0.73 0.26 0.19 78.9 23.9

IV
11.8 11.8 30.5 3.4 26.0 3.4 0.85 0.34 0.11 70.1 23.9

V
17.8 5.8 31.6 3.7 22.4 2.0 0.71 0.33 0.10 72.5 23.3

VI
5.8 17.8 23.3 0.8 20.2 0.8 0.87 0.55 0.22 41.6 18.4

When the OLR was increased to 0.8 kg COD/m3day, there was an improvement in the biomass concentration where the biomass concentration have increased to 30.5 3.4 g/L and 31.6 3.7 g/L in Stage IV and Stage V as compared to 25.2 1.8 g/L of biomass concentration in Stage III which run at the same HRT (24 hours) but with OLR 0.6 kg COD/m3day. The increase in OLR has caused an increment in the biomass concentration in the reactor. A slight increase in the biomass concentration was also observed along with the longer period of the anaerobic phase (Stage V), i.e. 18 hours.

The ratio of the volatile biomass (MLVSS) to total biomass (MLSS) reduced from Stage I to Stage III mainly due to decrease in the OLR as the HRT increased from 6 to 24 hours, whereas the MLVSS/MLSS ratio of the Stage III and Stage IV

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with 12 hours aerobic reaction phase was observed higher with the ratio of 0.73 and 0.85, respectively. The increment may be due to the increase of the OLR from 0.6 to 0.8 kg COD/m3day (Stage III to Stage IV). Increase in the OLR means more carbon sources were supplied to the microorganisms in the reactor. When more food is available, more growth will take place and this is indicated by the increase in the MLVSS/MLSS ratio.

However, when the anaerobic period of the HRT is extended, the MLVSS/MLSS ratio decreased (0.71). Decrease in MLVSS/MLSS ratio may indicate an increase of inorganic accumulation within the granulation biomass. The same observation was reported by Panswad et al. (2001a) that increase of inert solids in the biomass was observed when the system was exposed to high anoxic/anaerobic condition in the SBR cycle. When the duration of aeration phase was increased up to 18 hours, the biomass started to reduce again (Stage VI) and increase of VSS in the effluent was once again observed. This may give an indication that too long of aerobic reaction phase is not suitable for granular biomass system. Prolong of aeration time may result in instability of the reactor performance. The profile of biomass concentration of the reactor system throughout the experimental process is given in Figure 6.4.

The sludge retention time (SRT) of the biomass in the SBR system can be calculated by using Equation 6.1.

where, = = Solid retention time (d) Volatile solid concentration in the reactor system (g VSS/L),

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= =

Working volume of the SBR system (L), Biomass concentration of manually discharged mixture (g VSS/L)

= = = =

Manually discharge mixture volume (L), Effluent volatile solid concentration (g VSS/L) Effluent volume of the SBR operating cycle (L) Cycle time of the SBR operation (d)

Figure 6.4

Profile of biomass concentration at different stages of the experiment.

() MLSS, () MLVSS. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h)

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Since in the operating system there was no physical sludge discharging at any of the operating time, Eq. 6.1 can be simplified as Eq. 6.2 (Liu and Tay, 2007b).

X vss Vr X e Ve /t c

(6.2)

Based on Eq. 6.2, the SRT of the reactor system increased from 27.6 13.4 to 78.9 30.8 d when the length of the HRT increased from 6 to 24 hours (Stage I to Stage III). With HRT of 24 hours, increase of anaerobic reaction phase up to 18 hours (Stage IV to Stage V) has slightly increased the SRT from 70.1 23.9 to 72.5 23.3 d. The SRT value changes in each stage of the experiment. According to Wijffels and Tramper (1995), the favorable sludge age for high removal efficiency for COD and nitrification process is more than 4 days. Based on the SRT obtained, this granular system is capable of the simultaneous degradation of nitrification process and COD removal. Since the treatment goal is to remove recalcitrant dyeing compound, the SRT value of all stages evaluated in this experiment was in the acceptable range from degradation of xenobiotic compounds (Grady et al. 1999).

6.5.3 Effect of Hydraulic Retention Time on Physical Properties of the Granular Biomass

In this experiment, the HRTs were between 6 to 24 hours with variation time for anaerobic and aerobic conditions during the reaction phase. The effects of this variation on the physical properties of the granules are given in Table 6.6.

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The mean granular size in the reactor was the largest (i.e 843 44 m) during Stage I which was at the shortest HRT and highest OLR. When the HRT increases from 6 to 24 hours, the OLR was reduced from 2.5 to 0.6 kg COD/m3day. This condition may contribute to the smaller granules formation due to the reduction in the food supply. Furthermore, as the HRT increased from 6 to 24 hours, the mean granular size was reduced may also be due to the long exposure of the granules to the shear force imposed by high superficial air velocity during the increasing aerobic reaction phase. When the OLR were increased from 0.6 to 0.8 kg COD/m3day from Stage III to Stage IV, the mean granular size slightly increased due to increase in food supply.

Table 6.6: experiment

Reaction Phase Anaerobic (hours) Aerobic (hours) Mean size (m) SVI (mL/g) SV (m/h)

Physical properties of the granular biomass at different stages of

Stage I 2.8 2.8 II 5.8 5.8 III 11.8 11.8 IV 11.8 11.8 V 17.8 5.8 VI 5.8 17.8

843 44 13.1 0.4 41.3 3.1

590 55 18.8 1.5 35.1 0.8

440 40 21.4 1.6 24.5 1.1

567 79 16.8 1.3 28.4 1.3

575 46 15.5 1.3 33.4 2.5

385 22 24.8 0.9 21.3 0.5

At Stage V, even though the HRT was 24 hours, the mean granular size was observed to increase. This is due to the short aerobic reaction phase (i.e. 6 hours) as anaerobic reaction phase was prolonged up to 18 hours and with increase in the OLR. This shows that bigger granules could still be maintained in the reactor system at higher HRT provided that aerobic reaction phase is reduced by increasing the

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anaerobic reaction phase. This condition seems to be suitable for treating textile wastewater that requires both anaerobic and aerobic phases. With respect to the development of the granulation process, increase in the anaerobic reaction phase would cause changes in the EPS component within the granular sludge enhancing the granulation process. Aerobic granules may consist of layers of aerobic and facultative anaerobic granules (Jang et al., 2003 and Tay et al., 2002a). The aerobic microorganisms are able to produce more EPS as compared to the anaerobic microorganisms (Foster, 1991). As the aerobic microorganisms are responsible in producing the EPS, under anaerobic condition the facultative microorganisms suppressed the EPS production and encourage the consumption of EPS. Fermentation of EPS and disruption of microorganisms would take place inside the granules during anaerobic condition leading to reduction of the EPS in the granule. Reduction of the EPS in the inner part of the granules resulted in the reduction of the surface negative charges, the steric interaction and entanglement of the EPS and increase in hydrophobicity which means less water trapped (Foster, 1991). With the effect of shear force, the compacting process onto the granule would have taken place and the granules continue to grow. The increase of the granular size is stabilized when the balance between growth and detachment due to shear force effect is reached. The interaction between the production of EPS by the aerobic microorganisms during aerobic reaction phase may has been balanced with the consumption of the EPS by the anaerobic or facultative microbes during the anaerobic reaction phase that caused increase in the granular size during Stage V. This means having an intermittent reaction phase of anaerobic and aerobic process would be a good strategy for the application of granulation system for textile wastewater treatment. Balance in bioactivity of the production and consumption of EPS could maintain a reasonable amount of EPS within the granules (Li et al., 2006b) which are important for the successful development and growth of the granular biomass.

The SVI value of the granular sludge was used to evaluate the granular settling ability. It is anticipated that bigger granules would have higher settling velocity and hence, reduce the SVI value, indicating good settling ability. The SVI

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value changes with the same pattern as the granular biomass concentration as well as the mean particle size of the granules. As the granular particles decreased in size, the SVI value increased. The SVI value improved when the anaerobic reaction phase was prolonged in Stage V indicating such reaction pattern would help to develop granules with better settling profile. As stated earlier, according to Panswad et al. (2001a), inert biomass increased as the anoxic/anaerobic condition was prolonged. It could be possible that the accumulation of inert particles within the granules increased and resulted with improved SVI properties of the granular biomass.

Figure 6.5 shows the particle size distribution of granular biomass in the reactor at each stage of the experiment. The figure shows that the particle size distribution was clearly affected by the HRT and aeration time which imposed shear force to the granules. As shown earlier in Table 6.4, when the HRT increased, without increasing the concentration of substrate in the influent, will cause reduction in the OLR. This means less food is supplied into the reactor. The granular biomass in the reactor will be exposed to a longer starvation period when the HRT is increased. The starvation effect become more obvious when the aeration time is longer as the HRT increased. When there was no more food to be consumed (starvation phase), the microorganisms will undergo endogenous respiration where the EPS within the granules will be used as the alternative of the energy sources (Liu et al., 2005a). The granular biomass, then, would experience microbial decay and lyses. This would lead to increase in the granules porosity and weaken the granular structure. Empty holes in the granules would be observed with fragile granule structure. In these circumstances, the granules would easily defragment into smaller sizes under operational condition. Control over the granular sizes and the length of starvation time are among the important factors to be considered for maintaining performance stability of the granular reactor system.

Hydraulic retention time is an important parameter that control the contact time between the biomass and the wastewater in a reactor system. The HRT of a system must be long enough for the degradation process by the microorganisms to take place. However, in the application of granular biomass in the treatment system,

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the HRT should not be too long as it may cause the disintegration of the granules. According to Tay et al. (2002b) and Wang et al. (2005b), a short HRT is favorable for rapid granulation process, while too long HRTs may lead to granulation system failure due to high biomass lost (Pan et al., 2004). An optimum HRT of biogranulation systems would be able to stabilize the reactor performance with good biomass retention and high removal performance. According to Pan et al. (2004), the optimum HRT for aerobic granulation systems ranging from 2 to 12 hours enabled the formation and maintenance of stable aerobic granules with good settleability and microbial activities. However, the optimum HRT for the treatment of different types of wastewater may vary depending on the type of wastewater and the targeted degradation compound.

Figure 6.5 Distribution of size particles at different stages of the experiment. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h)

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Figure 6.6 shows the profile of SVI throughout the experiments. The SVI value in Stage V was reduced from 16.8 1.3 mL/g (in Stage IV) to 15.5 1.3 mL/g. This is expected to be due to the accumulation of more inert solids within the granules as shown with low levels of MLVSS/MLSS ratio in Stage V (0.71). Despite changes in HRT that caused decrease in the granular sizes, the SVI values of the whole experiments were good except for Stage VI. During Stage VI, the prolonged duration of the aerobic phase (i.e. 17.8 hours) which was operated at high superficial air velocity (2.5 cm/s), cause the granular biomass to rupture. At this stage, size of the granular biomass becomes smaller causing the settleability of the particles to reduce and was demonstrated with increase in SVI value.

II

III

IV

Figure 6.6

Profile of sludge volume index throughout the experiment. Stage I:

anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage

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III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h)

6.5.4 Effect of Hydraulic Retention Times on Chemical Oxygen Demand Removal

The profile for COD concentration in the influent, effluent and removal performance for all six stages of experiment is given in Figure 6.7. The biogranular system showed consistent COD degradation performance with 84.2 0.9% removals after about 50 days of start-up period (acclimatization phase). experimental process was increased from 6 hours to 24 hours. the OLR as mentioned earlier.
3 3

The overall

performance was almost consistent despite the fact that the duration of the This phenomenon may be due to the decreasing biomass concentration and also due to the decrease in When the OLR was increased from 0.6 kg COD/m day to 0.8 kg COD/m day on the 194th day of the experiment (Stage III to Stage IV), the COD removal efficiency increased from about 84.4 0.4% at the end of Stage III (day 193) to 90.7 0.2% at the end Stage IV(day 236).

Mohan et al. (2007b) reported that the performance efficiency of the system was found to be affected by the operating OLR. The SBR system operating at higher OLR resulted with a high substrate uptake rate at the end of the cycle period. This was also observed by Ong et al. (2005b).

An increase in the percentage of COD removal efficiency was also observed when the period of anaerobic phase was increased from 12 hours to 18 hours. The removal increased from 90.7 0.2 % in Stage IV to 94.1 0.6 % in Stage V. Psukphun and Vinitnantharat (2003) claimed that the increase in the non-aeration phase in the SBR system would cause an alteration in the population of anaerobic microorganisms in the system which is expected to produce good COD and color removal for textile wastewater. However, according to Kapdan and Oztekin (2006), when the duration of anaerobic phase is too long, the contribution of aerobic reaction

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phase might be decreased. This is possibly due to the toxic effect of aromatic amines produced during dye degradation.

Figure 6.7 Profile of COD removal performance of the reactor system at different stages of the experiment. () Influent COD; () Effluent COD, () COD removal. Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic (11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h)

Owing to the condition in the SBR system where different reaction phases occur in the same column, too long anaerobic reaction periods will cause high accumulation of aromatic amine in the same compartment. High concentrations of aromatic amines may inhibit the activity of aerobic microorganisms during the aerobic phase. In this study, eventhough the anaerobic reaction phase was extended up to 18 hours, there was no reduction in COD removal. This shows that there was

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no inhibition on the activity of aerobic microorganisms by the long accumulation of the byproduct produced from anaerobic degradation of the dye compound. It might be that the concentration of dye used during this experiment was not that high to produce enough concentration of the aromatic amines that may cause toxic effect towards the microorganisms within the biogranules. Furthermore, the biogranules might not be affected by the dyestuff degradation byproducts due to the structural form of the biogranules. The biogranules structure which consisted of EPS acts as a shield for microorganisms within the granules against any shock loading or toxic compound.

At the final stage (Stage VI) of the experiment, a surge drop of COD removal efficiency was observed. As the aeration time was increased from 6 to 18 hours, the COD removal reduced from 94.1 0.6% to 82.6 0.8%. The drop in the COD removal efficiency was due to the increase in biomass loss into the effluent. The MLSS in Stage VI was 23.3 0.8 g/L as compared to 31.6 3.7 g/L observed in the previous stages.

6.5.5 Effect of Hydraulic Retention Time on Color Removal

Color removal was observed to increase from 66.7 1.6 % to 76.5 0.8 % as the HRT increased from Stage I to Stage III. Increase in the HRT allows longer contact time between the granules and the wastewater resulting in better color removal. Furthermore, when the OLR was increased from 0.6 kg COD/m3day (Stage III) to 0.8 kg COD/m3day (Stage IV), a significant improvement in color removal from 76.5 0.8 % to 83.1 1.4 % was observed. This may be caused by the increase in the microbial population. Ong et al. (2005b) reported that the percentage of color removal efficiency increased by 16% in anaerobic and 50% in aerobic SBR reactor systems when the OLR rate was increased from 2.66 to 5.32 g COD/Lday. An increase from 82% to 90% of color removal efficiency was observed by

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Talarposhiti et al. (2001) when the COD loading was increased in a two-phase anaerobic packed bed reactor from 0.25 to 1 kg COD/m3day. Since more color removal took place in anaerobic condition (Banat et al., 1996; van der Zee et al., 2001a and Dos Santos et al., 2007), the percentage of color removal was once again increased from Stage IV (83.1 1.4%) to Stage V (86.5 0.5%) when the anaerobic reaction phase was extended from 12 to 18 hours of the 24 hours reaction cycle. Improved decolorization process that occurs during the anaerobic stage enhances the overall wastewater biodegradation since more readily biodegradable substances could be degraded in the following aerobic treatment (Stolz, 2001). Figure 6.8 shows the profile of the color removal performance.

II

III

IV

Figure 6.8 Profile of color removal performance of the reactor system at different stages of the experiment. () Influent color, () Effluent color, () Color removal. (100 ADMI 1 Pt-Co). Stage I: anaerobic (2.8 h): aerobic (2.8 h); Stage II: anaerobic (5.8 h): aerobic (5.8 h); Stage III and Stage IV: anaerobic (11.8 h): aerobic

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(11.8 h); Stage V: anaerobic (17.8 h): aerobic (5.8 h); Stage V: anaerobic (5.8 h): aerobic (17.8 h) With respect to the mechanisms that are involved in color degradation, the addition of electrondonating substrate could considerably improve the decolorization reductive rate (Bras et al., 2001, Dos Santos et al., 2005). Their studies using anaerobic and aerobic sequential wastewater treatment system indicated that the anaerobic stage was the main step for color degradation while the aerobic phase acted as the polishing step and enhancement in COD removal. Higher initial COD concentration did not improve color removal but caused deterioration in COD removal in the anaerobic-aerobic SBR system (Kapdan and Oztekin, 2006).

Psukphun and Vinitnantharat (2003) reported that the duration of the anaerobic phase should be long enough to obtain better COD and color removal. Increase in the HRT would provide enough time of the COD and intermetabolites of simulated textile wastewater in anaerobic or/and anaerobic/aerobic systems (Isik and Sponza, 2008). This means biodegradation of the azo bonds may require a certain contact time in order to achieve high removal efficiency. Depending only on the filling stage to provide anaerobic condition for the cleavage of azo bond compounds may not be adequate for textile wastewater treatment. However, the time requires for the cleavage of the azo bond may be affected by the complexity of the dye molecule structures. Higher contact time for the anaerobic reaction phase can be provided by having the anaerobic reaction stage during the react phase in the SBR cycle as proposed in this experimental study. The suitable contact time of anaerobic and aerobic reaction phase may provide high removal performance for the cleavage of the N=N bond (anaerobic condition) and mineralization of aromatic amines (aerobic phase). Furthermore, the reduction of COD is more effective during the aerobic stage as compared to the anaerobic reaction condition (Smith et al., 2007). From this study, it shows that having longer anaerobic (18 hours) and shorter aerobic (6 hours) reaction phase resulted with the highest removal for color and slight improvement in the efficiency of COD removal. The effect of HRT on the COD and color removal performance by the FGS biomass at different stages of the experiment is given in Table 6.7.

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Table 6.7: Profile of COD and color removal percentage at different stages of experiment
Reaction Phase Anaerobic (hours) Aerobic (hours) COD (%) Stage I 2.8 II 5.8 III 11.8 IV 11.8 V 17.8 VI 5.8

2.8

5.8

11.8

11.8

5.8

17.8

84.2 0.9

84.6 1.1

84.4 0.4

90.7 0.2

94.1 0.6

82.6 0.8

Color (%)

66.7 1.6

74.3 0.4

76.5 0.8

83.1 1.4

86.5 0.5

75.4 0.3

6.5.6 Effect of Hydraulic Retention Time on the Biokinetics of Facultative Anaerobic Granular Sludge during Biodegradation of Dye

The total solid biomass concentration in a biological reactor system is governed by the rate of substrates utilization and biomass production by the microorganisms. The rates of such processes which are known as the biokinetic parameters would give prediction on the performance of the biological process in wastewater treatment. The understanding and information on the rate of biological reactions and basic principles governing the growth of microorganisms are very important in developing an effective design and operation of the biological reactor system (Tchobanoglous et al., 2004).

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In this study, the biokinetic parameters of the FAnGS were also investigated in relation to the effect of different HRTs. The biokinetic parameters that were investigated are the overall specific biomass growth rate (overall), endogenous decay rate , observed biomass yield , and theoretical biomass yield All of

the calculations for the biokinetic parameters are according to the equations listed in Table 6.8 and are based on the reports by Liu and Tay (2007a) and Chen et al. (2008b). The results of biokinetic parameters for all stages in this experiment are given in Table 6.9.

When the experiment moved from Stage I to Stage III, the SRT was increased from 27.6 13.4 to 78.9 30.8 d, these have caused the overall to reduce from 0.036 to 0.013/d. The results are in accordance with the report stated by Li et al. (2006b) that sludge biomass will loose their bioactivity when the SRT is increased. The reduction of the overall as the HRT increased was also observed by Liu and Tay (2007a). As mentioned earlier, the OLR was reduced when the HRT was increased from 6 to 24 hours (from Stage I to Stage III). The reduction in the OLR may also contribute to the reduction of overall from Stage I to Stage III. The overall of Stage IV and Stage V was the same when the SRT of these two stages slightly increased from 70.1 23.9 to 72.5 23.3 d, respectively. The overall of Stage VI increased as the SRT was reduced to 41.6 18.4 d eventhough Stage VI was operated with the same HRT as Stage IV and V. The reduction of the SRT in Stage VI may be contributed by the increase in the sludge washout that was shown by the increase in the suspended solids concentration in the effluent discharge.

The rate of biomass lost due to endogenous respiration is represented by endogenous decay rate kd, as given in Equation 6.4. The OUR that was measured during the last 10 min or before the second aeration phase stop of one cycle operation was used to calculate the kd. As the HRT increased from 6 to 24 hours (Stage I to Stage III), the kd values reduced. However, since the reduction was also very small, the kd can be considered as constant when the HRT was increased. Furthermore, the kd value during 24 hours HRT of Stage III to V can also be considered constant (0.0075 to 0.0076/d). It can thus be concluded that the kd is considered constant

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throughout the experiment. The kd values calculated from this study were very small as compared to the kd values of aerobic granules (Chen et al., 2008b) and of the activated sludge (Tchobanoglous, 2004). Table 6.8: Coefficient of biokinetic parameters
Biokinetic Coefficient Overall specific biomass growth rate Units Formula Equation

Per day

(6.3)

= sludge retention time

Endogenous decay rate

Per day

= oxygen uptake rate (mg/L.h) = theoretical chemical oxygen demand which is assume as 1.42 mg O2/ mg biomass M= biomass concentration (mg VSS/L)

(6.4)

Observed biomass yield

mg VSS/mg COD

= Effluent volatile solid concentration (g VSS/L) Ci = COD concentration in the influent (mg/L) Ce = COD concentration in the effluent (mg/L)

(6.5)

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Theoretical biomass yield

mg VSS/mg COD

(6.6)

Table 6.9: Kinetic coefficients of FAnGS at different stages of the experiment

Kinetic coefficients of facultative granules Observed specific biomass growth rate (overall) (per day) Endogenous decay rate kd (per day) Observed biomass yield (Yobs) (mg VSS/ mg COD) Theoretical biomass yield Y (mg VSS/ mg COD)

Stage I

Stage II

Stage III

Stage IV

Stage V

Stage VI

0.036

0.024

0.013

0.014

0.014

0.024

0.0096

0.0086

0.0075

0.0075

0.0076

0.0060

0.316

0.298

0.242

0.269

0.217

0.412

0.399

0.395

0.385

0.410

0.338

0.515

The observed biomass yield Yobs is the ratio of the biomass production rate to the substrate removal rate and is calculated according to Equation 6.5. The Yobs is one of the most important parameter used in biological kinetic models. Equation 6.5 is derived from the equation below (Liu and Tay, 2007a):

where,

211

XVSS1 = XVSS2 = Ve tc Xe Ve = = = =

Volatile solid concentration at the beginning of cycle operation in SBR reactor (g VSS/L) Volatile solid concentration at the end of cycle operation in SBR reactor (g VSS/L) Working volume of the SBR system Cycle time of SBR operation (d) Effluent volatile solid concentration (g VSS/ L) Effluent volume in SBR operating cycle (L)

Equation 6.5 is simplified from Equation 6.7 when the reactor system reached steady state and the biomass was maintained at a constant value (Chen et al., 2008b). The Yobs can be used to describe the sludge productivity which relates to the net sludge production.

