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Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.7 (3&4) : 700-704. 2009 www.world-food.net
Helsinki, Finland
e-mail: info@world-food.net

Characterization of new algae isolated from textile wastewater plant


Jihane Cheriaa*, Fadhila Bettaieb, Ikbel Denden and Amina Bakhrouf *

Laboratory of Analysis, Treatment, Valorization of Environmental Pollution and Products "LR01ES16", Faculty of Pharmacy,
Department of Microbiology, Avicenne Street, 5000 Monastir, Tunisia. *e-mail: aminafdhila@yahoo.fr, jihanecheriaa@yahoo.fr

Received 11 May 2009, accepted 2 October 2009.

Abstract
A new microalga was isolated from a clarifier tank of an industrial textile plant. The main goal of this study was to identify and to determine optimal
culture conditions of the isolated alga and to test its involvement in reducing dye pollution. The pollution reduction of four industrial textile dyes
(indigo, remazol brilliant orange, direct blue and crystal violet) was evaluated with chemical oxygen demand (COD) exhaustion and with alga color
removal measured under shaking conditions. Our results showed that unicellular alga has both autotrophic and heterotrophic growth with a
photosynthetic activity producing pigments (chlorophyll a), thus the new isolated unicellular green alga may be Chlorella. For the green alga,
optimum culture conditions were pH 8, temperature 25°C and salinity at 15 g L-1. Furthermore, this alga has the capacity to reduce COD and color
removal of indigo textile dye at rates of 89% and 46%, respectively, within five days. The results suggest the importance of unicellular green alga as
an environmental preservation system.

Key words: Autotrophic, dyes, heterotrophic, green microalga, treatment, decolorization, salinity.

Introduction
There are more than 100,000 available dyes and different pigments study aims to identify a new unicellular alga in textile wastewater;
widely used in various stages of dyeing treatments and processing, secondly, to determine the effect of some parameters, pH, salinity
thus the pollution generated by dye materials is very important 1. and temperature, on alga growth and the color removal capacity
Dye presence, as little as 10 to 20 mg L-1, in water affects water of four industrial textile dyes (ITD), indigo (IND), direct blue (DB),
transparency and causes a part of aesthetic deterioration 2, 3. In remazol brilliant orange (Rbo) and crystal violet (CV) used in
many cases, color is the most easily detected parameter and, also, Tunisian textile dyeing industry.
constitutes the most quickly measurable unit 4. The use of microbial
consortia offers considerable advantages over the use of pure Materials and Methods
cultures in the synthetic dyes degradation 5. Indeed, color removal Water sampling:Samples of water were purchased from the
can be achieved by using different microorganisms belonging to secondary clarifier (SC) textile wastewater Tunisian plant. The
different taxonomic groups of bacteria, fungi such as white-rot main characteristics of water samples were indicated in Table 1.
fungi, yeasts and algae that have been reported for their ability to For the first time we isolated the alga on Chlorella agar medium,
decolorize a wide range of dyes 6. Although, bacteria play a key pH 4.5 ± 0.2 (Sigma, Product of India, C9720), in Petri plates
role in the biodegradation of organic pollutants, recent studies incubated at 25°C over one week under daylight. Cells were plated
have indicated that in addition to providing oxygen for aerobic on nutritive agar (Institut Pasteur, France) growth medium in Petri
bacterial biodegraders, microalgae can also biodegrade organic plates and allowed to develop colonies at same conditions. Green
pollutants directly 7, 8. It was reported that more than 30 azo colonies were selected for further analysis of morphology cells
compounds were biodegraded and decolorized by Chlorella and pigments production, to determine cells growth conditions
pyrenoidosa, Chlorella vulgaris and Oscillatoria tenuis in which and their ability to reduce dyes pollution.
azo dyes were decomposed into simpler aromatic amines 9.
Microalgae comprise diverse group of prokaryotic and eukaryotic Morphological observations: Fresh samples culture of isolated
organisms with great ecological importance. Based on numerous alga and a Hücker modified Gram stain, were preliminarily
biochemical and cellular differences, two major groups of green investigated light microscopy (LM), using a microscope
microalgae are recognized: Chlorophyta and Conjugaphyta 10. (Orthoplan, Leitz Wezler, Germany).
Microalgae contain about 50% of global organic carbon fixation11.
Many species are source of natural products such as pigments, Scanning electron microscopy (SEM): Cells algal cultures were
enzymes, unique fatty acids, vitamins 12 and continue to be used harvested by centrifugation (5000 rpm × 10 min), washed with
for biotechnological applications 13-15. At first step, the present PBS and fixed with 0.5% glutaraldehyde and 1% formaldehyde,