The results in Table 6.9 show that the sludge production is inversely related to the value of SRT as shown in Stage I to III. As SRT increased, the Yobs, value decreased. Since the biomass activity is reduced when the SRT increased, this has caused the reduction of the biomass yield. The results obtained from this experiment are in accordance with the ones reported by van Loosdrecht and Hence (1999). It is well known that the net sludge production in an activated sludge system decreases with increasing sludge age. The biokinetic parameters could give a good indication for the system performance. It can be used as a basis for the design and product optimization of a system reactor. The Yobs value of Stage IV to Stage V, decreased from 0.269 to 0.217 mg VSS/ mg COD as the SRT of those stages was increased from 70.1 23.9 to 72.5 23.3 d, respectively. Eventhough Stage IV and Stage V were operated with the same HRT, the ratio of anaerobic/aerobic reaction phase was different. It shows that when the ratio of anaerobic/aerobic time was increased, the Yobs decreased.

The theoretical

value is calculated using Equation 6.6. It is expected that

the theoretical Y value will be higher as compared to Yobs. The difference between

212

the

and theoretical Y value is contributed by endogenous metabolism, predation,

death and lysis process. The theoretical Y value obtained in this study shows the same pattern as given by the Yobs. The results for the Yobs and theoretical Y value obtained in this experiment are within the typical reported range of conventional activated sludge system (Al-Malack, 2006 and Tchobanoglous et al., 2004). 6.6 Conclusions

i.

The granular biomass concentration of the reactor system reduced as the HRT increased which is mainly due to the reduction in the OLR. However, with HRT of 24 hours, the biomass slightly increased when the period of anaerobic reaction phase was longer then the aerobic reaction. The ratio of MLVSS/MLSS increased during Stage V with anaerobic/aerobic reaction phase was set with 17.8/5.8 hour which may be due to the increased accumulation of inert particles within the granules. Eventhough with increase in the HRT, the concentration of granular biomass can be improved with increase in the anaerobic reaction time and reduction in the aerobic reaction time as shown in Stage V.

ii.

The size and the SVI of the FAnGS reduced as the HRT of the system increased (Stage I to Stage III) due to increase in the aeration time that resulted with the disintegration of the FAnGS. Too long aerobic reaction times exposed the granules under prolonged starvation condition causing instability of the granular structure that lead to disruption of the granules. The size and the SVI value were improved with the increase in the OLR (Stage IV). The size and the SVI were also improved with increase in the anaerobic and reduction in the aerobic reaction phases (Stage V).

iii.

The percentage of COD removal in this study was not likely affected by the increase in the HRT which was mainly due to the decrease in the granular biomass and OLR (Stage I to Stage III). However, the COD removal was improved with the increase in the anaerobic reaction phase shown in Stage V

213

as compared to Stage IV. The percentage of color removal has improved with the increase in the HRT.

iv.

Stage V (17.8 and 5.8 hours of anaerobic and aerobic reaction phase, respectively) can be considered as the best condition for the removal of color and the organic compound since the percentage of color and COD removal are the highest.

v.

Increase in the HRT resulted with an increase in the SRT. Since the SRT is inversely related to the overall, increase in the HRT will cause a reduction in the overall. Increase in the HRT has caused a reduction in the bioactivity of the granular sludge shown by the reduction of the overall, Yobs and Y values. A slight increase in the SRT was observed with increase in the anaerobic/aerobic time ratio. This has caused a reduction in the Yobs and Y values but the overall is considered constant. constant throughout the experiment. The kd is also considered

CHAPTER 7

EFFECT OF SUBSTRATE AND RIBOFLAVIN ON FACULTATIVE ANAEROBIC GRANULAR SLUDGE

7.1

Introduction

The biodegradation process of azo dyes can be influenced by many factors. Among them, the presence of redox mediator and the use of different compositions as the primary substrate were identified as factors that may give effect on the rate of dye degradation process (van der Zee et al., 2001b; Keck et al., 2002; van der Zee and Cervantes, 2009). Despite many studies conducted on biodegradation of azo dyes, the effectiveness of these two factors in enhancing the decolorization of textile wastewaters is still ambiguous. Most of the study on the effect of redox mediator focused on either single or mixed bacteria cultures in degrading the dye. Anaerobic granular sludge has been frequently used as the source of biomass in dye degradation under anaerobic condition while only few studies were conducted by using aerobic biomass (Keck et al., 1997 and Kudlich et al., 1997).

Furthermore, knowledge on the effect of the redox mediator and primary substrate concentration on the use of facultative granules for decolorization of the azo dye through the application of experimental design is still lacking. The presence

215

of substrate in the textile wastewater will release electrons during its degradation process. These electrons are required for the degradation of dye which will be mediated by the redox mediator. Redox mediators are responsible in transferring the electrons to the dye compounds. However, the understanding on the interactions between these factors is still indistinct and need to be explored further. In this study, the impacts of the redox mediator and primary substrate concentration were investigated. Mixed azo dye consisted of Sumifix Black EXA, Sumifix Navy Blue EXF and Synozol Red K-4B were again used as the model compound. A similar substrate which has been used in studies presented in Chapters 4 through 6 was used and riboflavin was used as the redox mediator. Riboflavin was chosen as the redox mediator since it represents the redox active moiety in ubiquitous enzyme cofactors such as flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which have been implicated in the azo dye reduction. FMN and FAD are not practical to be used since they are expensive biochemical materials. Riboflavin is an affordable vitamin which could be added into the bioreactor to stimulate azo dye reduction during anaerobic treatment (Field and Brady, 2003). findings on the study. This chapter presents the

7.2

Materials

Most of the chemical or reagents and equipment used in this study are as described in Section 4.2. In addition, a 150 mL serum bottle with rubber stopper was used as a batch test in this experiment. A sealer (E-Z Crimper-20 mm) was used to seal the serum bottle with a metal cap. A 30 mL syringe with needle was used for sampling purposes. Synthetic textile dyeing wastewater as described in Section 4.2.1 was used as the media solution of the experiment. The concentration of the mixed dyes in the wastewater was prepared at 100 mg/L. Sigma (St Louis, USA). according to the experimental design. Riboflavin was obtained from Substrate and riboflavin concentration were varied

216

7.2.1 Granular Precursor

A range of 0.3 to 0.6 mm of FAnGS biomass with an average granular size of 0.45 mm was used in this experiment. The FAnGS used in this study was developed as presented in Chapter 4. About 10% (v/v) of granules were inoculated into the serum bottle containing 130 mL of synthetic medium which give about 1.94 g/L of volatile suspended solids (VSS). The FAnGS was always kept in the synthetic dyeing wastewater prior to conducting experiments for acclimatization purposes.

7.3

Analytical Methods

7.3.1 Chemical Oxygen Demand and Color Removal

In this study, the removal efficiency of COD through the batch experiment using FAnGS was conducted according to the analytical methods described in Section 4.3.4.2. The percentage of color removal was estimated quantitatively by measuring the absorbance reduction at the maximum absorbance wavelength of the dyes used in the experiment. The individual dye was measured at 600 and 542 nm wavelength using a UV-visible spectrophotometer (Shimadzdu Model UV-2450). Distilled water was used as the blank.

217

7.4

Experimental Procedures

Screening of redox mediator was conducted as the preliminary test in order to determine the suitable range of redox concentration to be used in the experimental design study. Figure 7.1 show the experimental work carried out in this study.

Figure 7.1 Experimental works for the investigation on the effect of substrate concentration and redox mediator on COD and color removal via the aid of experimental design

218

7.4.1 Screening for Concentration of Redox Mediator

The screening process was conducted using batch experiment. Forty-five (45) mL of synthetic dye wastewater with the organic substrate concentration at 1500 mg/L and dye concentration of 100 mg/L were filled in a 50 mL centrifuge tube. Ten (10) % (v/v) of granular biomass was added into the synthetic media. Different concentrations of riboflavin ranging from 0.001 to 0.01 mM were added in each of the centrifuge tube. A half (0.5) mL of sample was taken hourly for an interval of 24 hours and measured for the color reduction. The experiments were conducted at room temperature with the samples kept under static condition. The samples were centrifuged at 5,000 rpm for five minutes to pellet down any suspended particles prior to color measurement.

7.4.2 Batch Experiment for Chemical Oxygen Demand and Color Removal Using Facultative Anaerobic Granular Sludge

Batch experiments were conducted to study the effects of substrate and redox mediator concentration on COD and color removal under anaerobic and aerobic conditions. One hundred and thirty (130) mL of synthetic wastewater was added to the serum bottle. Then, 10% (v/v) of granular biomass was added to the serum bottle. The bottles were capped with rubber stoppers and then were sealed with metal caps using a sealer (E-Z Crimper).

The anaerobic condition was established by purging the headspace with N2/CO2 (80%/20%) for 2 min into the serum bottle. By doing this, the concentration of DO was kept lower than 0.2 mg/L. After purging, the redox mediator was added into the wastewater sample in the serum bottle. The concentration of redox mediator

219

and substrate in the synthetic wastewater were based on the concentration set by the experimental design as will be explained in the next section. During the anaerobic reaction phase, the serum bottles were placed on an orbital shaker and were shaken at a speed of 100 rpm for continuous contact of granules with the synthetic wastewater. The experiment under anaerobic condition was conducted for twelve hours. After the anaerobic reaction phase was completed, the samples were then exposed to the aeration phase by supplying air bubbles at an air flow rate of 10 ml/h for another twelve hours.

Samples were taken every two hours during the 24 hours of the reaction period. During the anaerobic phase, the samples were taken using a 30 mL syringe with needle. To avoid introducing oxygen into the reactor during the anaerobic phase, the needle was plugged through the rubber cap after removing the top part of the metal cap.

7.4.3

2-Level Factorial and Central Composite Design Experiment

Two-level factorial and Central Composite Experimental design was used in this study. The concentrations of substrates and redox mediator were used as the variables while the removal of COD and color were the responses. The concentrations of the substrate and redox mediator were in the range of 500 to 3000 mg/L and 1 to 150 M, respectively. MinitabTM Statistical Software was used for the design and analysis of the factorial experiment while Design Expert Statistical Software was used for the CCD experiment.

Table 7.1 shows the experimental runs used in the factorial design and CCD in actual and coded values. The two variables of 2-level factorial design comprised

220

of four experimental runs (CC01 to CC04). Since the experiments were conducted in duplicate, a total of eight runs were carried out. The results provide the linear effect as well as the interaction effects of the variables. Star point (CC05 to CC08) and centre point (CC09 to CC013) values were added for the CCD experiments that present any non-linearity effect of the variables in the reaction process.

Table 7.1: Experimental runs of factorial design and CCD in actual and coded values (not in random order)
Factor 1 Run A: Substrate CC01 CC02 CC03 CC04 CC05 CC06 CC07 CC08 CC09 CC10 CC11 CC12 CC13 -1 (866.1) 1 (2633.8) -1 (866.1) 1 (2633.8) -1.414 (500) 1.414 (3000) 0 (1750) 0 (1750) 0 (1750) 0 (1750) 0 (1750) 0 (1750) 0 (1750) B: Riboflavin -1 (22.8) -1 (22.8) 1 (128.2) 1 (128.2) 0 (75.5) 0 (75.5) -1.414 (1) 1.414 (150) 0 (75.5) 0 (75.5) 0 (75.5) 0 (75.5) 0 (75.5) Factor 2

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7.5

Results and Discussion

7.5.1 Screening for Redox Concentration

The percentage of color removal from the mixed dye at different concentrations of riboflavin is shown in Figure 7.2. As indicated earlier, the mixed dye consisted of Sumifix Black EXA, Synozol Red K-4B and Sumifix Navy Blue EXF with the highest peak measured at wavelength of 480, 542 and 600 nm, respectively. Since riboflavin is a colored compound that has a peak wavelength absorbance at 475 nm, the reading for Sumifix Black EXA dye at 480 nm wavelength has been interfered by the color of the riboflavin. Hence, only the results of Sumifix Navy Blue EXF and Synozol Red K-4B are shown in the figure.

As shown in the figure, a very low concentration of riboflavin is capable of increasing the color removal of the mixed dye. Up to riboflavin concentration of 0.1 mM, increase in its concentration, increased the extent of color removal. The highest color removal (80%) was achieved when the concentration of redox mediator was at 0.1 mM. The color removal was then reduced when the concentration of the redox mediator was further increased. A minimum percentage of color removal was observed at the redox mediator dose of 1 mM. In the control sample where there was no addition of riboflavin, the color reduction can be considered as reasonably high with about 60% removal. This may be due to the presence of other accelerating dye degradation agent that may naturally have been produced in the sludge or in the granules (Dos Santos et al., 2006b).

Different concentrations of redox mediator have been used to accelerate the dye degradation process in previous studies. For example, 0.012 -0.024 mM and 1 mM of AQDS has been used by Dos Santos et al. (2004) in batch experiments. van der Zee et al. (2001a) has used AQDS at the concentration of 19 M to 155 M in a

222

continuous experiment for the degradation of the Reactive Red 2 by anaerobic granular sludge in an UASB system. Riboflavin in the range between 0.091 to 0.546 mM was used by Field and Brady (2003) in enhancing the reduction of Mordant Yellow 10 by anaerobic granular sludge in batch experiments.

Figure 7.2: Color removal at different concentrations of riboflavin. Absorbance at 600 nm (), absorbance at 542 nm ()

Most of the previous experiments were conducted at very low concentrations of redox mediator. A small amount of redox mediator is capable of achieving high color removal. Furthermore, the results for color removal would be interfered when high concentrations of redox mediator are applied since most of the redox mediators are colored chemical compounds. Only few types of redox mediator are in the form of colorless compounds such as methyl vilogen and NAD. However, as mentioned earlier, the application of NAD is limited due to its economical constraint.

223

As a result of the screening study, a further study on the effect of redox mediator on dye degradation was conducted at low concentrations of 1 to 150 M of riboflavin.

7.5.2 Factorial Design Analysis of Chemical Oxygen Demand Removal

The experimental results for the factorial runs are given in Table 7.2. The overall COD removal was higher under the anaerobic reaction phase reaching almost 80% as compared to 68% of the highest COD removal for the aerobic reaction phase. The lowest percentage of COD removal of anaerobic and aerobic reaction conditions was about 24% and 29%, respectively. The percentage of total COD removal varied between 66 to 86%.

The summary table of the ANOVA that shows the main and two-way interaction effect is given in Table 7.3. Figure 7.3 shows the Pareto chart generated by MINITAB for anaerobic, aerobic and total COD removal. The detailed results of the analysis constructed by the software are given in Appendices F1 to F3. The analyses show that at 90% confidence level (P-value less than 0.1), substrate and riboflavin have significant main and interactive effects on aerobic COD removal and total COD removal. As for COD removal during the anaerobic stage, only substrate was found to have a significant effect.

224

Table 7.2: Experimental results for factorial design analysis Run CC01 CC02 CC03 CC04 CC05 CC06 CC07 CC08 Anaerobic COD removal 32.9 79.4 28.8 78.4 25.9 78.9 24.4 77.1 Aerobic COD removal 67.8 29.7 52.2 27.9 68.3 28.8 55.3 37.5 Total COD removal 78.4 85.5 66.0 84.4 76.5 85.0 66.2 85.7

Table 7.3: The P-values of the estimated main and interaction effects of substrates and riboflavin for the percentage of COD removal
Anaerobic COD Significanta removal Aerobic COD removal Significanta Total COD removal Significanta

Effect

Main Substrate < 0.0001 Yes < 0.0001 Yes < 0.0001 Yes

Riboflavin 2-way interaction Substrate Riboflavin

a

0.373

No

0.001

Yes

< 0.0001

Yes

0.755

No

0.002

Yes

0.001

Yes

significant at = 0.1

225

Figure 7.3 The Pareto chart of COD removal for (a) anaerobic, (b) aerobic and (c) total removal (A: substrate; B: riboflavin; : 0.1)

226

7.5.2.1 Factorial Analysis: The Main Effect of Substrate on Chemical Oxygen Demand Removal

With respect to the experimental conditions used in this study, the substrate concentration shows highly significant effects for both the anaerobic and aerobic reaction phases with P-value of less than 0.0001. The effect was also significant for total COD removal where the P-value was also less than 0.0001. However, the direction of the effect was opposite when compared between the anaerobic and aerobic reaction phases.

During the anaerobic reaction phase, the estimated effect was +50.45 while during the aerobic reaction phase the value was -32.45. This means during the anaerobic reaction phase, the percentage of COD removal increased as the substrate was increased. Under the aerobic reaction phase, the percentage of COD removal decreased with increase in substrate concentration. Substrate concentration was found to cause a positive effect on total COD removal with an estimated effect of 13.4.

When the substrate concentrations were increased, more food was supplied to the microorganisms. Such condition was postulated to cause increment in the Increase in COD concentration of the suspended biomass in the serum bottle which leads to increase in the percentage of COD removal under anaerobic condition. removal from 73% to 88% has been also reported by Siman et al. (2004) when the OLR was increased from 1.5 to 5.4 g COD/Lday in an anaerobic sequencing biofilm batch reactor system.

The negative effect of substrate concentration on aerobic COD removal can be due to several reasons. Since the experiment for aerobic reaction phase was a continuation from the anaerobic reaction phase, the same sample was used for the

227

aerobic phase after the anaerobic reaction was completed.

Since most of the

substrate has been degraded during anaerobic reaction, so the amount of substrate left for aerobic reaction was lesser. In other words, there was only a little amount of substrate left to be used during the aerobic phase eventhough the initial concentration of substrate at the start of the experiment was high.

Another possible reason for the lower percentage of COD removal as the substrate increased under aerobic reaction phase was insufficient oxygen supplied. As the substrate increased, the demand for oxygen is expected to be greater in order to degrade more substrate. Since the rate of the aeration was not increased throughout the experiment, this might contribute to the decreasing COD removal since the supply of oxygen is not enough to oxidize the increasing concentration of substrate.

Another possibility might be due to the uncleavage dye compounds and the accumulation of non-mineralized or slow degradation of amines compounds as reported by Tan and Field (2000). It can be due to the toxic effect of the accumulation of aromatic amines, the byproduct produced during the increasing anaerobic degradation of the mixed dye presence in the synthetic media. Aromatic amines are known as toxic compounds (Weisburger, 2002) which may affect the microbial degradation activity of the aerobic microorganisms localized at the outer layer of the granules.

228

7.5.2.2 Factorial Analysis: The Main Effect of Riboflavin on Chemical Oxygen Demand Removal

The concentration of riboflavin did not have a significant effect on the percentage of COD removal for the anaerobic reaction phase since the P-value was 0.373. As the concentration of redox mediator increased, more of the N=N bond are expected to be cleaved (Field and Brady, 2003 and Mendez-Paz et al., 2005) and more amines were released during the anaerobic reaction phase. Since amine could not be further degraded under anaerobic condition, the concentration of COD did not reduce significantly.

However, for the aerobic reaction phase, the redox mediator has a significant effect on the percentage of COD removal with the P-value of 0.001. The percentage of COD removal during the aerobic reaction phase was reduced when the concentration of redox mediator was increased with the estimated effect of -7.95. As stated earlier, under anaerobic condition, when the redox mediator was increased, more cleavage of the azo bond will take place and hence increased the release of the amines. Since the aeration rate was kept constant throughout the experiment, as the concentration of redox mediator increased, the oxygen supply was not enough to degrade the increasing concentration of the amines thus causing reduction in COD removal. The presence of high concentrations of amines may also impose a toxicity effect to the microorganisms in the serum bottle that lead to reduction in aerobic COD removal.

The effect of increasing redox mediator on the percentage of total COD removal was also significant where the P-value is less than 0.0001 and the estimated effect was -5.78. This means, as the concentration of the redox mediator increased, the percentage of total COD removal was reduced. Based on the value of the estimated effect, it shows that the significant effect of redox mediator in total COD removal was mainly contributed by the aerobic reaction phase. The reasoning for the

229

reduction of the total COD removal was the same as explained for the reduced COD removal under aerobic condition. Figure 7.4 shows the main effect of substrate and riboflavin during the anaerobic and aerobic reaction phases and also for the total COD removal.

Figure 7.4 Main effect plot of substrate and riboflavin for (a) anaerobic, (b) aerobic and (c) total COD removal

230

7.5.2.3 Factorial Analysis: The Interaction Effect of Substrate and Riboflavin on Chemical Oxygen Demand Removal

The interaction effect between substrate and riboflavin was insignificant for percentage of COD removal in the anaerobic reaction phase but was significant for the aerobic reaction phase and the total COD removal with P-values of 0.02 and 0.001, respectively.

At low substrate concentration, increase in riboflavin is expected to result in increase in the production of amines. Amines that can be further degraded under aerobic condition contributed to the increased concentration of COD. Since there was no increment on the aeration rate, the amount of oxygen may not be enough for the microorganisms to degrade the increasing COD level resulting in decreasing percentage of COD removal as the riboflavin increased. Due to the same reason, increase in the concentration of riboflavin has also caused a reduction but only with a slight decrease in the percentage of COD removal at high concentrations of substrate.

Higher percentages of total COD removal were observed at higher concentrations of substrate (2633.88 mg COD/L) as compared to the lower concentrations of substrate (866.12 mg COD/L) at low concentrations of riboflavin. However, increase of riboflavin at higher substrate levels has barely caused any changes on the percentage of total COD removal as the riboflavin itself has caused an increase in the COD value (through the formation of amines). The high removal percentage during high substrate concentrations was due to the anaerobic degradation process.

The reduction of the total COD removal was observed at low substrate concentration as the riboflavin increased, mainly contributed by the increase in the concentration of the aromatic amines and the insufficiency of oxygen supply. Figure

231

7.5 shows the interaction plot of substrate and riboflavin for anaerobic, aerobic and total COD removal.

Figure 7.5 Interaction plot for the percentage of COD removal for (a) anaerobic, (b) aerobic and (c) total removal (Substrate: ---- 2633.88 m/L; ___ 866.12 mg/L; Centre point)

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7.5.3

Central Composite Design Analysis of Chemical Oxygen Demand Removal

Table 7.4 shows the experimental results of the CCD for COD removal. The analysis was carried out using full quadratic terms including linear, square and interaction. The results of the ANOVA for COD percentage removal at different reaction conditions are shown in Table 7.5. The detailed results for estimated regression coefficient and ANOVA table are given in Appendices F4 to F6.

Based on the P-value, the results showed that only the linear term of substrate (P-value 0.094) of the anaerobic reaction phase and the square term of substrate (Pvalue of 0.045) of the aerobic reaction phase are significant. This shows that substrate concentration only caused a linear effect on the COD removal during the anaerobic reaction phase and has a non-linear effect during the aerobic reaction phase. Both the models of COD removal under anaerobic and aerobic reaction phases are insignificant with R-squared values of 46% and 47.9%, respectively.

Figure 7.6 shows the contour and surface plot of the significance defined model for total COD removal. The figure clearly shows that as the substrate concentration increases, the percentage of COD removal also increases. With respect to the effect of riboflavin, at low substrate concentrations, increase in the concentration of riboflavin has caused a reduction in the total COD removal from 71.2% to 61.8%. At high concentrations of substrate, increase in the concentration of riboflavin have caused a slight increase in the total COD removal from 82.3 to 84.1%. The highest percentage removal of total COD was observed at substrate and riboflavin concentrations of 2165.8 mg/L and 23.4 M, respectively with 84.5% removal.

233

Table 7.4: Experimental results for CCD analysis

Run CC01 CC02 CC03 CC04 CC05 CC06 CC07 CC08 CC09 CC10 CC11 CC12 CC13 Anaerobic reaction phase 32.9 79.4 28.8 78.4 23.9 31.6 24.4 21.3 22.1 25.6 24.3 23.9 24.0 Aerobic reaction phase 67.8 29.7 52.2 27.9 32.0 66.8 73.7 73.1 75.4 72.5 73.7 72.6 73.6 Total COD removal 78.4 85.5 66.0 84.4 48.2 77.3 80.1 78.8 80.8 79.5 80.1 79.2 79.9

The best statistical model that can be used to represent the total COD removal for this process based on the experimental conditions in this study obtained from the response surface analysis is:

Total COD removal = +51.26 + 0.04 A - 0.23 B + 6.0710-5 AB - 8.5810 A + 5.9210 B

-6 2 -4 2

(7.1)

where: A = Substrate in mg/L B = Ribofalvin in uM

234

Table 7.5: Summary of the P-value of the response surface modeling analysis
Anaerobic COD removal Aerobic COD removal The P-valuea Substrate Riboflavin SubstrateSubstrate RiboflavinRiboflavin SubstrateRiboflavin R-squared value Lack of Fit 0.094 0.868 0.249 0.385 0.939 46% <0.0001 0.799 0.725 0.045 0.538 0.707 47.90% <0.0001 0.002 0.295 0.008 0.396 0.277 85.80% <0.0001 Total COD removal

Term

0.01 0.04: Highly significant; 0.05 0.1: significant; 0.1 0.2: less significant; < 0.2: insignificant (Vecchio, 1997)

7.5.4 Factorial Design Analysis of Color Removal

In the investigation of color removal, focus was made on the anaerobic reaction phase only. This is because when the measurements for color removal were made during the aerobic reaction phase, negative responses were obtained. The negative response was due to the resurgence of color in the samples under the aerobic reaction phase. As mentioned earlier, since the absorbance for Sumifix Black dye is at 480 nm which is near to the absorbance of the riboflavin which is at 475 nm, the measurement for Sumifix Black dye was interfered by the color of the riboflavin. Hence, further analysis and discussion only focused on Sumifix Navy Blue EXE and Synozol Red K-4B only.

235

(a)

(b) Figure 7.6 The relationship between substrate, riboflavin and percentage of total COD removal after 24 hours of experimental run, (a) Contour plot and (b) Responses surface plot

236

Table 7.6 shows the experimental results for factorial runs for the percentage of color removal measured at absorbance 600 nm and 542 nm for Sumifix Navy Blue EXF and Synozol Red K-4B, respectively. Table 7.7 shows the summary of the ANOVA of the factorial runs. The detailed results for the table of estimated effect and coefficient and ANOVA table are given in Appendices G1 to G4. The Pareto chart that shows the effects of substrate and riboflavin concentration on the percentage of color removal of Sumifix Navy Blue EXF and Synozol Red K-4B at five and twelve hours of the experimental runs are given in Figures 7.7 and 7.8, respectively. The results of color removal were investigated at the early and end stages of the experiment in order to observe any time dependent effect among these variables. Figure 7.8 shows both variables give significant effect on color removal except for the interaction effect at twelve hours experiment for Sumifix Navy Blue EXF. Figure 7.8 demonstrates that both variables are significant at both five and twelve hour experiments for Synozol Red K-4B.

Table 7.6: Experimental results for factorial design analysis

Sumifix Navy Blue EXF Run 5 hours CC01 CC02 CC03 CC04 CC05 CC06 CC07 CC08 74.7 80.0 77.2 83.5 75.0 79.7 76.5 84.0 12 hours 77.0 76.7 83.9 81.0 78.8 76.4 84.0 82.0 5 hours 68.5 77.5 62.3 78.0 69.3 76.1 62.8 79.4 12 hours 75.2 75.8 82.3 80.0 76.0 76.0 82.5 81.0 Synozol Red K-4B

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Table 7.7: The P-values of the estimated main and interaction effects of variables substrates and redox mediator for the percentage of color removal
Sumifix Navy Blue EXF Effect 5 hours Main effect Substrate <0.0001 Yes 0.022 Yes <0.0001 Yes 0.071 Yes Significanta 12 hours Significanta 5 hours Synozol Red K-4B Significanta 12 hours Significanta

Riboflavin Interaction effect Substrate Riboflavin

a

<0.0001

Yes

<0.0001

Yes

0.015

Yes

<0.0001

Yes

0.017

Yes

0.351

No

0.002

Yes

0.028

Yes

significant at = 0.1

7.5.4.1 Factorial Analysis: Main Effect of Substrate on Color Removal

The results of the factorial design analysis showed that at five hours after the experiment started, the substrate was observed to give a significant main effect for both dyes; both with P-values of less than 0.0001 and an estimated effect of +5.95 and +12.02 for Sumifix Navy Blue EXF and Synozol Red K-4B, respectively. The percentage of color removal increased as the concentration of substrate was increased from 866.1 mg/L to 2633.9 mg/L. The percentage of color removal was a bit higher by about 4% for Sumifix Navy Blue EXF as compared to Synozol Red K-4B.

238

Figure 7.7

Pareto chart of Sumifix Navy Blue EXF removal at (a) 5 and (b) 12

hours (: 0.1; A: Substrate; B: Riboflavin)

239

Figure 7.8 Pareto chart of Synozol Red K-4B removal at (a) 5 and (b) 12 hours (: 0.1; A: Substrate; B: Riboflavin)

240

The effect of substrate concentration in this study is in accordance with the results reported by other researchers. It has been reported that the percentage of color removal increased as the substrate supplement to the system was increased (Fu et al., 2002 and Sirianuntapiboon and Srisornsak, 2007). Dafale et al. (2008) measured the kinetic constant of dye degradation of azo dyes with and without the presence of substrate glucose and reported that the kinetic constant was increased fivefold for the decolorization of Remazol Black B with the addition of 2 g/L of glucose. The use of substrate is essential in obtaining a good percentage of dye degradation (Delee et al., 1998 and Ozsoy et al., 2005). It was reported that the azoreductase enzyme system is responsible for the decolorization of dyes by bacteria with the presence of substrate under anaerobic conditions (Yoo, 2002).

The presence of external carbon sources will donate or produce electrons after being oxidized under the catabolism process. The released electrons will be used for the formation of the reducing equivalent or the reduced co-factor (Carliell et al., 1995). These reducing equivalent or reduced co-factor will involve in the electron transfer to the N=N bond of dye chemical structure and resulted with the cleavage of the double bond. Therefore, when the concentration of substrate is increased, more electrons will be donated to the azo bond and this improved the percentage of color removal.

The effect of substrate concentration on color removal was different at twelve hours for both dyes. As the effect of substrate was found to cause a positive effect at five hours reaction, the effect was negative at twelve hours reaction (i.e -1.9 for Sumifix Navy Blue EXF and -0.8 for Synozol Red K-4B).

The negative effect is probably due to the accumulation of amines as they are colored compounds which can cause different color intensity. The presence of these colored amines may not affect the color removal at the early stage of the experiment since the intensity of color due to the presence of dye was still high and the amines

241

were still relatively low in concentration. However, as the degradation of the dyes continued and reached the maximum removal at the later stage of the experiment (twelve hours), more amines are being produced and accumulated. Hence, the color of the wastewater was no longer due to the dye compounds alone. At twelve hours, the wastewater in the serum bottle did not become colorless but has turned greenish. This greenish color may be due to the accumulation of the aromatic amines and resulted with a slight increase in color intensity of the wastewater.

7.5.4.2 Factorial Design Analysis: Main Effect of Riboflavin on Color Removal

In this study, the results showed that the concentration of riboflavin has a significant effect on the color removal of the mixed azo dyes. The P-values for the effect of riboflavin on the color removal of Sumifix Navy Blue EXF is less than 0.0001 for both five and twelve hours of the experimental period. The color removal of Synozol Red K-4B is also significant at five and twelve hours of the experiment with the P-values of 0.015 and less than 0.0001, respectively.

At twelve hours of the experiment, the effect of riboflavin shows nearly the same positive magnitude for both types of dyes with an estimated effect of +5.5 and +5.7 for Sumifix Navy Blue EXF and Synozol Red K-4B, respectively. This mean, at twelve hours of the experimental run, as the concentration of riboflavin increased, the percentage of color removal for both types of dyes were also increased.

The results obtained at five hours of the experiment showed that the estimated effect for Sumifix Navy Blue EXF removal is positive with magnitude of 2.95. However, the result obtained for the removal of Synozol Red K-4B shows a negative value of the estimated effect (-2.225). It shows that the addition of

242

riboflavin has caused a reduction in the percentage of Synozol Red K-4B removal at the earlier stage of the degradation process. It is possible that Synozol Red K-4B has a more complex chemical structure as compared to Sumifix Navy Blue EXF. Due to the complexity, the degradation of Synozol Red K-4B at five hours of the experiment was lesser than Sumifix Navy Blue. As discussed earlier in Section 7.4.1, riboflavin is a colored chemical compound; so, increase in the concentration of riboflavin will add color to the synthetic media. While the color of Synozol Red K-4B is not being sufficiently removed at five hours of the experiment, the addition of riboflavin has caused the overall color reduction for Synozol Red K-4B to decrease.

The addition of redox mediator has been reported to accelerate the transfer of electron from a primary electron donor to the azo dye bond that acted as the terminal electron acceptor (Kudlich et al., 1997 and Keck et al., 2002). Besides, the presence of redox mediator is also capable of minimizing the steric hindrance due to high density of the electrons in the azo bond dye molecule structure (Moir et al., 2001) and cause decreasing energy of the chemical reaction (Dos Santos et al, 2007). This would help to increase the dye degradation process.

Figures 7.9 and 7.10 show the main effect of substrate and riboflavin for both dyes at both 5 and 12 hours of anaerobic experimental condition.

7.5.4.3 Factorial Analysis: Interaction Effect

The concentration of substrate and riboflavin show a significant interaction effect for both dyes. However, for the removal of Sumifix Navy Blue EXF, the effect was not significant where the P-value is 0.351 at twelve hours of the experimental runs. The interaction effect at five hours for the removal of Sumifix

243

Navy Blue EXF gave the P-value of 0.017. Since Sumifix Navy Blue EXF is presumed to have a simpler molecular structure, the transfer of the reducing equivalence is not a limiting factor. The transfer of electron to the N=N bond of the Sumifix Navy Blue EXF was not so much dependent on the presence of the electron transfer. Furthermore, the amount of reducing equivalence produced due to the degradation of substrate could be high and easily being transferred to the dyes. Such condition may result with non-interaction effect between the two variables at twelve hours of the experimental conditions. The biodegradation of dye is a time dependent degradation process. This has been proven in the previous chapter (Chapter 6) where the percentage of color removal increase at the HRT increased. This could be explained by the increasing magnitude effect on the color removal between five and twelve hours of the reaction process.

Figure 7.9 condition

Main effect plot of substrate and riboflavin on the color removal of

Sumifix Navy Blue EXF at (a) 5 and (b) 12 hours of experiment under anaerobic

244

Figure 7.10

Main effect plot of substrate and riboflavin on color removal of

Synozol Red K-4B at (a) 5 and (b) 12 hours of experiment under anaerobic condition

The P-values of the interaction effect at five hours and twelve hours are 0.002 and 0.028, respectively, indicating significant interaction results. As for the Synozol Red K-4B, the dye is assumed to have more complex molecule structure as compared to Sumifix Navy Blue EXF. This has caused a reduction in color removal with the addition of riboflavin at the early stage (i.e five hours). At higher substrate concentration, more reducing equivalence would be released and this could be seen with a slight increase in the percentage of color removal for Synozol Red K-4B. At twelve hours, the presence of riboflavin has significantly accelerated the percentage of Synozol Red K-4B and Sumific Navy Bleu EXF removals at both low and high substrate concentrations.

245

The interaction effect for Sumifix Navy Blue EXF and Synozol Red K-4B at five and twelve hours of the experimental conditions are given in Figures 7.11 and 7.12, respectively.

Figure 7.11 Interaction of variables substrate and riboflavin for Sumifix Navy Blue EXF at (a) 5 and (b) 12 hours of the experimental conditions (Substrate: ---- 2366.88 m/L; ____ 866.12 mg/L; Centre point)

Figure 7.12 Interaction of variables substrate and riboflavin for Synozol Red K-4B at (a) 5 and (b) 12 hours of the experimental conditions (Substrate: ---- 2366.88 m/L;
____

866.12 mg/L; Centre point)

246

7.5.5 Central Composite Design Analysis of Color Removal

The color removal was measured at five and twelve hours of the experimental conditions for both type of dyes and the results are given in Table 7.8. The analysis was carried out using full quadratic terms including linear, square and interaction. The results of the ANOVA for color removal at different reaction conditions are shown in Table 7.9. The detailed results for estimated regression coefficient and ANOVA table are given in Appendices G5 to G9.

Table 7.8: Experimental results for CCD analysis

Sumifix Navy Blue EXF Run 5 hours CC01 CC02 CC03 CC04 CC05 CC06 CC07 CC08 CC09 CC10 CC11 CC12 CC13 74.7 80.0 77.2 83.5 79.8 82.3 78.9 83.3 82.8 82.4 87.3 83.2 85.8 12 hours 77 76.7 83.9 81.0 86.9 81.8 71.8 81.4 79.5 80.9 85.9 81.5 79.8 5 hours 68.5 77.5 62.3 78.0 66.5 73.9 68.8 82.3 76.2 79.7 82.5 79.1 79.8 12 hours 75.2 75.8 82.3 80.0 85.5 81.1 71.3 79.8 78.1 80.1 83.9 80.5 79.1 Synozol Red K-4B

247

Table 7.9: Summary of the P-value of the response surface modeling analysis
Sumifix Navy Blue EXF The P-value Term 5 hours Full Quadratic terms Substrate Riboflavin Substrate2 Riboflavin2 Substrate Riboflavin R-squared value Lack of Fit (LOFT)
a

Synozol Red K-4B

Linear + Square 0.0369 0.078 0.0275 0.0289 -

12 hours

5 hours

12 hours

0.0512 0.0998 0.0396 0.0413 0.8326

0.1221 0.0041 0.1717 0.0121 0.5543

0.0219 0.3008 0.0177 0.1988 0.4552

0.1769 0.0031 0.1322 0.0069 0.4624

74.36%

74.19%

83.49%

75.04%

85.59%

0.3865

0.5007

0.8885

0.0456

0.7938

0.01 0.04: Highly significant; 0.05 0.1: significant; 0.1 0.2: less significant; < 0.2: insignificant (Vecchio,

1997)

Statistical models were developed to relate the concentration of substrate and redox mediator with color removal. Since the effects of both variables are time dependent, the models were developed at five and twelve hours of the experimental condition. The modeling attempts were developed with the aid of Design Expert 7P.

Based on the P-value, the results associated with substrate concentration were significant at five hours of the experiment and became trivial at twelve hours. The results showed the same patterns for both linear and square terms. With regard to the

248

effect of redox mediator (riboflavin), the variable shows a significant effect for the color removal of Sumifix Navy Blue EXF at both five and twelve hours of the experimental conditions for both linear and square terms.

The color removal of Synozol Red K-4B for the effect of riboflavin was opposite as compared to the effect of substrate concentrations. The effect of the redox mediator was insignificant at five hours but became significant at twelve hours for both linear and square terms. The effect of substrate and redox mediator with respect to the interaction terms for Sumifix Navy Blue EXF and Synozol Red K-4B were all insignificant for both five and twelve hours. The R-squared values for all models were in the acceptable ranges (74-86%). For the color removal of Sumifix Navy Blue EXF at both five and twelve hours of the experiment, the P-values for the LOFT was insignificant with the value of 0.3865 and 0.8885, respectively.

For the color removal of Synozol Red K-4B, the P-value of the LOFT is only insignificant at twelve hours (P-value 0.7938) and significant (P-value 0.0456) at five hours of the experimental conditions. The significant P-value for the LOFT implies that the predictive understanding of the model is not statistically accurate and that the process appears to be too complex to model.

An attempt was made in order to improve the statistical model by removing all the insignificant terms for the full quadratic terms. However, the insignificant terms were only removed for the five hours reaction phase of Sumifix Navy Blue EXF. Removing the interaction term has improved the statistical model since the Pvalue of the LOFT has increased to 0.5007 as compared to 0.3865 for the full quadratic term model. All the insignificant terms for Synozol Red K-4B and the twelve hours reaction phase of Sumifix Navy Blue EXF were unchanged since removing those insignificant terms will cause reduction for the R-squared and Pvalue of the LOFT.

249

The best statistical models that can be used to represent the color removal process based on the experimental conditions in this study as attained from the CCD experimental design analysis are given in Table 7.10. The mathematical model of Sumifix Navy Blue EXF for five hour reaction phase was generated based on the reduced quadratic model.

Table 7.10: Mathematical models in terms of actual values

Dye Time (hours) Statistical model

5 Sumifix Navy Blue EXF 12

= 65.38 + 0.01A + 0.15B - 2.79 10-6A2 7.77 10-4B2

= 77.1 5.8 10-3A + 0.23B + 1.54 10-6A2 9.61 10-4B2 1.4A.B

5 Synozol Red K4B 12

= 49.0 + 0.02A + 0.09B 6.34 10-6A2 8.21 10-4B2 + 3.6A.B

= 75.3 5.34 10-3A + 0.22B + 1.54 10-6A2 9.62B2 1.56 10-5A.B

A: Substrate (mg/L); B: Riboflavin(M)

The predicted versus actual plots for the COD removal for Sumifix Navy Blue EXF and Synozol Red K-4B are shown in Figures 7.13 to 7.14. The observed points of the plots reveal that the actual values can be considered distributed relatively near to the straight line for the color removal of dyes at both five and twelve hours of the experimental conditions. The predicted and actual values obtained from this experiment are considered to be fit.

250

87.30

84.15

Predicted

81.00

77.85

74.70

(a)
74.70 77.85 81.00 84.15 87.30

Actual
86.90

83.13

Predicted

79.35

75.58

71.80

(b)
71.80 75.58 79.35 83.13 86.90

Actual

Figure 7.13: Predicted versus actual data for Sumifix Navy Blue EXF removal at (a) 5 hours and (b) 12 hours

251

82.50

77.45

Predicted

72.40

67.35

62.30

(a)
62.30 67.35 72.40 77.45 82.50

Actual
85.50

81.84

Predicted

78.19

74.53

70.88

(b)
70.88 74.53 78.19 81.84 85.50

Actual

Figure 7.14

Predicted versus actual data for Synozol Red K-4B removal at (a) 5

hours and (b) 12 hours

252

The response surface and contour plots based on the significant model are given in Figures 7.15 to 7.18. The response surface and contour plots show that the reactions that took placed in the experiment changed with time with respect to the observed variables for both dyes. The reactions were also different as the structure of the dyes differed. Figure 7.15 shows the surface plot and response surface plots of substrate and riboflavin for the percentage of Sumifix Navy Blue EXF at five hours reaction time with a reduced quadratic model. The response was found to be a symmetrical mound shape. The maximum prediction of the percentage of color removal was indicated by the surface confined in the smallest curve of the contour diagram. The maximum percentage of color removal was 85% that occurred when the concentration of substrate and riboflavin were at 2111.8 mg/L and 96.1 M, respectively.

Based on Figure 7.16, the surface plot indicating the best predicted decolorization for Sumifix Navy Blue EXF (85.3%) at twelve hours reaction phase was obtained with substrate and riboflavin concentrations of 866.1 mg/L and 128.2 M, respectively. The predicted value agrees with the actual removal (82.9) which deferred by only 3% obtained experimentally under the same condition.

The pattern of the contour and response surface plot for the color removal of Synozol Red K-4B as shown in Figures 7.17 to 7.18 are almost the same as shown for Sumifix Navy Blue EXF except at five hours of the reaction period. The pattern of the plots for Synozol Red K-4B at five hours appears as an elliptical shape (Figure 7.17). At low substrate concentration, increase in the concentration of riboflavin did not cause significant change on the color removal. At the lowest concentration of riboflavin (22.8 mg/L), the percentage of color removal was about 67.83%. The percentage of color removal slightly increased to 70.22% when the concentration of riboflavin reached the centre point (75.5 m) before reducing again to 67.83% when the concentration increased to the highest value of riboflavin (128.2 m). The highest percentage of color removal was 81.12% that occurred at substrate and riboflavin concentrations of 2238.4 mg/L and 104.07 m, respectively.