700 Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009
dehydrated by successive passages through 50, 80 and 100% Results and Discussion
ethanol (three times), washed with water and spread over Isolation and morphology of microalga: Alga cells cultivated
supporting glass squares and dried at room temperature 16, 17. on nutrient agar medium gave green colonies. The size of the
microalgal colonies was between 0.1-0.2 cm with circular shapes
Qualitative analysis of pigments: The profile pigment analysis (Fig. la), similar to colonies obtained by eubacteria. The examination
was studied to verify if the isolated alga is photosynthetic or not. of exponential culture by light microscopy showed spherical to
Alga cells were cultivated on nutritive agar medium, during seven oval shapes with a thin cell wall, a bright green single parietal
days at 25°C. The extraction of pigments was carried out from chloroplast, the multiplication mode is by autosporulation and
colonies obtained after culture as described by Jin et al. 12. The the autospores are released through a rupture of the mother cell
absorbance of the supernatant was measured at (400-800 nm) by wall (Fig. 1b-c). The observation by transmission electronic
using a UV-Visible spectrophotometer (Beckman Coulter, Inc. microscopy confirmed the oval shape with a 3 µm diameter cells
Fullerton, USA). size (Fig. ld). The isolated microalga was able to grow in nutrient
broth, media adapted to eubacteria. This result showed that
Optimization of cell growth conditions: To found the optimal isolated microalgae can grow heterotrophically. The ability of alga
T°C, pH and salinity growth conditions of the isolated microalgae to grow both autotrophically and heterotrophically was also
species, cells were cultivated on Chlorella broth flasks at variable showed by Oh-hama and Miyachi 21.
temperatures (20, 25, 30, 35 and 40°C) and at pH tested (5, 5.5, 6,
6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10) in an orbital incubator at 110 rpm for
seven days. The pH of Chlorella broth was adjusted with HCl
and NaOH solutions (1 N). To test the effect of salinity on alga
cells growth, sodium chloride salt was used at different
concentrations (0, 5, 10, 15, 20, 25, 30, 35 and 40 g L-1). All
experiments were carried into 250 ml erlenmeyer flasks containing
100 ml Chlorella broth medium. The pellet cells were suspended
in Chlorella broth (Sigma, Product of India, C9845) medium at a
density ranged between 105-106 cells/ml. Cells were incubated at
25°C with agitation at 110 rpm in an orbital incubator (SI-600 shaker,
Lab Companion-Jeio Tech, Korea) under daylight. After three days
of each culture period, the growth was determined
spectrophotometrically at 665 nm as pigments extracted by
methanol (90°C) from the cells mass according to De Marsac and
Houmard 18 procedure,
Figure 1. Algae isolated from textile wastewater: (a) color appearance of
Test of unicellular alga activity on industrial textile dyes: The
alga colonies on nutritive agar plates grown at 25°C, under daylight, (b) alga
new isolated microalgae from textile wastewater were used as cells under culture conditions (c) appearance of cells algae after Gram stain,
biological material to reduce pollution of ITD. We tested four modified by Hücker method (b and c) were observed by light microscopy
different dyes: indigo (indigoid dye), remazol brilliant orange Rbo at 1000× magnification, (d) SEM showing the size of algae cells.
(azo-dye), direct blue (reactive dye) and crystal violet (basic dye),
purchased from industrial textile corporations (SITEX, Ksar-Hellal, Pigments production: The analysis of profile pigments (Fig. 2)
Tunisia), which showed maximum absorbance (λmax), respectively, produced by the isolated microalga cells after culture on nutrient
at 602, 617, 594 and 592 nm. The industrial textile dyes were agar medium, showed two maximum peaks obtained at 420- 460
prepared and used at 10 mg L-1 in erlenmeyer flasks containing nm corresponding to the carotenoid. Peaks of absorbance were
100 ml of microalgae seeded in sterile Chlorella broth medium between 400-500 nm and 600-700 nm correspond to chlorophyll
adjusted between 105-106 cells/ml. Growth of unicellular green alga (Chla) and chlorophyll (Chlb), respectively. As indicated by
was evaluated daily during eight-days by using a Jeffrey et al. 22 and from the profile (Fig. 2), we could deduce that
spectrophotometer at 665 nm wavelength. This growth was the isolated Chlorella-like alga can produce also carotenoid. It
compared to cells cultivated in medium without dyes. The should be noted that, carotenoid, Chla and Chlb are produced
percentage of chemical oxygen demand (COD) removal was under heterotrophic condition. Moreover, photosynthetic
calculated as following % COD = [(COD at t0) – (COD at t1)]/ (COD activity producing oxygen by alga can relieve biological oxygen
at t0) × 100. The COD was measured after removing algal cells by demand in wastewater 19. According to Jenkins et al. 23, few works
centrifugation at 3000 rpm during 10 minutes 19. The decolorization indicate the presence of photosynthetic algae in water charged
experiments were performed at the same time. Aliquot was with color because of the weak penetration of solar rays. Indeed,
centrifuged at 5000 rpm during 10 minutes in order to precipitate we often isolated microalgae in textile industrial wastewater after
the cell mass 20 and the supernatants were evaluated at respective treatment from the SC.
maximum absorbance (λmax) of the tested dyes. The percentage
of decolorization was calculated as following % decolorization = Optimal conditions of algal cells growth: The optimal culture
[(Absorbance at t0) - (Absorbance at t1)]/ (Absorbance at t0) × conditions of the green unicellular alga growth were temperature
100. At t1 the COD and color absorbance were measured after five 25-28°C, pH 8.0, salinity at 15 g L-1 equivalent to 0.25 M of NaCl
days. (Fig. 3A-C). Growth of alga cells depends on temperature range,

Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009 701
0.8 0.16
(A)
0.7 0.14

Absorbance at 665 nm
Relative absorption

0.6 0.12
0.5 0.10
0.4 0.08
0.3 0.06
0.2 0.04
0.1 0.02
0 0
0 4 8 2 4 8 0 6 0 4 20 25 30 35 40
40 42 44 47 496 520 54 56 592 616 64 664 688 712 73 76 78
Temperature (°C)
Wavelenght at nm
Figure 2. UV-visible profile of total pigment extracted by methanol 0.6
solvent from algae isolated from textile wastewater plant. The peaks at

Absorbance at 665 nm
0.5 (B)
420-460 nm are corresponding to carotenoid and the peaks located in
400-500 nm and 600-700 nm to Chl a with Chl b. 0.4

0.3
pH and salinity concentration. Optimal pH for the alga growth is
8; this result explains in partly its presence in SITEX textile 0.2
wastewater, because the average value of the pH is 7.9 (Table 1). 0.1
Sodium chloride is also a key growth factor in medium for microalgae.
0
The isolated green alga may be qualified as halotolerant species; 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
cells may grow at 2-2.5% of NaCl. These culture conditions confirm pH
the previous reports. Indeed, Häubner et al. 24 demonstrated that 0.35
selected aeroterrestrial microalgal strains grew between 1 and 30°C (C)
Absorbance at 665 nm