253

128.179

101.84

Riboflavin Riboflavin (M)

75.5

5
83.574 80.7615 82.1678

49.1603

79.3552 77.949
22.8205 866.117 1308.06 1750 2191.94 2633.8

80.7615

(a)

Substrate (mg/L)
Substrate

87.3

84.15

Color Removal (%)

81

77.85

74.7

128.179 101.84 75.5 1750 49.1603 22.8205 866.117 1308.06 2191.94

2633.88

(b)

Riboflavin Riboflavin (M)

Substrate Substrate (mg/L)

Figure 7.15 (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Sumifix Navy Blue EXF removal at 5 hours (Reduced Quadratic Model)

254

128.179

101.84

Riboflavin (M)

83.7339 82.121
75.5

80.5081

49.1603

78.8952

77.2823

22.8205

(a)

866.117

1308.06

1750

2191.94

2633.8

Substrate (mg/L)

86.9

Color Removal (%)

83.125

79.35

75.575

71.8

128.179 2633.88 101.84 75.5 1750 1308.06 866.117 2191.94

(b)

B: Riboflavin Riboflavin (M)

49.1603 22.8205

A: Substrate Substrate (mg/L)

Figure 7.16: (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Sumifix Navy Blue EXF removal at 12 hours

255

128.179

70.043

101.84

Riboflavin (M) B: Riboflavin

75.5

72.2577 74.4724

5
78.9017

49.1603

76.687 70.043 74.4724

22.8205 866.117 1308.06 1750 2191.94 2633.8

(a)

Substrate (mg/L)
A S b t t

82.5

Color removal (%)

77.45

72.4

67.35

62.3

128.179 2633.88 101.84 75.5 1750 1308.06 866.117 2191.94

Riboflavin B: Riboflavin (M)

49.1603 22.8205

A: Substrate Substrate
(mg/L)

(b)

Figure 7.17: (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Synozol Red K-4B removal at 5 hours

256

128.179

101.84

82.2693

80.7635 80.7635

Riboflavin (M) B: Riboflavin

75.5

79.2578

49.1603

77.752

76.2462

22.8205 866.117 1308.06 1750 2191.94 2633.8

A: Substrate Substrate (mg/L)

(a)

85.5

Color Removal (%)

81.95

78.4

74.85

71.3

128.179 2633.88 101.84 75.5 1750 1308.06 866.117 2191.94

Riboflavin B: Riboflavin (M)

49.1603 22.8205

A: Substrate Substrate (mg/L)

(b) Figure 7.18: (a) Contour and (b) 3D response surface plots representing relationship between the concentrations of substrate, riboflavin and color removal of Synozol Red K-4B removal at 12 hours

257

Figure 7.18 shows the contour and response surface plots for color removal of Synozol Red K-4B at 12 hours reaction. At this reaction time, the plots have changed to more of a saddler plot. The plot shows that as the concentration of redox mediator increased, the percentages of color removal were also increased. The highest and lowest removal percentages at this hour were predicted to be 83.8% and 74.7%. The highest color removal was observed when keeping the substrate and riboflavin concentrations at 866.1 mg/L and 128.18 m, respectively. The lowest color removal occurred at riboflavin concentration of 22.8 m but with the same substrate concentration. This may give an indication that at this hour the concentration of riboflavin affects the color removal as compared to the concentration of substrate. The percentages of color removal obtained from the actual experiment were 82.3% and 75.2% at the same substrate and riboflavin concentrations. The percentages of color removal given by the statistical model were only deferred by less than 1% indicating high prediction efficiency of the developed model. Both results indicate the accuracy of the model developed.

7.6

Conclusions

i.

Only a small amount of riboflavin as the redox mediator is required to accelerate a high percentage of color removal. Too much of the compound may overshadow the color removal since riboflavin itself is a colored compound.

ii.

The effect of substrate concentration was significant for COD removal at both anaerobic and aerobic reaction phases as well as for total COD removal. The magnitude and direction of the effect were different among the reaction phases. The effect of substrate was positive for the COD removal under anaerobic condition and also for total COD removal. However, the effect was negative under aerobic condition.

258

iii.

The interaction effect was insignificant for COD removal under anaerobic condition but significant for aerobic reaction and total COD removal.

iv.

The effect of riboflavin was insignificant for COD removal under anaerobic condition but was significant for the reaction under aerobic condition and total COD removal. The direction of the effect was negative for COD removal under all experimental conditions.

v.

The effect of substrate on the color removal of Sumifix Navy Blue EXF and Synozol Red K-4B was significant for both five and twelve hours of the reaction phase. However, the magnitude and direction of the effect were opposite when compared between the two reactions. The five hour reaction demonstrates a positive effect while negative for twelve hours reaction.

vi.

The effect of riboflavin was also significant at both five and twelve hours reaction phases for color removal of both dyes. All responses were positive with increasing magnitude as the reaction time move from five to twelve hours of reactions. Except for the five hours reaction phase for Synozol Red K-4B, riboflavin was negatively affecting the percentage of color removal.

vii.

The interaction effect of substrate and riboflavin were significant for both 5 and twelve hours of the reaction for Synozol Red K-4B. As for Sumifix Navy Blue EXF, the interaction effect was only significant for five hours of the reaction. The direction of the interaction effect was positive for five hours reaction but negative at twelve hours reaction phase. This applied for both dyes.

CHAPTER 8

CONCLUSIONS AND RECOMENDATIONS

Granulation system is a feasible treatment process that can be used to treat recalcitrant pollutant containing wastewater such as those found in the textile dyeing wastewater. This study was aimed at developing granular biomass that is capable of treating such wastewater. Synthetic wastewater comprising of a mixed dye of Sumifix Navy Blue EXF and Synozol Red K-4B and Sumifix Black EXA was used in this study. The biogranules were developed using a mixture of sludge from a sewage treatment plant and a textile mill wastewater treatment plant, anaerobic granules from a paper mill, and with the addition of a specialized dye degrader microbes customized to treat dye containing wastewater. Different types of study were conducted in several types of reactor. However, the different reactors shared a common feature, i.e. intermittent anaerobic and aerobic conditions in which the FAnGS were developed. With the aid of statistical experimental design, the effects of some variables were assessed.

The following are the conclusions that can be derived from this study. The recommendations to improve the findings of the study are given in the following section.

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8.1

Conclusions

Several conclusions that could be derived from the experimental results of this study are given below:

a.

Successful development of FAnGS has been demonstrated in the IFAnGSBioRec system with specialized features of the intermittent anaerobic and aerobic reaction mode during the reaction process. The FAnGS developed from the mixture of sludge and anaerobic granules were compact, strong in structure and possessed good settling properties. Such properties have increased the concentration of the biomass in the reactor which was observed to improve the performance of the system. The anaerobic granules seeding have created a noticeable different on the morphological features of the granules by having fragmented anaerobic granules within the FAnGS as compared to granules developed without the addition of anaerobic granular seeding.

b.

Under intermittent anaerobic and aerobic reaction phase strategy, the FAnGS was capable of eliminating pollutants that required both anaerobic and aerobic treatment conditions. Within 6 hours of HRT, the FAnGS was able to remove more than 95% ammonia, 62% color and 94% COD. Based on OUR/SOUR and SMA analyses, the granules seem to have facultative, anaerobic and aerobic microorganisms that degrade under both anaerobic and aerobic conditions.

c.

Several aerobic and facultative anaerobic types of microorganisms were identified within the granules and they are Bacillus cereus, Pseudomonas veronii, three species of Pseudomonas genus and Enterobacter sp. They are among those considered in the literature as dye degrader microbes.

261

d.

Further investigation on the isolated microorganisms within the FAnGS showed that they are capable of forming aggregates and exhibit reasonably high to moderate range of surface hydrophobicity. compared to individual microorganisms. The percentage of aggregation and surface hydrophobicity of the mixed culture are higher when

e.

Through the aid of factorial design and response surface modeling, substrate concentration, pH and temperature imposed significant effect on the coaggregation and surface hydrophobicity. Substrate concentration shows a positive significant effect on coaggregation and surface hydrophobicity while pH caused a negative effect. As the substrate concentration increased, the percentage of coaggregation and surface hydrophobicity also increased. As the pH value increased from acidic to alkaline, the percentage of coaggregation and surface hydrophobicity decreased. the surface hydrophibicity. process. The temperature caused a positive effect on the coaggregation process but a negative effect on Among the three variables, the significant interaction was only observed between pH and temperature for coaggregation While for surface hydrophobicity, the interaction effect was significant between pH and substrate concentration and between pH and temperature. The 3-way interaction effect of substrate concentration, pH and temperature was only significant for surface hydrophobicity. Based on the central composite analyses, substrate concentration and pH have a non-linear effect on coaggregation process and surface hydrophobicity.

f.

Increase in the HRT has affected the biomass profile and the physical properties of the granules. Not only the concentration of the granular biomass has reduced, the size and settling properties of the FAnGS have also reduced with the increase in the HRT. Due to the reduction of the biomass profile, the removal performance for COD was also reduced. However, the removal percentage of color improved as the HRT increased.

g.

In the application of FAnGS with intermittent reaction phase, increase in the anaerobic reaction time with minimum aerobic reaction time can be

262

considered as a good strategy to maintain and improve the performance of the granular reactor system particularly for the removal of color for textile wastewater treatment.

h.

Changes in the HRT have also affected the microbial activity of the granular biomass. Increase in the HRT has caused a reduction in the microbial activity with the reduction in the overall, Yobs and Y values. More stable condition of the granular biomass can be achieved through the increase in ratio of anaerobic reaction time to aerobic reaction time. The kd value also remains unchanged.

i.

Substrate concentration imposed a significant effect for COD removal. In the anaerobic condition, more COD is being removed as the substrate concentration increased. However, the result was opposite for COD removal under aerobic condition. The direction of total COD removal followed the removal under anaerobic condition but with lesser magnitude. and non-linear under aerobic condition. Substrate concentration has a linear effect on COD removal under anaerobic condition The effects of substrate concentration on total COD removal were found to be non-linear.

j.

The amount of redox mediator that is required to accelerate the color removal is very small. Within the range used in the experiment, the concentration of redox mediator did not give any significant effect for anaerobic COD removal. However, the effect was highly significant for aerobic and total COD removal. The concentration of riboflavin was negatively affecting the removal of COD under aerobic condition and total COD removal. aerobic and total COD removal. The concentration of substrate and riboflavin also response interactively for

263

k.

With respect to color removal, the effect of substrate caused a positive significant effect at the early stage of the experiment but the opposite direction was observed at the later stage. Hence, the effect of substrate concentration on color removal was also considered as time dependent. The non-linear effect of substrate on color removal was only significant during the early stage of the experiment.

l.

The riboflavin has a significant positive effect on the color removal of Sumifix Navy Blue EXF throughout the experiment. The negative significant effect of riboflavin was found on Synozol Red K-4B at the early stage of the experiment and change to positive at the later stage of the experiment. The effect of riboflavin was found to be non-linear for the color removal of Sumifix Navy Blue EXF throughout the experiment. As for Synozol Red K4B, the non-linear effect of riboflavin was only significant at the later stage of the experiment.

m.

The application of experimental design (i.e. factorial design and response surface) could provide more reliable results where a significant effect either as the main or/and interaction can be obtained and quantitatively identified. The use of experimental design is a more effective approach that could provide more information on the association of the variables towards the responses through less experimental work as compared to one-factor-at-atime approach. However, the selection of values and the variables that will be included in the statistical model may affect the result of the experiment. A misleading conclusion may occur when the selection of values and variables are not carefully made. The development of the statistical model from the experimental design is also dependent on the complexity of the process. A process can become difficult to model when the outcomes of the mechanisms involved in the process are not directly associated with the investigated variables.

264

8.2

Recommendations

In order to improve the performance of the FAnGS in treating textile wastewater, further studies are needed and are given as follows:

a.

Since the size of the granules is an important aspect in the degradation of the dye, a study focusing on the effect of the granules size on dye degradation is needed in order to obtain maximum removal rate.

b.

Knowledge on the kinetics of decolorization and COD removal with the effect of the environmental factors such as temperature, pH, co-substrate, with and without the presence of redox mediator are severely lacking. The results of kinetic studies would be able to assist on the design and operation of the reactor system. This information is important in order to develop an efficient biodegradation process for the dye containing wastewater.

c.

In this study, the presence of aromatic amines with its autoxidation effect has caused interference in the quantification of the color removal. A study approach on the fate of the aromatic amines with respect to the detection, degree of mineralization and autoxidation as well as the toxicity effect to the microorganisms and the surrounding environment are important aspects that need to be investigated. Further investigations are needed to overcome the problem of recolorization so that true measurement on the color removal under aerobic condition could be precisely determined.

d.

The application of biogranules treatment approach is affected by many factors. Cost effect analyses with the exploitation on the affecting variables are required in order to obtain the most economical application of biogranular treatment system at the most optimal condition.

APPENDIX A: DATA AND EXAMPLES OF CALCULATIONS A-1: Organic Loading Rate , where X = COD concentration of the influent (mg/L) Vadd= Volume of influent added in each cycle operation (mL) Vtotal = Total working volume of the experiment (mL) T = Hydraulic retention time (hour). X FAnGS development Stage I II III IV V VI 1270 Vadd 2 Vtotal 4 T 6 OLR (kg/m3d) 2.54

X 1270 1270 1270 1604 1604 1604

Vadd 2 2 2 2 2 2

Vtotal 4 4 4 4 4 4

T 6 12 24 24 24 24

OLR (kg/m3d) 2.54 1.27 0.635 0.802 0.802 0.802

A-2: Superficial Air Velocity

Air flow rate (L/min) 5

Diameter of column (m) 0.08

Surface area (m2) 5.024 x10-3

Superficial air velocity (cm/s) 1.66

Air flow rate (L/min) 7.5

Diameter of column (m) 0.08

Surface area (m2) 5.024 x10-3

Superficial air velocity (cm/s) 2.5

302

A-3: Oxygen Uptake Rate

where OUR DOa DOb T = = = = Oxygen uptake rate (mg/L.h) Initial dissolved oxygen (mg/L) End dissolved oxygen (mg/L) Time (min)
Stage I DOb 2.00 1.97 1.98 1.99 1.99 2.00 2.00 1.99 1.97 1.99 1.99 1.98 2.00 1.99 2.00 2.00 1.96 4.92 2.00 2.00 1.98 1.99 1.98 2.00 2.00 2.40 Stage II DOb 3.30 2.33 2.99 2.96 2.97 2.98 2.99 3.00 2.98 2.97 3.00 3.00 2.99 3.00 2.98 2.68 3.00 2.69 3.00 2.92 2.98 2.33 3.00 3.00 3.00 2.56 3.00 3.00 3.00

Time 32 57 90 77 103 108 180 230 250 280 340 500 530 600 640 680 720 75 109 450 540 740 780 830 870 900

DOa 6.79 7.28 7.67 7.12 7.48 7.59 7.80 7.81 7.40 7.47 7.55 7.78 7.80 7.57 7.40 7.20 7.30 6.10 6.38 7.60 7.50 7.40 7.20 7.50 7.40 7.10

OUR 538.65 335.37 227.60 239.84 191.88 186.33 116.00 91.10 78.19 70.46 58.87 41.76 39.40 33.48 30.38 27.53 26.70 56.64 144.66 44.80 36.80 26.32 24.09 23.86 22.34 18.80

Time 210 91 71 101 119 172 210 298 385 498 617 698 818 926 936 1110 1129 1136 1186 190 470 850 930 1200 1300 1350 1623 1500 1240

DOa 7.85 5.85 8.18 7.72 8.00 7.75 8.14 7.51 8.13 8.13 7.78 8.56 8.29 7.74 8.15 8.17 9.16 8.48 8.38 8.19 8.25 8.12 7.94 8.49 8.59 8.41 8.70 8.70 7.30

OUR 78.00 139.25 263.15 169.66 152.17 99.84 88.29 54.48 48.16 37.30 27.89 28.68 23.33 18.43 19.88 17.81 19.64 18.35 16.33 99.85 40.37 24.52 19.12 16.47 15.48 15.60 12.64 13.68 12.48

303

Stage III DOb 3.00 1.80 2.41 3.30 2.00 2.00 2.00 2.00 2.00 2.00 2.41 2.41 2.41 2.41 2.41 2.41 2.41 2.42 2.42 2.42 2.44 2.48 3.00 2.58 2.58 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 Stage IV DOa DOb 7.00 2.00 6.90 2.00 7.20 1.98 7.60 2.00 7.30 2.00 7.30 1.98 7.30 2.00 7.40 2.00 7.30 2.00 7.30 2.00 7.40 1.98 7.40 2.00 7.40 1.98 7.40 1.98 7.50 1.98 7.50 2.00 7.30 2.00 7.40 2.00 7.30 1.98 7.50 2.00 7.40 2.00 7.60 2.00 7.50 1.98 7.50 2.00 7.60 1.98 7.71 7.82 7.30 7.50 7.70 7.60 7.60 7.40 7.30 7.50 7.40 7.80 7.60 7.70 7.50 7.50 7.30 7.50 7.80 7.60 7.40 7.70 1.98 2.00 1.98 2.00 2.00 2.00 1.98 2.00 2.00 2.00 2.00 1.98 1.98 1.98 1.98 2.00 2.00 1.98 2.00 2.00 2.00 2.00

Time 500 150 185 400 207 370 400 400 500 600 550 640 760 800 1200 900 1100 1220 1920 2200 2300 2400 2300 430 1800 1900 2200 2000 2300 2500 2000 2750 2800

DOa 7.50 7.50 7.40 7.50 7.30 7.80 7.40 7.50 7.50 7.50 7.40 7.60 7.70 7.70 7.80 7.70 7.80 7.50 7.80 7.70 7.80 7.80 7.80 7.50 7.76 8.06 7.74 7.98 7.85 7.81 8.00 8.20 8.00

OUR 32.40 136.80 97.10 37.80 92.17 56.43 48.60 49.50 39.60 33.00 32.66 29.19 25.06 23.81 16.17 21.16 17.64 14.99 10.09 8.64 8.39 7.98 7.51 41.19 10.36 9.59 7.76 8.96 7.59 6.93 9.00 6.95 7.07

Time 85 169 240 173 293 415 500 660 690 890 835 880 940 970 990 1000 1020 1090 1100 1180 1190 1200 1400 1500 1650 235 375 420 420 800 1000 640 840 900 650 690 870 910 980 990 930 950 1300 1440 1600 1700 1800

OUR 211.76 104.38 78.30 116.53 65.12 46.15 38.16 29.45 27.65 21.44 23.37 22.09 20.76 20.12 20.07 19.80 18.71 17.83 17.41 16.78 16.34 16.80 14.19 13.20 12.26 87.78 55.87 45.60 47.14 25.65 20.16 31.61 23.14 21.20 30.46 28.17 24.08 22.23 21.01 20.07 21.29 20.08 15.29 14.50 12.60 11.44 11.40

304

Stage V DOb 2.00 2.00 1.98 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Stage VI DOa DOb 7.91 3.00 8.11 3.00 8.90 2.99 8.70 3.00 8.80 2.99 8.90 2.92 9.20 2.31 8.90 2.99 8.70 2.84 9.30 3.00 9.00 2.99 9.22 2.99 9.14 2.36 9.60 2.99 9.44 2.98 9.10 2.89 8.98 2.99 8.50 2.99 9.40 2.98 9.20 2.97 8.90 2.80 9.10 2.90 9.20 2.97 9.30 2.80 7.60 7.40 6.01 8.59 8.41 8.20 8.37 8.22 8.30 8.30 8.41 8.20 8.23 8.12 8.10 8.00 8.00 8.10 8.20 8.20 8.20 3.50 3.00 2.98 2.91 3.00 2.98 2.99 3.00 2.49 2.33 3.00 2.98 2.99 3.00 2.98 2.99 2.98 2.98 3.00 3.00 3.00

Time 129 107 125 190 170 160 165 180 260 290 350 390 480 580 630 780 860 900 860 1000 1100 1050 124 170 300 350 450 430 600 800 900 980 1080 1100 1600 1900

DOa 6.83 8.11 7.01 7.81 8.32 8.05 7.59 7.45 7.67 7.41 7.37 7.22 7.60 7.60 7.44 7.30 7.60 7.40 7.20 7.41 7.37 7.17 6.50 7.20 7.10 7.10 7.30 7.40 7.30 7.40 7.20 7.60 7.30 7.60 7.70 7.40

OUR 134.79 205.57 144.86 110.08 133.84 136.13 121.96 109.00 78.51 67.16 55.23 48.18 42.00 34.76 31.09 24.46 23.44 21.60 21.77 19.48 17.57 17.73 130.65 110.12 61.20 52.46 42.40 45.21 31.80 24.30 20.80 20.57 17.67 18.33 12.83 10.23

Time 500 720 166 678 820 932 1179 1100 1070 1110 1500 1950 1700 1620 1890 1650 1780 1600 1680 1640 1570 1690 1640 1690 194 480 300 600 720 740 1260 1300 900 1050 2100 2100 2000 2100 2200 2200 2300 2200 2400 2500 2590

OUR 35.4 25.6 128.2 30.3 25.5 23.1 21.0 19.3 19.7 20.4 14.4 11.5 14.4 14.7 12.3 13.5 12.1 12.4 13.8 13.7 14.0 13.2 13.7 13.8 76.1 33.0 36.4 34.1 27.1 25.4 15.4 14.5 23.2 20.5 9.3 8.9 9.4 8.8 8.4 8.2 7.9 8.4 7.8 7.5 7.2

305

A-4: Sludge Retention Time

X vss Vr X e Ve /t c

where, = = = = = = Solids retention time (d) Volatile solids concentration in the reactor system (g VSS/L) Working volume of the SBR system (L) Effluent volatile solids concentration (g VSS/L) Effluent volume of the SBR operating cycle (L) Cycle time of the SBR operation (d)

Stage 32.9 I 29.8 32.9 24.5 II 26.7 22.3 20.9 III 17.9 16.6 26.7 IV 22.3 29.1 24.3 V 20.3 22.6 20.2 VI 19.4 21 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 0.30 0.52 0.20 0.27 0.43 0.22 0.20 0.40 0.19 0.52 0.36 0.30 0.30 0.44 0.25 0.59 0.31 0.75 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 6 6 6 12 12 12 12 12 12 12 12 12 12 12 12

SRT 27.4 14.3 41.1 45.4 31.0 50.7 104.5 44.8 87.4 51.3 61.9 97.0 81.0 46.1 90.4 34.2 62.6 28.0

average

sd

27.6

13.4

42.4

10.2

78.9

30.8

70.1

23.9

72.5

23.3

41.6

18.4

306

A-5: Sludge Volume Index

where, SVI Bedvolume d.w 4 1000 = = = = = Sludge volume index (mL/g SS) Volume of settled biomass in reactor (L) Dry weight of biomass in reactor (g SS/L ) Working volume (L) Conversion factor (L to mL)

Stage

Average dry weight 34.7

Bedheight (cm) at 5 min settling 37.0 37.0 37.0 43.0 43.0 43.0 43.0 43.0 43.0 41.0 41.0 41.0 39.0 39.0 39.0 46.0 46.0 46.0

A (cm2) 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14

Bedvolume (cm3) 1855.17 1855.17 1855.17 2156.01 2156.01 2156.01 2156.01 2156.01 2156.01 2055.73 2055.73 2055.73 1955.45 1955.45 1955.45 2306.43 2306.43 2306.43

Bedvolume (L) 1.855 1.855 1.855 2.156 2.156 2.156 2.156 2.156 2.156 2.056 2.056 2.056 1.955 1.955 1.955 2.306 2.306 2.306

SVI (mL/g MLSS) 13.4 12.6 13.4 20.5 18.3 17.7 22.8 19.7 21.7 17.1 18.2 15.5 16.1 14.0 16.5 24.1 25.7 24.4

SVI (average)

SD

36.7 34.5 26.3

13.1

0.4

II

29.5 30.4 23.6

18.8

1.5

III

27.3 24.8 30.1

21.4

1.6

IV

28.3 33.1 30.4

16.9

1.3

34.8 29.6 23.9

15.5

1.3

VI

22.4 23.6

24.8

0.9

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Day 0 3 5 7 14 17 21 24 28 30 32 33 36 37 38 39 43 46 47 50 56 72 78 84 88 92 95 99 103 106 111 119 124 129 135 142 148 155 161 169 175 187 193 201 209 215 224 230 236 Stage Average dry weight 23.0 15.4 17.5 14.1 15.7 19.2 23.1 25.9 26.5 27.3 28.1 26.5 28.8 28.9 29.8 31.3 30.8 32.6 33.3 32.1 33.3 33.7 34.8 35.3 35.5 35.4 35.3 35.3 35.1 33.3 33.2 30.8 29.7 29.9 28.7 28.7 27.9 27.2 25.6 24.8 25.3 25.2 25.4 26.6 28.4 27.7 30.5 30.5 30.5 Bedheight (cm) at 5 min settling 39.5 38.0 38.0 46.0 42.0 41.0 43.0 40.0 40.1 30.5 24.0 25.0 27.5 37.0 37.5 38.5 42.0 42.0 34.5 35.5 35.0 35.0 29.0 31.0 33.0 36.5 38.0 37.0 41.5 41.0 41.0 44.0 42.0 42.0 44.0 43.0 44.0 44.0 42.0 44.0 44.0 43.0 39.0 39.0 38.0 38.0 40.0 40.0 41.0 A (cm2) 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 Bedvolume (cm3) 1980.52 1905.31 1905.31 2306.43 2105.87 2055.73 2156.01 2005.59 2010.61 1529.26 1203.36 1253.50 1378.85 1855.17 1880.24 1930.38 2105.87 2105.87 1729.82 1779.96 1754.89 1754.89 1454.05 1554.33 1654.61 1830.10 1905.31 1855.17 2080.80 2055.73 2055.73 2206.15 2105.87 2105.87 2206.15 2156.01 2206.15 2206.15 2105.87 2206.15 2206.15 2156.01 1955.45 1955.45 1905.31 1905.31 2005.59 2005.59 2055.73 Bedvolume (L) 1.981 1.905 1.905 2.306 2.106 2.056 2.156 2.006 2.011 1.529 1.203 1.253 1.379 1.855 1.880 1.930 2.106 2.106 1.730 1.780 1.755 1.755 1.454 1.554 1.655 1.830 1.905 1.855 2.081 2.056 2.056 2.206 2.106 2.106 2.206 2.156 2.206 2.206 2.106 2.206 2.206 2.156 1.955 1.955 1.905 1.905 2.006 2.006 2.056 SVI (mL/g MLSS) 21.53 30.91 27.19 40.78 33.45 26.77 23.35 19.36 18.97 14.00 10.72 11.83 11.97 16.05 15.77 15.42 17.08 16.15 13.00 13.86 13.17 13.02 10.45 11.01 11.65 12.92 13.49 13.14 14.82 15.44 15.47 17.91 17.73 17.61 19.21 18.78 19.74 20.28 20.57 22.24 21.79 21.39 19.25 18.38 16.77 17.19 16.44 16.44 16.83

II

III

IV

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Day 241 248 255 262 268 276 282 286 292 299 307 314 320 328 Stage Average dry weight 29.6 30.4 30.5 30.8 30.5 31.4 31.6 29.4 24.8 23.7 20.3 22.7 23.0 23.3 Bedheight (cm) at 5 min settling 41.0 40.0 38.0 38.0 40.0 39.0 39.0 42.0 43.0 43.0 42.0 44.0 45.0 46.0 A (cm2) 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 50.14 Bedvolume (cm3) 2055.73 2005.59 1905.31 1905.31 2005.59 1955.45 1955.45 2105.87 2156.01 2156.01 2105.87 2206.15 2256.29 2306.43 Bedvolume (L) 2.056 2.006 1.905 1.905 2.006 1.955 1.955 2.106 2.156 2.156 2.106 2.206 2.256 2.306 SVI (mL/g MLSS) 17.36 16.49 15.64 15.47 16.44 15.57 15.47 17.92 21.73 22.74 25.93 24.25 24.52 24.75

VI

A-6: Removal Performance (COD, Ammonia and Color Removal)

COD (mg/L) Influent Effluent % Removal Influent Color(ADMI) Effluent % Removal Ammonia (mg/L) Influent Effluent % Removal

Day

1 3 8 11 14 17 21 24 28 31 34 52 55 59 66

1270 1260 1275 1265 1255 1260 1270 1255 1280 1275 1250 1265 1260 1270 1270

390 278 241 187 237 258 160 136 161 181 89 104 89 120 80

69.3 78.0 81.1 85.2 81.1 79.5 87.4 89.1 87.4 85.8 92.9 91.8 92.9 90.6 93.7

1010 1010 1005 1020 1050 1010 1020 1020 1030 1020 1020 1040 1030 1020 1010

761 703 495 458 426 403 473 370 373 429 407 339 433 418 388

24.6 30.4 50.7 55.1 59.4 60.1 53.6 63.8 63.8 58.0 60.1 67.4 58.0 59.0 61.6

35.0 37.2 34.7 33.3 29.4 37.5 35.9 39.5 40.9 30.2 31.9 41.2 49.8 40.7 32.3

11.6 15.1 2.2 3.2 1.7 1.5 1.4 0.9 1.3 2.0 1.1 1.3 3.7 2.5 1.5

66.8 59.5 93.7 90.5 94.3 95.9 96.0 97.7 96.9 93.3 96.4 96.7 92.6 93.9 95.2

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Day 0 3 5 14 17 21 24 28 30 32 36 38 39 43 46 47 50 56 72 78 84 88 92 95 99 103 106 111 119 124 129 135 142 148 155 161 171 180 190 195 202 209 215 224 230 236 241 248 255 Influent COD 1270 1265 1268 1260 1270 1275 1277 1270 1272 1266 1270 1269 1270 1271 1268 1273 1275 1280 1274 1272 1269 1266 1270 1270 1281 1268 1271 1274 1277 1270 1270 1275 1275 1270 1270 1270 1264 1276 1275 1605 1600 1608 1608 1611 1605 1600 1608 1606 1608 Effluent COD a 345 325 290 261 268 240 222 226 212 211 210 209 192 206 207 211 203 198 199 198 193 186 199 199 191 193 193 200 203 217 202 201 189 198 212 199 193 202 185 116 149 125 172 153 162 146 127 132 119 b 357 331 287 258 262 231 226 234 212 197 204 207 208 215 209 201 205 224 214 216 212 189 192 194 199 188 184 203 194 206 202 196 207 202 215 216 211 198 196 169 155 159 145 150 141 152 137 117 93 c 348 329 316 256 236 223 221 231 215 214 199 226 217 208 202 205 194 229 186 197 193 195 187 203 214 209 194 187 202 202 196 193 215 197 230 231 202 193 207 149 166 174 148 147 135 149 125 114 116 Average COD (Effluent) 350 328 298 258 255 231 223 230 213 208 204 214 206 210 206 206 201 217 200 204 199 190 193 199 201 197 191 197 200 208 200 197 204 199 219 215 202 197 196 144 157 153 155 150 146 149 130 121 109 COD removal a 72.8 74.3 77.1 79.3 78.9 81.2 82.6 82.2 83.3 83.3 83.5 83.5 84.9 83.8 83.7 83.4 84.1 84.5 84.4 84.4 84.8 85.3 84.3 84.3 85.1 84.8 84.8 84.3 84.1 82.9 84.1 84.2 85.2 84.4 83.3 84.3 84.7 84.2 85.5 92.8 90.7 92.2 89.3 90.5 89.9 90.9 92.1 91.8 92.6 b 71.9 73.8 77.4 79.5 79.4 81.9 82.3 81.6 83.3 84.4 83.9 83.7 83.6 83.1 83.5 84.2 83.9 82.5 83.2 83.0 83.3 85.1 84.9 84.7 84.5 85.2 85.5 84.1 84.8 83.8 84.1 84.6 83.8 84.1 83.1 83.0 83.3 84.5 84.6 89.5 90.3 90.1 91.0 90.7 91.2 90.5 91.5 92.7 94.2 c 72.6 74.0 75.1 79.7 81.4 82.5 82.7 81.8 83.1 83.1 84.3 82.2 82.9 83.6 84.1 83.9 84.8 82.1 85.4 84.5 84.8 84.6 85.3 84.0 83.3 83.5 84.7 85.3 84.2 84.1 84.6 84.9 83.1 84.5 81.9 81.8 84.0 84.9 83.8 90.7 89.6 89.2 90.8 90.9 91.6 90.7 92.2 92.9 92.8 Average COD removal 72.4 74.0 76.5 79.5 79.9 81.9 82.5 81.9 83.2 83.6 83.9 83.1 83.8 83.5 83.8 83.8 84.3 83.0 84.3 84.0 84.3 85.0 84.8 84.3 84.3 84.5 85.0 84.6 84.4 83.6 84.3 84.6 84.0 84.3 82.8 83.0 84.0 84.5 84.6 91.0 90.2 90.5 90.4 90.7 90.9 90.7 91.9 92.5 93.2 sd 0.5 0.3 1.3 0.2 1.3 0.7 0.2 0.3 0.1 0.7 0.4 0.8 1.0 0.4 0.3 0.4 0.5 1.3 1.1 0.8 0.9 0.4 0.5 0.4 0.9 0.9 0.4 0.6 0.4 0.6 0.3 0.4 1.1 0.2 0.8 1.3 0.7 0.4 0.9 1.7 0.6 1.5 0.9 0.2 0.9 0.2 0.4 0.6 0.9

310

262 268 276 282 286 292 299 307 314 320 328 1608 1607 1603 1600 1605 1608 1605 1606 1606 1600 1608 108 117 99 106 124 146 218 210 236 290 296 122 87 83 90 141 169 209 226 234 280 275 117 125 99 88 138 150 220 222 239 261 270 116 110 94 94 134 155 216 219 237 277 280 93.3 92.7 93.8 93.4 92.3 90.9 86.4 86.9 85.3 81.9 81.6 92.4 94.6 94.8 94.4 91.2 89.5 87.0 85.9 85.4 82.5 82.9 92.7 92.2 93.8 94.5 91.4 90.7 86.3 86.2 85.1 83.7 83.2 92.8 93.2 94.1 94.1 91.6 90.4 86.6 86.3 85.3 82.7 82.6 0.5 1.3 0.6 0.6 0.6 0.8 0.4 0.5 0.2 0.9 0.9

Day
0 3 5 14 17 21 24 28

Influent Color 1051 1054 1047 1050 1049 1055 1050 1055 1052 1052 1050 1051 1050 1055 1050 1049 1055 1050 1048 1052 1052 1051 1050 1050 1054 1055 1059 1055 1052 1055 1050 1050 1052 1050 1054

Effluent Color a 796 736 620 562 543 567 617 544 482 465 547 490 558 554 446 461 383 377 327 294 311 327 344 321 344 350 325 319 307 332 282 273 270 247 284 b 802 729 601 569 541 572 629 554 498 472 562 479 562 560 467 471 392 386 321 322 306 343 349 326 370 344 331 318 309 329 288 270 266 254 277 c 790 740 640 548 536 586 637 552 485 472 571 496 563 536 453 473 407 395 319 315 313 322 334 289 337 322 309 291 290 310 266 291 275 271 310

30 32 36 38 39 43 46 47 50 56 72 78 84 88 92 95 99 103 106 111 119 124 129 135 142 148 155

Average Color (Effluent ) 796 735 620 560 540 575 628 550 488 470 560 488 561 550 455 468 394 386 322 310 310 330 342 312 350 339 322 309 302 324 279 278 270 257 290

Color removal a 24.3 30.2 40.8 46.5 48.2 46.3 41.2 48.4 54.2 55.8 47.9 53.4 46.9 47.5 57.5 56.1 63.7 64.1 68.8 72.1 70.4 68.9 67.2 69.4 67.4 66.8 69.3 69.8 70.8 68.5 73.1 74 74.3 76.5 73.1 b 23.7 30.8 42.6 45.8 48.4 45.8 40.1 47.5 52.7 55.1 46.5 54.4 46.5 46.9 55.5 55.1 62.8 63.2 69.4 69.4 70.9 67.4 66.8 69 64.9 67.4 68.7 69.9 70.6 68.8 72.6 74.3 74.7 75.8 73.7 c 24.8 29.8 38.9 47.8 48.9 44.5 39.3 47.7 53.9 55.1 45.6 52.8 46.4 49.2 56.9 54.9 61.4 62.4 69.6 70.1 70.2 69.4 68.2 72.5 68 69.5 70.8 72.4 72.4 70.6 74.7 72.3 73.9 74.2 70.6

Average color removal 24.3 30.3 40.8 46.7 48.5 45.5 40.2 47.9 53.6 55.3 46.7 53.5 46.6 47.9 56.6 55.4 62.6 63.2 69.3 70.5 70.5 68.6 67.4 70.3 66.8 67.9 69.6 70.7 71.3 69.3 73.5 73.5 74.3 75.5 72.5

sd 0.6 0.5 1.9 1.0 0.4 0.9 1.0 0.5 0.8 0.4 1.2 0.8 0.3 1.2 1.0 0.6 1.2 0.9 0.4 1.4 0.4 1.0 0.7 1.9 1.6 1.4 1.1 1.5 1.0 1.1 1.1 1.1 0.4 1.2 1.6

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161 171 180 190 195 202 209 215 224 230 236 241 248 255 262 268 276 282 286 292 299 307 314 320 328 1053 1050 1050 1056 1053 1050 1050 1057 1050 1054 1050 1052 1050 1048 1047 1050 1050 1050 1049 1050 1050 1059 1055 1050 1050 249 268 257 259 250 181 160 186 159 174 191 157 162 175 146 143 140 141 232 222 232 268 236 270 258 266 277 248 242 237 190 161 189 155 194 179 161 155 176 150 154 135 145 215 214 235 264 248 254 260 284 282 265 250 258 180 171 194 171 180 162 183 172 159 160 150 158 134 234 212 246 254 263 279 255 266 276 257 250 248 183 164 190 162 183 177 167 163 170 152 149 144 140 227 216 238 262 249 268 258 76.4 74.5 75.5 75.5 76.3 82.8 84.8 82.4 84.9 83.5 81.8 85.1 84.6 83.3 86.1 86.4 86.7 86.6 77.9 78.9 77.9 74.7 77.6 74.3 75.4 74.7 73.6 76.4 77.1 77.5 81.9 84.7 82.1 85.2 81.6 83 84.7 85.2 83.2 85.7 85.3 87.1 86.2 79.5 79.6 77.6 75.1 76.5 75.8 75.2 73 73.1 74.8 76.3 75.5 82.9 83.7 81.6 83.7 82.9 84.6 82.6 83.6 84.8 84.7 85.7 85 87.2 77.7 79.8 76.6 76 75.1 73.4 75.7 74.7 73.7 75.6 76.3 76.4 82.5 84.4 82.0 84.6 82.7 83.1 84.1 84.5 83.8 85.5 85.8 86.3 86.7 78.4 79.4 77.4 75.3 76.4 74.5 75.4 1.7 0.7 0.8 0.8 1.0 0.6 0.6 0.4 0.8 1.0 1.4 1.3 0.8 0.9 0.7 0.6 1.1 0.5 1.0 0.5 0.7 0.7 1.3 1.2 0.3

A-7: Coaggregation

where, CAg% = CA0 = CAi = Percentage of coaggregation The absorbance of cultured media at 0 h The absorbance of cultured media after centrifugation

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CA0 = The absorbance of cultured media at 0 h Cai = The absorbance of cultured media after centrifugation 1 hour 71 40 50 38 39 39 49 48 29 38 35 34 37 43 48 45 2 hour 59 38 42 39 42 42 40 37 30 43 33 31 31 45 47 48 3 hour 63 33 52 40 51 32 34 32 34 40 35 33 41 49 46 37 4 hour 64 37 44 37 42 28 30 27 33 44 37 35 39 53 47 32 5 hour 68 40 62 46 39 40 28 28 39 41 38 36 52 57 48 39

No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 hour 117 69 115 98 68 62 85 81 86 85 115 126 83 97 117 93

2 hour 113 72 131 114 68 70 80 80 107 102 207 178 81 94 111 89

3 hour 115 68 135 128 69 85 84 83 97 99 210 211 82 88 123 96

4 hour 127 67 165 147 65 79 83 80 110 100 234 188 85 97 118 94

5 hour 135 72 189 128 74 71 80 76 111 108 243 244 85 93 113 92

% of AGG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 hour 39.3 42.6 56.9 60.8 43.1 36.5 42.4 41.3 65.9 55.0 69.5 73.1 55.1 55.6 59.4 51.9

2 hour 47.8 47.0 67.6 65.4 37.7 40.5 50.6 53.9 71.7 57.7 84.2 82.3 62.2 52.2 57.3 46.3

3 hour 45.2 52.0 61.5 69.0 26.1 62.6 59.8 61.9 64.6 59.8 83.1 84.6 50.3 44.8 62.7 61.8

4 hour 49.6 45.3 73.1 75.0 36.0 64.5 64.3 65.9 69.8 55.8 84.0 81.5 54.6 45.4 59.9 66.4

5 hour 49.6 44.7 67.2 64.1 46.8 44.1 65.6 62.9 65.2 61.9 84.5 85.4 38.8 38.7 57.4 57.6

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A-8: Surface Hydrophobicity

where, SHb% = A0 = = Ai Percentage of surface hydrophobicity The absorbance of sample before mixing with xylene The absorbance of sample after extraction with xylene.

No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

A0
0.625 0.456 0.647 0.868 0.217 0.233 0.366 0.393 0.629 0.61 1.255 1.128 0.426 0.413 0.771 0.673

Ai
0.506 0.357 0.428 0.613 0.136 0.137 0.221 0.248 0.427 0.418 0.579 0.541 0.339 0.338 0.705 0.627

% of Hydrophobicity 19.0 21.7 33.8 29.4 37.3 41.2 39.6 36.9 32.1 31.5 53.9 52.0 20.4 18.2 8.6 6.8

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APPENDIX B: MOLECULAR PROCEDURE OF 16S SEQUENCE ANALYSIS B-1: Genomic DNA Isolation Each of the selected bacteria (BS1FAnGS, BS6FAnGS, BS7FAnGS, BS8FAnGS, BS10FAnGS, BS11FAnGS and BS12FAnGS) was extracted separately in order to obtain the DNA genomic of the bacteria. The genomic DNA was extracted from the cell pellets by using genomic DNA extraction and purification kit (Promega). Each of the bacteria was culture in nutrient broth for 24 hours before 1 ml of each bacterium cultures with optical density (OD at 660 nm) of more than 0.5 was taken and centrifuged at 13,000-16,000xg for 2 minutes in order to obtain the cell pellet. The supernatant was removed before 600 l of Nuclei Lysis Solution was added to the pelleted cell. The cells were gently resuspended using pipetters. The solution was incubated at 80oC for 5 minutes in order to lyses the cells. The solution was then left to cool at room temperature. Following this, 3 l of RNase solution was added to the cell lysate before the tube was inverted for 2-5 times to well mix the solution. The solution was incubated at 37oC for 30 minutes. Then the sample was to cool again at room temperature. After that, 200 l of Protein Precipitation solution was added to the RNase-treated cell lysate. The solution was vortex vigorously at high speed for 20 second to mix the Protein Precipitation solution with the cell lysate. The sample was then incubated in ice for 5 minutes before centrifugation at 13,000-16,000 g for 3 minutes. The supernatant containing the DNA was transferred to a clean 1.5 mL microcentrifuge, which contain 600 l of room temperature isopropanol. The solution was gently mixed by inversion until the thread-like strands of DNA form appeared as a visible mass. The sample was then centrifugation at 13000-16,000 g for 2 minutes. The supernatant was carefully poured off after centrifugation. Then the tube was drained on clean adsorbent paper before 600 l of 70% ethanol was added into the tube. Then the tube was gently inverted for several times for washing the DNA pellet. Then the sample was centrifuged again at 13,000-16,000 g for 2 minutes. The ethanol was aspirate carefully. The tube was drained on clean

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absorbent paper and the pellet was air-dry for 15 minutes. Then 100 l of DNA Rehydration solution was added to the tube.
o

The DNA was rehydrated by

incubating at 65 C for 1 hour. The successful isolated DNA solution was stored at 4oC.

B-2:

Analysis of Genomic DNA The analysis of the genomic DNA of each of the bacteria was measured

qualitatively via agarose gel electrophoresis. 1% (w/v) of agarose was dissolved in 1 x TAE buffer (Table B-1) in microwave oven until there were no solid particles in the solution and the solution is completely homogenous. Table B-1 showed the TAE buffer that was prepared as concentrated stock solution (50x). The solution was allowed to cool to approximately 50oC and then was poured into the gel base sealed at both with masking tapes. A suitable comb was placed vertically at one end of gel base. The comb was removed after the gel agar was left solidified for 30 minutes. The gel was then submerged using 1 x TAE running buffer in the electrophoresis tank. The sample was mixed with 1 L of loading dye which consisted of bromophenol blue 0.25% (w/v), SDS 1% (w/v) and glycerol 80 % (v/v). The sample and 5 l of universal DNA marker (Gene Ruler Ladder Mix Marker) were loading into the performed wells. The gel was run at 80 V and 45 W for 60 minutes. Lastly, the gel was stained for 30 minutes in running buffer containing 0.5 g/mL ethidium bromide (EtBr) before the extracted DNA was examined and photographed on a UV transilluminator (TFX-35 Vilber Lourmat).

B-3:

Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) is a method for amplification of DNA in

vitro where through polymerase chain reaction the DNA can be multiply up to a billion fold (Madigan et al., 2000). The PCR copies the DNA using basic elements of natural DNA synthesis and replication processes (McPherson and Moller, 2006).

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Table B-1: Composition of the TAE buffer (50x) Components Tris-Base Glacial acetic acid 0.5 M EDTA (pH 8.0) Deionized water Volume 242.0 g 57.1 ml 1000 mL Top up until 1000 ml

The extracted DNAs were then amplified via PCR. In the PCR amplification process, universal primers that normally consisted of at least 17 to 25 nucleotides were used since they are homologous to broadly conserved sequences. Primers are needed that act as sites for initiation of DNA synthesis by the DNA polymerase. These primers define the region of the template DNA that need to be copied. Primers or also known amplimers are complementary to the regions of known sequence on opposite strands of the DNA template (McPherson and Moller, 2006). The universal primer used in this amplification is listed in Table B-2. The PCR process consisted of three distinct steps that are governed by temperature. The first step is known as the denaturation. At this stage, the doublestranded template DNA is denatured by heating typically at 94oC. Here the doublestranded DNA is separated into two complementary single strands. The second step is the annealing process where the oligonucleotide primers will be hybridized to the DNA template. The temperature used during this stage normally is between 40-72oC. The last step is the DNA synthesis process where the thermostable DNA polymerase will be effectively synthesis the DNA at reaction temperature of 72oC (McPherson and Moller, 2006). Table B-2: Reverse and forward of universal primers Primer 16S-Reverse primer 16S-Forward primer Sequence 5- cgg cta cct tgt tac gac tt - 3 5- gag ttt gat cct ggc tca g - 3 The

100 l of the reaction solution was prepared in PCR reaction tube.

reaction solution used for the amplification process was shown in Table B-3. The

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preparation of the reaction solutions were carried out on ice. The samples were resuspended gently to homogenize the mixtures well. Then samples were placed in a thermocycler (Perkin Elmer GeneAmp PCR System 9700) where polymerase chain reaction took place. Parameters for PCR cycle were shown in Table B-4. Table A-B: Composition of PCR reaction solution Components Genomic DNA 16S-Reverse primer 16S-Forward primer Buffer MgCl2 dNTP mix Taq polymerase dH2O Total Volume (l ) 5 3 3 10 8 2 1 68 100

B-4:

PCR Product Purification PCR product was purified using Promega WizardSV gel and PCR clean-up

system. According to this procedure an equal volume of Membrane Binding Solution and PCR reaction were mixed homogenously. The prepared PCR product was After poured to SV minicolumn assembled to a clean 2 mL collection tube.

incubation at room temperature for 1 minute, the sample was centrifuged at 16,000 x g for 1 minute. The flow-through liquid was discarded and the minicolumn was reinserted back into the collection tube. Then 700 L of Membrane Wash Solution that has been diluted with ethanol for washing the column were added into the minicolumn. The mixed solution was centrifuge at 16,000 x g for 1 minute. The washing procedure was repeated once again with 500 L of Membrane Wash Solution and centrifuged at 16,000 x g for 5 minutes. All the flow-through was discarded. The column assembly was

318

recentrifuged for another 1 minute with the microcentrifuge lid opened for evaporation of any ethanol residual. Then the minicolumn was transferred to a clean 1.5 mL microcentrifuge tube and was added with 50 L of Nuclease-Free Water. The solution was incubated for 1 minute at room temperature before centrifuge at 16,000 x g for 1 minute. Finally the minicolumn was discarded and the eluted purified DNA was store 4oC or -20oC.

Table B-4: Parameter of PCR cycle Parameter Pre-denaturation Denaturation Annealing Extension Final Extension Preservation Temperature (oC) 94 94 55 72 72 4 Time 4 min 1 min 45 s 1 min 7 min Infinite

B-5:

Purified DNA Estimation Yields of the purified DNA were determined using agarose electrophoresis

analysis. minutes.

Agarose with 1% (w/v) was used and was run at 80 V and 45 W for 60 Then, the gel was stained in running buffer plus 0.5 g/ml ethidium

bromide (EtBr) for 30 minutes. The result of the DNA electrophoresis was examined using UV transilluminator (TFX-35 Vilber Lourmat) and photographs were taken using digital camera. Gene Ruler Ladder Mix Marker was used as the universal marker.

B-6:

Sequencing of the 16S rRNA Gene and Homology Analysis The purified DNA (PCR product) was sent to Vivantis Laboratories Sdn Bhd

for sequencing purposes. The identification of selected bacteria were determined via comparing the 16S rDNA sequences obtained in this study to the GenBank database of National Center for Biotechnology Information (NCBI) websites

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(http://www.ncbi.nlm.nihgov/BLAST).

The nucleotides were compared through

Basic Local Alignment Search Tool (BLASTn) search program.

APPENDIX C:

MORPHOLOGY OF BACTERIA

Appendix C-1: Morphology of Bacteria Colony

Figure C-1 Example of classification of bacteria colony morphology (Wiley et al., 2008)

Appendix C-2: Morphology of Bacteria Cell

Figure C-2 Example of classification of bacteria cellular morphology (Lester and Birkett, 1999)

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APPENDIX D: MOLECULAR DATA ANALYSIS D1:

BLASTn Analysis Result for the Determination of the Alignment Scores

of the Full Sequence of 16S rDNA for BS1FAnGS BS1FAnGS Full length sequence (1394 nucleotides)
CGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATG CCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATACC GCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATG AGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATC CGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGA CTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCC AGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGG GAGGAAGGGCAGTTACCTAATACGTGATTGTTTTGACGTTACCGACAGAATAAG CACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAA TCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTAGTTAAGTTGGATGTGAA ATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACTGACTAGAGTATGGT AGAGGGTGGTGGAAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGG AACACCAGTGGCGAAGGCGAACCACCTGGACTGATACTGACACTGAGGTGCGA AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT GTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAG TTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGG GCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTT ACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAA CATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGG CACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA GTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAG AGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCAGAAAACCGATCGTAGTCCGG ATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATC AGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA TGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGG

Nucleotides marked in RED had been edited for sequence analysis. Top 10 Blast hits sequence on the full length sequence from NCBI
Accession AY144583.1 AF064460.1 FJ594447.1 AB365063.1 EU099375.1 Description Max score Total score 2555 2555 2551 2549 2549 Query coverage 100% 100% 100% 100% 100% E Max value ident 0.0 0.0 0.0 0.0 0.0 99% 99% 99% 99% 99% Links

Pseudomonas veronii strain UFZ-B547 16S 2555 ribosomal RNA gene, partial sequence Pseudomonas veronii 16S ribosomal RNA 2555 gene, complete sequence Pseudomonas sp. BS2(2009) 16S ribosomal 2551 RNA gene, partial sequence Pseudomonas sp. Pi 3-62 gene for 16S 2549 rRNA, partial sequence Pseudomonas sp. J7 16S ribosomal RNA 2549 gene, partial sequence

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Accession AY179328.1 AF539745.1 AF195777.1 FM162562.1 FJ184354.1 Description Max score Total score 2549 2549 2547 2543 2543 Query coverage 100% 100% 100% 100% 100% E Max value ident 0.0 0.0 0.0 0.0 0.0 99% 99% 99% 99% 99% Links

Pseudomonas veronii 16S ribosomal RNA 2549 gene, partial sequence Pseudomonas veronii UFZ-B549 ribosomal RNA gene, partial sequence 16S 2549

Pseudomonas sp. H1 16S ribosomal RNA 2547 gene, partial sequence Pseudomonas veronii partial 16S rRNA 2543 gene, strain MT4 Uncultured soil bacterium clone T7_3 16S 2543 ribosomal RNA gene, partial sequence

Phylogenetic Analysis
13 4 2 4 5 11

AF064460 Pseudomonas veronii AF539745 Pseudomonas veronii UFZ-B549 FJ594447 Pseudomonas sp. BS2 BS1FAnGS FM162562 Pseudomonas veronii AF195777 Pseudomonas sp. H1 AB365063 Pseudomonas sp. Pi 3-62 gene AY144583 Pseudomonas veronii strain U... AY179328 Pseudomonas veronii

63 30

EU099375 Pseudomonas sp. J7 FJ184354 Uncultured soil bacterium cl...

The phylogenetic tree showed the interrelationship between BS1FAnGS and top 10 Blast hits from NCBI.

D2: BLASTn Analysis Result for the Determination of the Alignment Scores of Forward and Reverse Sequences of Partial 16S rDNA for BS6FAnGS

BS6FAnGS - Forward sequence (338 nucleotides)

CCTGCCCATAAGACTGGCATAACTCCGGGAAACCGGGGCTAATACCGGATAAAA TTTTGAACCGCATGGTTCGAAATTGAAAGGCGTATTCGATTGTCACTTATGGATG GACCCGCGTCGCGTTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGGAACGATG CGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGCACTGAGACTCGGCCCAGA CTCCTACGGGAGGCATCAACAGGGAATCTTCCGCAATGGACGAAAGTCTGACGG AGCAACGCCGCGCGAGTGATTGAGGCTTTCTGGTCGTAAAACTCTGGTGTTATG GAAGAACAAGTGC

Nucleotides marked in RED had been edited for sequence analysis.

322

Top 10 Blast hits sequence on the forward sequence from NCBI

Accession FM873953.1 EU558974.1 EF422070.1 EF113618.1 FJ598018.1 FJ528077.1 FJ598437.1 FJ598436.1 EU429664.1 FJ581461.1

Description Uncultured bacterium partial 16S rRNA gene, clone MB04A04 Bacillus sp. cp-h31 16S ribosomal RNA gene, partial sequence Bacillus cereus strain S10 16S ribosomal RNA gene, partial sequence Bacillus thuringiensis strain IYM2 16S ribosomal RNA gene, partial sequence Bacillus cereus strain Bc6301 16S ribosomal RNA gene, partial sequence Bacillus sp. BM1-4 16S ribosomal RNA gene, partial sequence Bacillus sp. PM-3 16S ribosomal RNA gene, partial sequence Bacillus cereus strain PM-2 16S ribosomal RNA gene, partial sequence Bacillus thuringiensis serovar ostriniae 16S ribosomal RNA gene, partial sequence Bacillus cereus strain HWB1 16S ribosomal RNA gene, partial sequence

Max score 525 525 525 521 520 520 520 520 520 520

Total score 525 525 525 521 520 520 520 520 520 520

Query coverage 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

E value 5e146 5e146 5e146 6e145 2e144 2e144 2e144 2e144 2e144 2e144

Max Links ident 94% 94% 94% 94% 94% 94% 94% 94% 94% 94%

Phylogenetic Analysis
33 58 10 3 4 3 2 4 13

BS6FAnGS -For FM873953 Uncultured bacterium EF422070 Bacillus cereus strain S10 FJ598436 Bacillus cereus strain PM-2 FJ598437 Bacillus sp. PM-3 FJ581461 Bacillus cereus strain HWB1 FJ528077 Bacillus sp. BM1-4 EF113618 Bacillus thuringiensis strai... EU558974 Bacillus sp. cp-h31 FJ598018 Bacillus cereus strain Bc6301 EU429664 Bacillus thuringiensis serov... FM162562 Pseudomonas veronii (outgroup)

The phylogenetic tree showed the interrelationship between BS6FAnGS _For and top 10 Blast hits from NCBI.

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BS6FAnGS- Reverse sequence (595 nucleotides)

CCGCGATTACTAGCGATTCCAGTTTCATGTAGGCGAGTTGCAGCCTACAATCCAA ACTGAAAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCACCTCTTTGTA CCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGAC GTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACT TAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACA TCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCTCCCGAA GGAGAAGCCCTATCTCTAGGGTTTTCAGAGGATGTCAAGACCTGGTAAGGTTCT TCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAA TTCCTTTGAGTTTCAGCCTTGCGGCCGGACTCCCCAGGCGGAGTGCTTAATGCGT TAACTTCAGCACTAAAGGGCGGAAACCCTCTAACACTTAACACTCATCGTTTAC GGCGTGGACTACCAGGGTATCTAATCCCTGTTTGCTCCCCACGCTTTCGC

Nucleotides marked in RED had been edited for sequence analysis. BS6FAnGS- Reverse complementary sequence
GCGAAAGCGTGGGGAGCAAACAGGGATTAGATACCCTGGTAGTCCACGCCGTA AACGATGAGTGTTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGC ATTAAGCACTCCGCCTGGGGAGTCCGGCCGCAAGGCTGAAACTCAAAGGAATTG ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAA GAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTT CGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGT TGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGT ACAAAGAGGTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTTTCAGT TTGGATTGTAGGCTGCAACTCGCCTACATGAAACTGGAATCGCTAGTAATCGCG G

Top 10 Blast hits sequence on the reverse sequence from NCBI

Accession FM209283.1 EU239120.1 AY461756.2 AY461752.2 DQ067207.1 EU429669.1 EU429668.1 EU429666.1 Description Bacillus cereus partial 16S rRNA gene, strain TC4 Bacillus cereus strain KNUC260 16S ribosomal RNA gene, partial sequence Bacillus sp. H-15 16S ribosomal RNA gene, partial sequence Bacillus sp. H-11 16S ribosomal RNA gene, partial sequence Bacillus sp. A36 16S ribosomal RNA gene, partial sequence Bacillus thuringiensis serovar toumanoffi 16S ribosomal RNA gene, partial sequence Bacillus thuringiensis serovar thuringiensis 16S ribosomal RNA gene, partial sequence Bacillus thuringiensis serovar cameroun 16S ribosomal RNA gene, partial sequence Max score 1066 1061 1061 1061 1061 1059 1059 1059 1059 Total score 1066 1061 1061 1061 1061 1059 1059 1059 1059 Query coverage 100% 100% 100% 100% 99% 100% 100% 100% 100% E value 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Max Links ident 98% 98% 98% 98% 98% 98% 98% 98% 98%

EU429665.1 Bacillus thuringiensis serovar berliner 16S

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Accession Description ribosomal RNA gene, partial sequence EU429662.1 Bacillus thuringiensis serovar galleriae 16S ribosomal RNA gene, partial sequence 1059 1059 100% 0.0 98% Max score Total score Query coverage E value Max Links ident

Phylogenetic Analysis
1 0 0

EU239120 Bacillus cereus strain KNUC260 AY461752 Bacillus sp. H-11 EU429666 Bacillus thuringiensis serov...

EU429662 Bacillus thuringiensis serov... AY461756 Bacillus sp. H-15

2 2 11 1

EU429665 Bacillus thuringiensis serov... FM209283 Bacillus cereus EU429669 Bacillus thuringiensis serov... EU429668 Bacillus thuringiensis serov... BS6FAnGS-Rev

63

DQ067207 Bacillus sp. A36 FM162562 Pseudomonas veronii (outgroup)

The phylogenetic tree showed the interrelationship between BS6FAnGS_Rev and top 10 Blast hits from NCBI.

D3: BLASTn Analysis Result for the Determination of the Alignment Scores of Forward and Reverse Sequences of Partial 16S rDNA for BS7FAnGS BS7FAnGS - Forward sequence (383 nucleotides)
TAACACATGCGAGTCGAGCGGATGAAGGGAGCTTGCTCTCTGATTCAGCGGCGG ACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTCCGAAA GGGACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCT CACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTAGGTGGGGTAATGGCTCA CCTAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTG AGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGG GCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAA TGCACT

Nucleotides marked in RED had been edited for sequence analysis.

325

Top 10 Blast hits sequence on the forward sequence from NCBI

Accession

Description

Max score 697

Total score 697

Query coverage 100%

E Max Links value ident 0.0 99%

Uncultured proteobacterium clone MFC-B162FJ393104.1 E02 16S ribosomal RNA gene gene, partial sequence EU352759.1 EU170480.1 EU170479.1 EF593111.1 EU312076.1 EU287480.1 EF660333.1 AB007999.1 EF379150.1 Pseudomonas citronellolis strain NK 2.C2-1 16S ribosomal RNA gene, partial sequence Pseudomonas aeruginosa strain L-4 16S ribosomal RNA gene, partial sequence Pseudomonas sp. LF-1 16S ribosomal RNA gene, partial sequence Pseudomonas citronellolis 16S ribosomal RNA gene, partial sequence Pseudomonas sp. Pds-5 16S ribosomal RNA gene, partial sequence Pseudomonas sp. J9(2007) 16S ribosomal RNA gene, partial sequence Pseudomonas sp. LFJS3-9 16S ribosomal RNA gene, partial sequence Pseudomonas sp. WAS2 gene for 16S rRNA, partial sequence Uncultured bacterium clone AA4 32 16S ribosomal RNA gene, partial sequence

686 686 686 682 680 680 680 680 676

686 686 686 682 680 680 680 680 676

100% 100% 100% 99% 98% 100% 100% 100% 98%

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

98% 98% 98% 98% 99% 98% 98% 98% 98%

Phylogenetic Analysis
19 62 60 22 62

EU170480 Pseudomonas aeruginosa strai... EF379150 Uncultured bacterium EU170479 Pseudomonas sp. LF-1 16S EF593111 Pseudomonas citronellolis AB007999 Pseudomonas sp. WAS2 EU352759 Pseudomonas citronellolis st...

50 65 88

EU287480 Pseudomonas sp. J9 EF660333 Pseudomonas sp. LFJS3-9 BS7FAnGS-For FJ393104 Uncultured proteobacterium c... EU312076 Pseudomonas sp. Pds-5

The phylogenetic tree showed the interrelationship between BS7FAnGS_For and top 10 Blast hits from NCBI.

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BS7FAnGS -Reverse sequence (620 nucleotides)

TGGTGACCGTCCCCCCGAAGGTTAGACTAGCTACTTCTGGAGCAACCCACTCCC ATGGGGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGACATTC TGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGAT CCGGACTACGATCGGTTTTGTGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTC TGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACT TGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCC ACCTTAACGCGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAAC CCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGATT CCCGAAGGCACTCCCACATCTCTGCAGGATTCCGGACATGTCAAGGCCAGGTAA GGTTCTTCGCGTTGCTTCAAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCC CGTCAATTCATTTGAGTTTTAACCTTGCGGGCCGTACTCCCCAGGCGGTCGACTT ATCGCGTTAGCTGCGCCACTA

Nucleotides marked in RED had been edited for sequence analysis. BS7FAnGS -Reverse complementary sequence
TAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCCGCAAG GTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTT TAATTTGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCCGGAATCCTG CAGAGATGTGGGAGTGCCTTCGGGAATCGGAACACAGGTGCTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTC CTTAGTTACCAGCGCGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAAC CGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTAC ACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAA TCCCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAG TCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAG TCTAACCTTCGGGGGGACGGTCACCA

Top 10 Blast hits sequence on the reverse sequence from NCBI

Accession EU287480.1 AF039488.1 DQ339153.1 EU043324.1 DQ926686.1

Description Pseudomonas sp. J9(2007) 16S ribosomal RNA gene, partial sequence Pseudomonas sp. 273 small subunit ribosomal RNA gene, partial sequence Pseudomonas sp. RLD-1 16S ribosomal RNA gene, partial sequence Pseudomonas sp. SBR3-tpnb 16S ribosomal RNA gene, partial sequence Uncultured bacterium clone N3 16S ribosomal RNA gene, partial sequence

Max score 1110 1105 1099 1096 1094

Total score 1110 1105 1099 1096 1094

Query coverage 100% 100% 100% 98% 100%

E value 0.0 0.0 0.0 0.0 0.0

Max Links ident 99% 98% 98% 99% 98%

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Accession EU312076.1 FJ470323.1 DQ136054.2 EU170480.1 EU170479.1 Description Pseudomonas sp. Pds-5 16S ribosomal RNA gene, partial sequence Uncultured bacterium clone SLB16 16S ribosomal RNA gene, partial sequence Bacterium PT09 16S ribosomal RNA gene, partial sequence Pseudomonas aeruginosa strain L-4 16S ribosomal RNA gene, partial sequence Pseudomonas sp. LF-1 16S ribosomal RNA gene, partial sequence Max score 1092 1088 1088 1088 1088 Total score 1092 1088 1088 1088 1088 Query coverage 99% 100% 100% 100% 100% E value 0.0 0.0 0.0 0.0 0.0 Max Links ident 98% 98% 98% 98% 98%

Phylogenetic Analysis
74 78 67 58 50

AF039488 Pseudomonas sp. 273 DQ926686 Uncultured bacterium clone N3 DQ339153 Pseudomonas sp. RLD-1 EU312076 Pseudomonas sp. Pds-5 EU287480 Pseudomonas sp. J9 BS7FAnGS- Rev EU043324 Pseudomonas sp. SBR3-tpnb FJ470323 Uncultured bacterium clone S... DQ136054 Bacterium PT09

79

98 69

EU170480 Pseudomonas aeruginosa strai... EU170479 Pseudomonas sp. LF-1

The phylogenetic tree showed the interrelationship between BS7FAnGS_Rev and top 10 Blast hits from NCBI.

D4: BLASTn Analysis Result for the Determination of the Alignment Scores of Forward and Reverse Sequences of Partial 16S rDNA for BS10FAnGS BS10FAnGS Forward sequence (538 nucleotides)
GGCCCTACACATGCGAGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCG GCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCG GAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCG GGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATG GCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGG AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGAC AATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGAT TGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTACCTAATACGAGATTGTTTT GACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAAT ACAGAGGGTGCAAGCGTTAATCCCAATTACTGGGCGTAAAGCGCGCGTAGGTGG

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Nucleotides marked in RED had been edited for sequence analysis. Top 10 Blast hits sequence on the forward sequence from NCBI
Accession DQ536516.1 EF379138.1 DQ264528.1 EU086550.1 FM162562.1 FJ434132.1 FJ184354.1 FJ184352.1 FJ184350.1 FJ184346.1 Description Pseudomonas trivialis strain BIHB 745 16S ribosomal RNA gene, partial sequence Uncultured bacterium clone Mat Z4 19 16S ribosomal RNA gene, partial sequence Uncultured bacterium clone BANW559 16S ribosomal RNA gene, partial sequence Bacterium THCL4 16S ribosomal RNA gene, partial sequence Pseudomonas veronii partial 16S rRNA gene, strain MT4 Pseudomonas sp. IMER-A2-24 16S ribosomal RNA gene, partial sequence Uncultured soil bacterium clone T7_3 16S ribosomal RNA gene, partial sequence Uncultured soil bacterium clone T7_7 16S ribosomal RNA gene, partial sequence Uncultured soil bacterium clone T7_14 16S ribosomal RNA gene, partial sequence Uncultured soil bacterium clone T8_5 16S ribosomal RNA gene, partial sequence Max score 966 965 965 963 961 961 961 961 961 961 Total score 966 965 965 963 961 961 961 961 961 961 Query coverage 100% 99% 99% 99% 99% 99% 99% 99% 99% 99% E value 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Max Links ident 99% 99% 99% 99% 99% 99% 99% 99% 99% 99%

Phylogenetic Analysis
3 0 1

EF379138 Uncultured bacterium clone M... FJ184350 Uncultured soil bacterium cl... EU086550 Bacterium THCL4

FJ184354 Uncultured soil bacterium cl... DQ536516 Pseudomonas trivialis strain...

1 6 48 10

DQ264528 Uncultured bacterium clone B... FM162562 Pseudomonas veronii FJ184352 Uncultured soil bacterium cl... FJ434132 Pseudomonas sp. IMER-A2-24

FJ184346 Uncultured soil bacterium cl... BS10FAnGS

AE005177 Escherichia coli (outgroup)

The phylogenetic tree showed the interrelationship between BS10FAnGS_For and top 10 Blast hits from NCBI.

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BS10FAnGS -Reverse sequence (787 nucleotides)

GTCCCCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGA CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCG ATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTA CGATCGGTTTTCTGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGTACCGA CCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCA TCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATTAC GTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGG CACCAATCCATCTCTGGAAAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCG CGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTC ATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAG CTGCGCCACTAAAGAGCTCAAGGCTCCCAACGGCTAGTTGACATCGTTTACGGC GTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTG TCAGTATCAGTCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGC ATTTCACCGCTACACAGGAAATT

Nucleotides marked in RED had been edited for sequence analysis

BS10FAnGS -Reverse complementary sequence

AATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGA AGGCGACCACCTGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAA CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGG GAGCCTTGAGCTCTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGA GTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGG TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACA TCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTG CTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGA GCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGCACTCTAAGGAGAC TGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTT ACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCG CGAGGTGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAAC TCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCA CCAGAAGTAGCTAGTCTAACCTTCGGGGGGAC

Top 10 Blast hits sequence on the reverse sequence from NCBI

Accession EU934227.1 AY512607.1 AY263479.1 AY144583.1 Description Pseudomonas sp. LaGso27g, 16S ribosomal RNA gene, partial sequence Pseudomonas sp. A3YXyl2-4 16S ribosomal RNA gene, partial sequence Pseudomonas sp. R1enr 16S ribosomal RNA gene, partial sequence Pseudomonas veronii strain UFZ-B547 16S ribosomal RNA gene, partial sequence Max score 1443 1443 1443 1443 Total score 1443 1443 1443 1443 Query coverage 100% 100% 100% 100% E value 0.0 0.0 0.0 0.0 Max Links ident 99% 99% 99% 99%

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Accession AY882021.1 AF058286.1 DQ339583.1 AF064460.1 FJ594447.1 FJ517635.1 Description Pseudomonas sp. GD100 16S ribosomal RNA gene, partial sequence Pseudomonas mandelii 16S ribosomal RNA gene, complete sequence Pseudomonas sp. Enf22 16S ribosomal RNA gene, partial sequence Pseudomonas veronii 16S ribosomal RNA gene, complete sequence Pseudomonas sp. BS2(2009) 16S ribosomal RNA gene, partial sequence Pseudomonas sp. DM2 16S ribosomal RNA gene, partial sequence Max score 1443 1443 1443 1443 1437 1437 Total score 1443 1443 1443 1443 1437 1437 Query coverage 100% 100% 100% 100% 100% 100% E value 0.0 0.0 0.0 0.0 0.0 0.0 Max Links ident 99% 99% 99% 99% 99% 99%

Phylogenetic Analysis
13 0 2

BS10FAnGS Rev FJ594447 Pseudomonas sp. BS AF058286 Pseudomonas mandelii

DQ339583 Pseudomonas sp. Enf22 AY263479 Pseudomonas sp. R1enr

11

3 10

EU934227 Pseudomonas sp. LaGso27g AY512607 Pseudomonas sp. A3YXyl2-4 AY144583 Pseudomonas veronii strain U...
8

AF064460 Pseudomonas veronii AY882021 Pseudomonas sp. GD100 FJ517635 Pseudomonas sp. DM2

The phylogenetic tree showed the interrelationship between BS10FAnGS_Rev and top 10 Blast hits from NCBI.

D5: BLASTn Analysis Result for the Determination of the Alignment Scores of the Full Sequence of 16S rDNA for BS11FAnGS BS11FAnGS -Full sequence (1429 nucleotides)
TGCGGCAGGGCTACACATGCAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGG TGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGG ATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGG GGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGG TGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCA GCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGG AATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAG GCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGCTGAGGTTAATAAC

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CTCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAG CCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGC ACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACT GCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGT AGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCC TGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAG GCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTT TCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGT CGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT ATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAA ACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCT ACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGC GGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATG AAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGG TAGCTTAACCTTCGGGAGGGCGCTCACCATC

Nucleotides marked in RED had been edited for sequence analysis. Top 10 Blast hits sequence on the full length sequence from NCBI
Accession EU781735.1 EU331414.1 EU221358.1 DQ068845.1 DQ068880.1 AB244469.1 Description Enterobacter sp. VET-7 16S ribosomal RNA gene, partial sequence Enterobacter sp. L3R3-1 16S ribosomal RNA gene, partial sequence Enterobacter asburiae strain J2S4 16S ribosomal RNA gene, partial sequence Uncultured bacterium clone 5s2 16S ribosomal RNA gene, partial sequence Uncultured bacterium clone bb2s4 16S ribosomal RNA gene, partial sequence Enterobacter cloacae gene for 16S rRNA, partial sequence, strain: NC1111 Max score 2603 2603 2603 2591 2588 2586 Total score 2603 2603 2603 2591 2588 2586 Query coverage 99% 99% 99% 99% 99% 99% E Max Links value ident 0.0 0.0 0.0 0.0 0.0 0.0 99% 99% 99% 99% 99% 99%

Uncultured Enterobacteriaceae bacterium AB114621.1 gene for 16S rRNA, partial sequence, clone:ER-9 AM184307.1 FJ445214.1 EF655641.1 Pantoea agglomerans partial 16S rRNA gene, strain WAB1969 Pantoea sp. NIIST-186 16S ribosomal RNA, partial sequence Uncultured bacterium clone B12 16S ribosomal RNA gene, partial sequence

2586

2586

99%

0.0

99%

2582 2580 2580

2582 2580 2580

99% 99% 99%

0.0 0.0 0.0

99% 99% 99%

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Phylogenetic Analysis
60 50 27

DQ068845 Uncultured bacterium clone 5s2 FJ445214 Pantoea sp. NIIST-186 EF655641 Uncultured bacterium clone B12 EU221358 Enterobacter asburiae strain...

50

AB244469 Enterobacter cloacae gene DQ068880 Uncultured bacterium clone b...

12 51

EU331414 Enterobacter sp. L3R3-1 AM184307 Pantoea agglomerans BS11FAnGS

21 44

EU781735 Enterobacter sp. VET-7 AB114621 Uncultured Enterobacteriacea...

The phylogenetic tree showed the interrelationship between BS11FAnGS and top 10 Blast hits from NCBI.

D6: BLASTn analysis result for the determination of the alignment scores of the full sequence of 16S rDNA for BS12FAnGS BS12FAnGS -Full Length sequence (1403 nucleotides)
GTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAA TGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCTAATA CCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGA TGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGA TCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCA GACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGA TCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTT GGGAGGAAGGGCAGTTACCTAATACGTGATTGCTTTGACGTTACCGACACAATA AGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTT AATCTTAATTACTGGTCATAAAGCGCGCGTAGGTGGGTTTGTTAAGTTGGATGTG AAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACTGACTAGAGTATG GTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGG AACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAA AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATG TCAACTAGCCGTTGGGAGCCTTGAGCTTTTAGTGGCGCAGCTAACGCATTAAGTT GACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGG CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTA CCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAAC ATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA AGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGC ACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAG TCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGA GGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGA TCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCA

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GAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACCCCGCCCGTCACACCAT GGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCCTCGGGAGGACGGTACCA

Nucleotides marked in RED had been edited for sequence analysis. Top 10 Blast hits sequence on the full length sequence from NCBI
Accession AJ583501.3 DQ136048.2 AB056120.1 AM421982.1 AY014806.1 FJ179366.1 EU086570.1 AB334768.1 DQ536516.1 AY599721.1 Description Pseudomonas extremaustralis 16S rRNA gene, strain CT14-3 Bacterium PT03 16S ribosomal RNA gene, partial sequence Pseudomonas veronii gene for 16S rRNA, strain:INA06 Pseudomonas sp. NJ-61 16S rRNA gene, strain NJ-61 Pseudomonas sp. NZ024 16S ribosomal RNA gene, partial sequence Pseudomonas trivialis strain BIHB 750 16S ribosomal RNA gene, partial sequence Bacterium TLCL3 16S ribosomal RNA gene, partial sequence Pseudomonas veronii gene for 16S ribosomal RNA, partial sequence Pseudomonas trivialis strain BIHB 745 16S ribosomal RNA gene, partial sequence Pseudomonas sp. TB3-6-I 16S ribosomal RNA gene, partial sequence Max score 2536 2536 2536 2531 2531 2525 2525 2525 2525 2525 Total score 2536 2536 2536 2531 2531 2525 2525 2525 2525 2525 Query coverage 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% E value 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Max Links ident 99% 99% 99% 99% 99% 99% 99% 99% 99% 99%

Phylogenetic Analysis
17 51 49 12 33

AJ583501 Pseudomonas extremaustralis AY014806 Pseudomonas sp. NZ024 DQ136048 Bacterium PT03 AB056120 Pseudomonas veronii gene AM421982 Pseudomonas sp. NJ-61

37

BS12FAnGS AY599721 Pseudomonas sp. TB3-6-I

93 35

FJ179366 Pseudomonas trivialis strain... DQ536516 Pseudomonas trivialis strain... EU086570 Bacterium TLCL3
53

AB334768 Pseudomonas veronii gene EF422070 Bacillus cereus (outgroup)

The phylogenetic tree showed the interrelationship between BS12FAnGS and top 10 Blast hits from NCBI.

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VIVANTIS TECHNOLOGIES SDN. BHD. (587389-D)
Suite 2-B, No. 23, Jalan U1/15, Hicom Glenmarie Industrial Park, 40150, Shah Alam, Selangor, Malaysia. Tel: 603-5569 5785 Fax: 603-5569 5786 URL: http://www.vivantis.com E-mail: info@vivantis.com

Service Report Microorganism Identification By 16S/18S rRNA Sequencing

Customer Name Institute/Company : Dr. Azmi Aris/ Ms Khalida Muda : Dept of Environmental Eng. Faculty of Civil Eng. UTM
Comment Phylogenetic analysis using both 5 (forward) and 3 (reverse) sequences showed that this bacteria is closely related to Pseudomonas veronii Phylogenetic analysis using both 5 (forward) and 3 (reverse) sequences showed that this bacteria is closely related to Bacillus cereus Phylogenetic analysis using both 5 (forward) and 3 (reverse) sequences showed that this bacteria is closely related to Pseudomonas sp. Phylogenetic analysis using both 5 (forward) and 3 (reverse) sequences showed that this bacteria is closely related to Pseudomonas sp. Phylogenetic analysis using both 5 (forward) and 3 (reverse) sequences showed that this bacteria is closely related to Enterobacter sp. Phylogenetic analysis using both 5 (forward) and 3 (reverse) sequences showed that this bacteria is closely related to Pseudomonas sp. Remark Sequencing results, phylogenetic trees, multiple alignment and top 10 sequences match were supplied. Sequencing results, phylogenetic trees, multiple alignment and top 10 sequences match were supplied. Sequencing results, phylogenetic trees, multiple alignment and top 10 sequences match were supplied. Sequencing results, phylogenetic trees, multiple alignment and top 10 sequences match were supplied. Sequencing results, phylogenetic trees, multiple alignment and top 10 sequences match were supplied. Sequencing results, phylogenetic trees, multiple alignment and top 10 sequences match were supplied.

Date: 12th February 2009

Sample ID

Our Label

1. BS1FAnGS

G1

2. BS6FAnGS

02

3. BS7FAnGS

03

4. BS10FAnGS

N2

5. BS11FAnGS

N3

6. BS12FAnGS

N4

In order to view the sequencing result, the Chromas software need to be used. The Chromas freeware is available at http://www.technelysium.com.au/chromas_lite.html Prepared by:

Chan Toh Theng Lab Manager Vivantis Technologies Sdn. Bhd.

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APPENDIX E: FACTORIAL DESIGN AND RESPONSE SURFACE METHODOLOGY DATA ANALYSIS FOR COAGGREGATION AND SURFACE HDROPHOBICITYASSAY E-1: Factorial Design Analysis for Coaggregation Assay (1 hour)

Fractional Factorial Fit: CAgg_1h versus Substrate, pH, Temperature Estimated Effects and Coefficients for CAgg_1h (coded units) Term Constant Substrat pH Temperat Substrat*pH Substrat*Temperat pH*Temperat Substrat*pH*Temperat Effect 7.775 -9.725 15.325 -6.600 -2.200 -0.650 1.325 Coef 53.025 3.887 -4.862 7.663 -3.300 -1.100 -0.325 0.663 SE Coef 1.006 1.006 1.006 1.006 1.006 1.006 1.006 1.006 T 52.70 3.86 -4.83 7.62 -3.28 -1.09 -0.32 0.66 P 0.000 0.005 0.001 0.000 0.011 0.306 0.755 0.529

Analysis of Variance for CAgg_1h (coded units) Source Main Effects 2-Way Interactions 3-Way Interactions Residual Error Pure Error Total DF 3 3 1 8 8 15 Seq SS 1559.53 195.29 7.02 129.57 129.57 1891.41 Adj SS 1559.53 195.29 7.02 129.57 129.57 Adj MS 519.843 65.097 7.023 16.196 16.196 F 32.10 4.02 0.43 P 0.000 0.051 0.529

E-2:

Factorial Design Analysis for Coaggregation Assay (2 hour)

Fractional Factorial Fit: CAgg_2h versus Substrate, pH, Temperature Estimated Effects and Coefficients for CAgg_2h (coded units) Term Constant Substrat pH Temperat Substrat*pH Substrat*Temperat pH*Temperat Substrat*pH*Temperat Effect 11.350 -15.375 12.925 -7.475 -4.775 -4.100 -4.500 Coef 57.775 5.675 -7.687 6.463 -3.737 -2.388 -2.050 -2.250 SE Coef 1.318 1.318 1.318 1.318 1.318 1.318 1.318 1.318 T 43.83 4.31 -5.83 4.90 -2.84 -1.81 -1.56 -1.71 P 0.000 0.003 0.000 0.001 0.022 0.108 0.159 0.126

Analysis of Variance for CAgg_2h (coded units) Source Main Effects 2-Way Interactions 3-Way Interactions Residual Error Pure Error Total DF 3 3 1 8 8 15 Seq SS 2129.07 381.95 81.00 222.41 222.41 2814.43 Adj SS 2129.07 381.95 81.00 222.41 222.41 Adj MS 709.69 127.32 81.00 27.80 27.80 F 25.53 4.58 2.91 P 0.000 0.038 0.126

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E-3:

Factorial Design Analysis for Coaggregation Assay (3 hour)

Fractional Factorial Fit: CAgg_3h versus Substrate, pH, Temperature Estimated Effects and Coefficients for CAgg_3h (coded units) Term Constant Substrat pH Temperat Substrat*pH Substrat*Temperat pH*Temperat Substrat*pH*Temperat Effect 17.375 -11.225 9.200 -1.775 0.800 -6.900 -1.700 Coef 59.363 8.687 -5.612 4.600 -0.887 0.400 -3.450 -0.850 SE Coef 2.417 2.417 2.417 2.417 2.417 2.417 2.417 2.417 T 24.56 3.59 -2.32 1.90 -0.37 0.17 -1.43 -0.35 P 0.000 0.007 0.049 0.094 0.723 0.873 0.191 0.734

Analysis of Variance for CAgg_3h (coded units) Source Main Effects 2-Way Interactions 3-Way Interactions Residual Error Pure Error Total DF 3 3 1 8 8 15 Seq SS 2050.12 205.60 11.56 747.75 747.75 3015.04 Adj SS 2050.12 205.60 11.56 747.75 747.75 Adj MS 683.37 68.53 11.56 93.47 93.47 F 7.31 0.73 0.12 P 0.011 0.561 0.734

E-4:

Factorial Design Analysis for Coaggregation Assay (4 hour)

Fractional Factorial Fit: CAgg_4h versus Substrate, pH, Temperature Estimated Effects and Coefficients for CAgg_4h (coded units) Term Constant Substrat pH Temperat Substrat*pH Substrat*Temperat pH*Temperat Substrat*pH*Temperat Effect 18.637 -9.637 5.462 -4.637 -2.088 -6.562 1.238 Coef 61.944 9.319 -4.819 2.731 -2.319 -1.044 -3.281 0.619 SE Coef 2.134 2.134 2.134 2.134 2.134 2.134 2.134 2.134 T 29.02 4.37 -2.26 1.28 -1.09 -0.49 -1.54 0.29 P 0.000 0.002 0.054 0.236 0.309 0.638 0.163 0.779

Analysis of Variance for CAgg_4h (coded units) Source Main Effects 2-Way Interactions 3-Way Interactions Residual Error Pure Error Total DF 3 3 1 8 8 15 Seq SS 1880.31 275.72 6.13 583.03 583.03 2745.18 Adj SS 1880.31 275.72 6.13 583.03 583.03 Adj MS 626.769 91.907 6.126 72.878 72.878 F 8.60 1.26 0.08 P 0.007 0.351 0.779

337

E-5: Factorial Design Analysis for Coaggregation Assay (5 hours)

Fractional Factorial Fit: CAgg_5h versus Substrate, pH, Temperature Estimated Effects and Coefficients for CAgg_5h (coded units) Term Constant Substrat pH Temperat Substrat*pH Substrat*Temperat pH*Temperat Substrat*pH*Temperat Effect 19.362 -13.837 5.562 -0.587 0.713 -12.287 -0.737 Coef 58.406 9.681 -6.919 2.781 -0.294 0.356 -6.144 -0.369 SE Coef 0.4839 0.4839 0.4839 0.4839 0.4839 0.4839 0.4839 0.4839 T 120.69 20.01 -14.30 5.75 -0.61 0.74 -12.70 -0.76 P 0.000 0.000 0.000 0.000 0.561 0.483 0.000 0.468

Analysis of Variance for CAgg_5h (coded units) Source Main Effects 2-Way Interactions 3-Way Interactions Residual Error Pure Error Total DF 3 3 1 8 8 15 Seq SS 2389.30 607.34 2.18 29.98 29.97 3028.79 Adj SS 2389.30 607.34 2.18 29.98 29.97 Adj MS F 796.432 212.56 202.447 54.03 2.176 0.58 3.747 3.747 P 0.000 0.000 0.468

E-6:

Factorial Design Analysis for Surface Hydrophobicity Assay

Fractional Factorial Fit: SHb (%) versus Substrate, pH, Temperature Estimated Effects and Coefficients for SHb (coded units) Term Constant Substrat pH Temperat Substrat*pH Substrat*Temperat pH*Temperat Substrat*pH*Temperat Effect 4.95 -8.05 -4.43 -11.25 -0.18 -20.82 -5.13 Coef 30.15 2.47 -4.02 -2.21 -5.62 -0.09 -10.41 -2.56 SE Coef 0.4889 0.4889 0.4889 0.4889 0.4889 0.4889 0.4889 0.4889 T 61.66 5.06 -8.23 -4.53 -11.50 -0.18 -21.30 -5.24 P 0.000 0.001 0.000 0.002 0.000 0.862 0.000 0.001

Analysis of Variance for SHb (coded units) Source Main Effects 2-Way Interactions 3-Way Interactions Residual Error Pure Error Total DF 3 3 1 8 8 15 Seq SS 435.54 2241.09 105.06 30.60 30.60 2812.30 Adj SS 435.54 2241.09 105.06 30.60 30.60 Adj MS F 145.181 37.96 747.032 195.30 105.063 27.47 3.825 3.825 P 0.000 0.000 0.001

338

E-7: Response Surface Modeling Analysis (Transforms) (Full Quadratic Terms) (5 hours)

for

Coaggregation

Assay

Response Surface Regression: Coaggregation versus Substrate, pH and Temperature Transform: Power Lambda: 2.05 Constant: 0 Analysis of variance table [Partial sum of squares - Type III]
Source Sum of Squares df 9 1 1 1 1 1 1 1 1 1 10 5 5 19 Mean Square 7.007E+006 1.128E+007 1.623E+007 5.639E+006 2.362E+005 56360.44 7.442E+006 1.322E+007 5.244E+006 1.521E+006 9.734E+005 1.848E+006 98249.40 18.81 0.0029 F Value 7.20 11.59 16.67 5.79 0.24 0.058 7.65 13.58 5.39 1.56 p-value Prob > F 0.0024 0.0067 0.0022 0.0369 0.6329 0.8147 0.0200 0.0042 0.0427 0.2397 significant

Model 6.307E+007 A-Substrate 1.128E+007 B-pH 1.623E+007 C-Temperature 5.639E+006 AB 2.362E+005 AC 56360.44 BC 7.442E+006 2 A 1.322E+007 2 B 5.244E+006 C2 1.521E+006 Residual Lack of Fit significant Pure Error Cor Total Std. Dev. Mean C.V. % PRESS 9.734E+006 9.242E+006 4.912E+005 7.280E+007 986.59 4133.89 23.87 `7.119E+007 Coefficient Estimate 4113.74 908.95 -1090.06 642.60 -171.85 83.93 -964.48 957.62 -603.20 -324.93

R-Squared Adj R-Squared Pred R-Squared Adeq Precision Standard Error 402.38 266.97 266.97 266.97 348.81 348.81 348.81 259.89 259.89 259.89 95% CI Low 3217.19 314.11 -1684.90 47.75 -949.05 -693.27 -1741.68 378.56 -1182.26 -903.99 95% CI High

0.8663 0.7460 0.0221 11.147

Factor Intercept A-Substrate B-pH C-Temperature AB AC BC A2 B2 C2

df 1 1 1 1 1 1 1 1 1 1

VIF

5010.30 1503.80 -495.21 1237.45 605.36 861.14 -187.27 1536.69 -24.13 254.14

1.00 1.00 1.00 1.00 1.00 1.00 1.02 1.02 1.02

339

E-8: Response Surface Modeling Analysis for Coaggregation (Transforms) (5 hours) (Linear + Square + pH x Tempareture)

Assay

Response Surface Regression: Coaggregation versus Substrate, pH and Temperature Transform: Power Lambda: 2. Constant: 0 Analysis of variance table [Partial sum of squares - Type III]
Source Model A-Substrate B-Ph C-Temperature BC A2 B2 Residual Lack of Fit Pure Error Cor Total Std. Dev. Mean C.V. % PRESS Sum of Squares 3.912E+007 7.112E+006 1.047E+007 3.590E+006 4.676E+006 9.044E+006 3.152E+006 7.416E+006 7.105E+006 3.116E+005 4.654E+007 df 6 1 1 1 1 1 1 13 8 5 19 Mean Square 6.520E+006 7.112E+006 1.047E+007 3.590E+006 4.676E+006 9.044E+006 3.152E+006 5.705E+005 8.881E+005 62320.34 14.25 0.0048 significant F Value 11.43 12.47 18.36 6.29 8.20 15.85 5.53 p-value Prob > F 0.0002 significant 0.0037 0.0009 0.0262 0.0133 0.0016 0.0352

755.31 3363.73 22.45 2.509E+007

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.8406 0.7671 0.4608 13.947

Factor Intercept A-Substrate B-pH C-Temperature BC A2 B2

Coefficient Estimate 3143.25 721.65 -875.70 512.68 -764.56 788.26 -465.39

df 1 1 1 1 1 1 1

Standard Error 261.49 204.39 204.39 204.39 267.04 197.98 197.98

95% CI Low 2578.33 280.10 -1317.25 71.13 -1341.47 360.55 -893.10

95% CI High 3708.17 1163.19 -434.15 954.23 -187.65 1215.98 -37.67

VIF 1.00 1.00 1.00 1.00 1.01 1.01

340

E-9: Response Surface Modeling Analysis for Surface Hydrophobicity Assay ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares - Type III]
Source Model A-Substrate B-pH C-Temperature AB AC BC A2 B2 C2 Residual Lack of Fit significant Pure Error Cor Total Sum of Squares 9065.62 267.37 1137.96 3.10 265.65 6.30 822.15 1559.95 5472.23 290.17 2881.90 2862.19 19.72 11947.53 df 9 1 1 1 1 1 1 1 1 1 10 5 5 19 Mean Square 1007.29 267.37 1137.96 3.10 265.65 6.30 822.15 1559.95 5472.23 290.17 288.19 572.44 3.94 145.17 < 0.0001 F Value 3.50 0.93 3.95 0.011 0.92 0.022 2.85 5.41 18.99 1.01 p-value Prob > F 0.0321 0.3582 0.0750 0.9194 0.3596 0.8854 0.1221 0.0423 0.0014 0.3393 significant

Std. Dev. Mean C.V. % PRESS

16.98 51.96 32.67 21749.41

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.7588 0.5417 -0.8204 5.870

Factor

Coefficient Estimate

df 1 1 1 1 1 1 1 1 1 1

Standard Error 6.92 4.59 4.59 4.59 6.00 6.00 6.00 4.47 4.47 4.47

95% CI Low 60.01 -5.81 -19.36 -9.76 -19.14 -14.26 -23.51 -20.37 -29.45 -14.45

95% CI High 90.86 14.66 1.11 10.71 7.61 12.49 3.24 -0.44 -9.52 5.48

VIF

Intercept 75.43 A-Substrate 4.42 B-pH -9.13 C-Temperature 0.48 AB -5.76 AC -0.89 BC -10.14 A2 -10.40 2 B -19.49 2 C -4.49

1.00 1.00 1.00 1.00 1.00 1.00 1.02 1.02 1.02

341

APPENDIX F: FACTORIAL DESIGN AND RESPONSE SURFACE METHODOLOGY DATA ANALYSIS FOR COD REMOVAL F1: Factorial Design Analysis for COD Removal
Fractional Factorial Fit: ANA_COD versus Substrate, RM
Estimated Effects and Coefficients for ANA_COD (coded units) Term Constant Substrate RM Substrate*RM Effect 50.450 -2.100 0.700 Coef 53.225 25.225 -1.050 0.350 SE Coef 1.048 1.048 1.048 1.048 T 50.78 24.07 -1.00 0.33 P 0.000 0.000 0.373 0.755

Analysis of Variance for ANA_COD (coded units) Source Main Effects 2-Way Interactions Residual Error Pure Error Total DF 2 1 4 4 7 Seq SS 5099.22 0.98 35.15 35.15 5135.35 Adj SS 5099.22 0.98 35.15 35.15 Adj MS F 2549.61 290.14 0.98 0.11 8.79 8.79 P 0.000 0.755

Estimated Coefficients for ANA_COD using data in uncoded units Term Coef Constant 28.1989 Substrate 0.0589095 RM -0.00113257 Substrate*RM 6.625473E-07

F2: Factorial Design Analysis for Aerobic COD Removal Fractional Factorial Fit: AER_COD versus Substrate, RM
Estimated Effects and Coefficients for AER_COD (coded units) Term Constant Substrate RM Substrate*RM Effect -32.45 -7.95 6.35 Coef 44.68 -16.22 -3.97 3.17 SE Coef 0.4131 0.4131 0.4131 0.4131 T 108.15 -39.28 -9.62 7.69 P 0.000 0.000 0.001 0.002

Analysis of Variance for AER_COD (coded units) Source Main Effects 2-Way Interactions Residual Error Pure Error Total DF 2 1 4 4 7 Seq SS 2232.41 80.64 5.46 5.46 2318.51 Adj SS 2232.41 80.64 5.46 5.46 Adj MS F 1116.20 817.73 80.64 59.08 1.37 1.36 P 0.000 0.002

342

F3: Factorial Design Analysis for Total COD Removal Fractional Factorial Fit: Total COD versus Substrate, RM
Estimated Effects and Coefficients for Total (coded units) Term Constant Substrate RM Substrate*RM Effect 12.800 -6.350 5.000 Coef 78.175 6.400 -3.175 2.500 SE Coef 0.2767 0.2767 0.2767 0.2767 T 282.53 23.13 -11.47 9.04 P 0.000 0.000 0.000 0.001

Analysis of Variance for Total (coded units) Source Main Effects 2-Way Interactions Residual Error Pure Error Total DF 2 1 4 4 7 Seq SS 408.325 50.000 2.450 2.450 460.775 Adj SS 408.325 50.000 2.450 2.450 Adj MS F 204.162 333.33 50.000 81.63 0.613 0.612 P 0.000 0.001

F4: Central Composite Design Analysis for Anaerobic COD Removal Response Surface Regression: Anaerobic COD removal versus Substrate, RM
The analysis was done using coded units. Estimated Regression Coefficients for Anaerobic COD removal Term Constant Substrate RM Substrate*Substrate RM*RM Substrate*RM S = 19.51 Coef 23.940 13.374 -1.186 9.299 6.849 0.775 SE Coef 8.723 6.896 6.896 7.395 7.395 9.753 T 2.744 1.939 -0.172 1.257 0.926 0.079 P 0.029 0.094 0.868 0.249 0.385 0.939

R-Sq = 46.0%

R-Sq(adj) = 7.5%

Analysis of Variance for Anaerobic COD removal Source Regression Linear Square Interaction Residual Error Lack-of-Fit Pure Error Total DF 5 2 2 1 7 3 4 12 Seq SS 2270.79 1442.09 826.30 2.40 2663.31 2657.02 6.29 4934.10 Adj SS 2270.79 1442.09 826.30 2.40 2663.31 2657.02 6.29 Adj MS F 454.159 1.19 721.044 1.90 413.151 1.09 2.402 0.01 380.473 885.673 563.05 1.573 P 0.400 0.220 0.388 0.939 0.000

343

F5: Central Composite Design Analysis for Aerobic COD Removal Response Surface Regression: Aerobic COD removal versus Substrate, RM
The analysis was done using coded units. Estimated Regression Coefficients for Aerobic COD removal Term Constant Substrate RM Substrate*Substrate RM*RM Substrate*RM S = 17.63 Coef 73.56 -1.65 -2.28 -16.33 -4.33 3.45 SE Coef 7.883 6.232 6.232 6.683 6.683 8.813 T 9.332 -0.264 -0.366 -2.443 -0.648 0.391 P 0.000 0.799 0.725 0.045 0.538 0.707

R-Sq = 47.9%

R-Sq(adj) = 10.7%

Analysis of Variance for Aerobic COD removal Source Regression Linear Square Interaction Residual Error Lack-of-Fit Pure Error Total DF 5 2 2 1 7 3 4 12 Seq SS 2000.32 63.36 1889.35 47.61 2174.92 2169.46 5.45 4175.23 Adj SS 2000.32 63.36 1889.35 47.61 2174.92 2169.46 5.45 Adj MS F 400.063 1.29 31.679 0.10 944.674 3.04 47.610 0.15 310.702 723.155 530.56 1.363 P 0.366 0.904 0.112 0.707 0.000

F6: Central Composite Design Analysis for Total COD Removal Response Surface Regression: Total COD removal versus Substrate, RM
The analysis was done using coded units. Estimated Regression Coefficients for Total COD removal Term Constant Substrate RM Substrate*Substrate RM*RM Substrate*RM S = 4.794 Coef 79.900 8.332 -1.917 -6.706 1.644 2.825 SE Coef 2.144 1.695 1.695 1.818 1.818 2.397 T 37.267 4.916 -1.131 -3.690 0.904 1.179 P 0.000 0.002 0.295 0.008 0.396 0.277

R-Sq = 85.8%

R-Sq(adj) = 75.7%

Analysis of Variance for Total COD removal Source Regression Linear Square Interaction Residual Error Lack-of-Fit Pure Error Total DF 5 2 2 1 7 3 4 12 Seq SS 974.42 584.75 357.75 31.92 160.88 159.38 1.50 1135.30 Adj SS 974.416 584.746 357.747 31.923 160.881 159.381 1.500 Adj MS F 194.883 8.48 292.373 12.72 178.874 7.78 31.923 1.39 22.983 53.127 141.67 0.375 P 0.007 0.005 0.017 0.277 0.000

344

APPENDIX G: FACTORIAL DESIGN AND RESPONSE SURFACE METHODOLOGY DATA ANALYSIS FOR COLOR REMOVAL G1: Factorial Design Analysis for Color Removal (Sumifix Navy Blue_600 nm_5 hour) Fractional Factorial Fit: 600_5h versus Substrate, RM
Estimated Effects and Coefficients for 600_5h (coded units) Term Constant Substrate RM Substrate*RM Effect 5.9500 2.9500 0.9500 Coef 78.8250 2.9750 1.4750 0.4750 SE Coef 0.1199 0.1199 0.1199 0.1199 T 657.45 24.81 12.30 3.96 P 0.000 0.000 0.000 0.017

Analysis of Variance for 600_5h (coded units) Source DF Main Effects 2 2-Way Interactions 1 Residual Error 4 Pure Error 4 Total 7 Seq SS 88.2100 1.8050 0.4600 0.4600 90.4750 Adj SS 88.2100 1.8050 0.4600 0.4600 Adj MS F 44.1050 383.52 1.8050 15.70 0.1150 0.1150 P 0.000 0.017

G2: Factorial Design Analysis for Color Removal (Sumifix Navy Blue_600 nm_12 hour) Fractional Factorial Fit: 600_12 versus Substrate, RM
Estimated Effects and Coefficients for 600_12 (coded units) Term Constant Substrate RM Substrate*RM Effect -1.9000 5.5000 -0.5500 Coef 79.9750 -0.9500 2.7500 -0.2750 SE Coef 0.2604 0.2604 0.2604 0.2604 T 307.11 -3.65 10.56 -1.06 P 0.000 0.022 0.000 0.351

Analysis of Variance for 600_12 (coded units) Source DF Main Effects 2 2-Way Interactions 1 Residual Error 4 Pure Error 4 Total 7 Seq SS 67.7200 0.6050 2.1700 2.1700 70.4950 Adj SS 67.7200 0.6050 2.1700 2.1700 Adj MS 33.8600 0.6050 0.5425 0.5425 F 62.41 1.12 P 0.001 0.351

345

G3: Factorial Design Analysis for Color Removal (Synozol Red K-4B_542 nm _5 hour) Fractional Factorial Fit: 542_5h versus Substrate, RM
Estimated Effects and Coefficients for 542_5h (coded units) Term Constant Substrate RM Substrate*RM Effect 12.025 -2.225 4.125 Coef 71.738 6.013 -1.112 2.063 SE Coef 0.2741 0.2741 0.2741 0.2741 T 261.68 21.93 -4.06 7.52 P 0.000 0.000 0.015 0.002

Analysis of Variance for 542_5h (coded units) Source DF Main Effects 2 2-Way Interactions 1 Residual Error 4 Pure Error 4 Total 7 Seq SS 299.103 34.031 2.405 2.405 335.539 Adj SS 299.103 34.031 2.405 2.405 Adj MS F 149.551 248.73 34.031 56.60 0.601 0.601 P 0.000 0.002

G4: Factorial Design Analysis for Color Removal (Synozol Red K-4B_542 nm _12hour) Fractional Factorial Fit: 542_12h versus Substrate, RM
Estimated Effects and Coefficients for 542_12h (coded units) Term Constant Substrate RM Substrate*RM Effect -0.8000 5.7000 -1.1000 Coef 78.6000 -0.4000 2.8500 -0.5500 SE Coef 0.1639 0.1639 0.1639 0.1639 T 479.46 -2.44 17.38 -3.35 P 0.000 0.071 0.000 0.028

Analysis of Variance for 542_12h (coded units) Source DF Main Effects 2 2-Way Interactions 1 Residual Error 4 Pure Error 4 Total 7 Seq SS 66.2600 2.4200 0.8600 0.8600 69.5400 Adj SS 66.2600 2.4200 0.8600 0.8600 Adj MS F 33.1300 154.09 2.4200 11.26 0.2150 0.2150 P 0.000 0.028

346

G5: Response Surface Modeling Design Analysis for Color Removal (Sumifix Navy Blue EXF_600 nm_5 hours) (Full Quadratic Term)

ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares - Type III] Sum of Squares 105.45 28.64 18.67 0.25 33.1 32.3 36.36 18.04 18.32 141.81 Mean Square 21.09 28.64 18.67 0.25 33.10 32.34 5.19 6.01 4.58 F Value 4.06 5.51 3.60 0.048 6.37 6.23 p-value Prob > F 0.0475 0.0512 0.0998 0.8326 0.0396 0.0413 significant

Source Model A-Substrate B-Riboflavin AB A2 B2 Residual Lack of Fit Pure Error Cor Total

df 5 1 1 1 1 1 7 3 4 12

1.31

0.3865 not significant

Std. Dev. Mean C.V. % PRESS

2.28 81.63 2.79 156.89

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.7436 0.5605 -0.1063 4.849

Factor Intercept A-Substrate B-Riboflavin AB A2 B2

Coefficient Estimate 84.30 1.89 1.53 0.25 -2.18 -2.16

df 1 1 1 1 1 1

Standard Error 1.02 0.81 0.81 1.14 0.86 0.86

95% CI Low 81.89 -0.013 -0.38 -2.44 -4.22 -4.20

95% CI High 86.71 3.80 3.43 2.94 -0.14 -0.11

VIF

1.00 1.00 1.00 1.02 1.02

347

G6: Response Surface Modeling Design Analysis for Color Removal (Sumifix Navy Blue EXF_600 nm_5 hours) (Reduced Quadratic Term-Linear + Square Terms) ANOVA for Response Surface Reduced Quadratic Model Analysis of variance table [Partial sum of squares - Type III] Sum of Squares 105.20 28.64 18.67 33.10 32.34 36.61 18.29 18.32 141.81 Mean Square 26.30 28.64 18.67 33.10 32.34 4.58 4.57 4.58 F Value 5.75 6.26 4.08 7.23 7.07 p-value Prob > F 0.0176 0.0369 0.0780 0.0275 0.0289 significant

Source Model A-Substrate B-Riboflavin A2 B2 Residual Lack of Fit Pure Error Cor Total

df 4 1 1 1 1 8 4 4 12

1.00

0.5007 not significant

Std. Dev. Mean C.V. % PRESS

2.14 81.63 2.62 116.48

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.7419 0.6128 0.1786 5.847

Factor Intercept A-Substrate B-Riboflavin A2 B2

Coefficient Estimate 84.30 1.89 1.53 -2.18 -2.16

df 1 1 1 1 1

Standard Error 0.96 0.76 0.76 0.81 0.81

95% CI Low 82.09 0.15 -0.22 -4.05 -4.03

95% CI High 86.51 3.64 3.27 -0.31 -0.29

VIF 1.00 1.00 1.02 1.02

348

G7: Response Surface Modeling Design Analysis for Color Removal (Sumifix Navy Blue EXF_600 nm_12 hour) ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares - Type III] Sum of Squares Mean Square 31.72 13.55 76.73 1.69 10.16 49.45 4.38 1.36 6.65 F Value 7.23 3.09 17.50 0.39 2.32 11.28 p-value Prob > F 0.0109 0.1221 0.0041 0.5543 0.1717 0.0121 significant

Source

df 5 1 1 1 1 1 7 3 4 12

Model 158.58 A-Substrate 13.55 B-Riboflavin 76.73 AB 1.69 2 A 10.16 B2 49.45 Residual 30.69 Lack of Fit Pure Error Cor Total 4.08 26.61 189.26

0.20

0.8885 not significant

Std. Dev. Mean C.V. % PRESS

2.09 80.62 2.60 70.59

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.8379 0.7220 0.6270 9.821

Factor Intercept A-Substrate B-Riboflavin AB A2 B2

Coefficient Estimate 81.52 -1.30 3.10 -0.65 1.21 -2.67

df 1 1 1 1 1 1

Standard Error 0.94 0.74 0.74 1.05 0.79 0.79

95% CI Low 79.31 -3.05 1.35 -3.13 -0.67 -4.54

95% CI High 83.73 0.45 4.85 1.83 3.09 -0.79

VIF 1.00 1.00 1.00 1.02 1.02

349

G8: Response Surface Modeling Design Analysis for Color Removal (Synozol Red K-4B_542 nm _5 hours

ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares - Type III] Sum of Squares 377.90 154.57 22.42 11.22 170.80 36.16 125.71 105.54 20.17 503.61 Mean Square 75.58 154.57 22.42 11.22 170.80 36.16 17.96 35.18 5.04 F Value 4.21 8.61 1.25 0.62 9.51 2.01 p-value Prob > F 0.0437 0.0219 0.3008 0.4552 0.0177 0.1988

Source Model A-Substrate B-Riboflavin AB A2 B2 Residual Lack of Fit Pure Error Cor Total

df 5 1 1 1 1 1 7 3 4 12

significant

6.98

0.0456

significant

Std. Dev. Mean C.V. % PRESS

4.24 75.01 5.65 782.01

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.7504 0.5721 -0.5528 5.778

Factor Intercept A-Substrate B-Riboflavin AB A2 B2

Coefficient Estimate 79.46 4.40 1.67 1.68 -4.95 -2.28

df 1 1 1 1 1 1

Standard Error 1.90 1.50 1.50 2.12 1.61 1.61

95% CI Low 74.98 0.85 -1.87 -3.34 -8.75 -6.08

95% CI High 83.94 7.94 5.22 6.69 -1.16 1.52

VIF 1.00 1.00 1.00 1.02 1.02

350

G9: Response Surface Modeling Design Analysis for Color Removal (Synozol Red K-4B_542 nm _12 hours ANOVA for Response Surface Quadratic Model Analysis of variance table [Partial sum of squares - Type III] Sum of Squares Mean Square 28.92 7.85 67.98 2.10 10.10 49.59 3.48 1.68 4.83 F Value 8.31 2.26 19.54 0.60 2.90 14.25 0.35 p-value Prob > F 0.0074 0.1769 0.0031 0.4624 0.1322 0.0069 0.7938 significant

Source

df 5 1 1 1 1 1 7 3 4 12

Model 144.60 A-Substrate 7.85 B-Riboflavin 67.98 AB 2.10 2 A 10.10 B2 49.59 Residual 24.35 Lack of Fit 5.04 Pure Error 19.31 Cor Total 168.95

not significant

Std. Dev. Mean C.V. % PRESS

1.87 79.44 2.35 66.03

R-Squared Adj R-Squared Pred R-Squared Adeq Precision

0.8559 0.7529 0.6092 10.475

Coefficient Factor Estimate Intercept 80.34 A-Substrate -0.99 B-Riboflavin 2.92 AB -0.72 A2 1.21 B2 -2.67

df 1 1 1 1 1 1

Standard Error 0.83 0.66 0.66 0.93 0.71 0.71

95% CI Low 78.37 -2.55 1.36 -2.93 -0.47 -4.34

95% CI High 82.31 0.57 4.47 1.48 2.88 -1.00

VIF 1.00 1.00 1.00 1.02 1.02

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