with optimum rates at 20-23°C. Optimal pH has been reported at 0.30


7±0.2. Chlorella vulgaris grows significantly at a high pH (8.0), 0.25
populations of Chlorella vulgaris co-immobilized with 0.20
Azospirillum brasilense grow more remarkably compared to their
0.15
growth at a lower pH. Furthermore, salinity improved microalgae
growth. Salt tolerance of algae Dunaliella tertiolecta has been 0.10
reported to be in the range of 0.17-1.5 M of NaCl 25. However, 0.05
other investigators working on Dunaliella tertiolecta have gone 0
far beyond these limits by quoting a NaCl tolerance range of 0.05- 0 5 10 15 20 25 30 35 40
Concentration of NaCl gL -1
3 M 26. According to the results obtained, we could draw that this
unicellular green alga belongs to Chlorella genus. The optimal
Figure 3. Growth curves of green alga at different conditions of (A)
values (pH, salinity and temperature) obtained for alga growth temperatures, (B) pH and (C) salt concentrations.
resemble to the physicochemical parameters of the textile
wastewater plant from which alga cells were isolated. In this case,
dyes). So, little concentration of dyes has negative effect on
alga can be considered as acclimated.
Chlorella cells growth. Nevertheless, the industrial dyes effect
was different from one type to another. Indeed, growth of
Effects of green alga on textile dyes: Using these optimized
isolated alga decreased significantly with crystal violet, remazol
parameters, we have studied the effect of industrial dyes on
brilliant orange and direct blue compared to indigo dye as
the growth of isolated green microalga in Chlorella broth. The
shown in Fig. 4. While limited growth of green microalga was
growth curves of alga cells (Fig. 4) shows that alga growth was
accompanied by COD reduction (Fig. 5), the high percentage
remarkably reduced compared to the control group (alga without
of COD removal for direct blue was 60.1% and 46.7% for indigo.
The percentage of decolorization for tested dyes (Fig. 6),
Table 1. Characteristics of the secondary clarifier of showed that alga color removal efficiency values varied from
industrial Tunisian textile plant. dye to another, indigo (89.3%); direct blue (79%); remazol
brilliant orange (75.3%) and crystal violet (72.5%). Results
Parameter Range Mean value showed that isolated alga contribute to remove color with a
T (°C) 20-32 27 significant reduction of COD in different samples of industrial
pH 7.7-8.1 7.9 textile dyes. The active role of isolated alga belonging to
SS (mg L-1) 9-35 18 Chlorella genus, to reduce the pollution of indigo dye may be
COD (mg L-1) 120-240 161 due to the acclimatization of alga since it was isolated from an
BOD5 (mg L-1) 12-40 21 effluent charged in indigo dye. Indeed, several groups showed
Salinity (mg L-1) 4916-7432 6432 that microalgae may serve as a solution to emerging
Unit Color Pt-co 67-225 147 environmental problems such as greenhouse effect and waste
SS suspended solids, BOD5 Biochemical oxygen demand during 5 days, COD Chemical
treatments 27-29. Biological remediation by unicellular green
oxygen demand, T Temperature. microalga was different from one dye to another; this may be

702 Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009
attributed to the adsorption or/and to the biodegradation by 100
microalgae. Consequently, the dye chemical structure, the 90

% Average of decolorization
pollutant charge induced by each dye and the acclimated period 80
were very important to enhance bioremediation as demonstrated 70
by Acuner and Dilek 30. However, biodegradation depends 60
strongly on the nature and properties of the molecule 50
considered; properties in turn depend on molecule/environment 40
interaction 31. The color removal of azo-dyes, such as remazol 30
brilliant orange, was related to the presence of an enzyme; in 20
fact several works reported that Chlorella and Oscillatoria 10
algae both have been demonstrated to reduce azo dyes through 0
the activity of unspecific, soluble cytoplasmic reductases IND Rbo D.B. CV
known as azo reductases. These enzymes convert azo dyes Dyes
into aromatic amines leading to a decrease of environmental Figure 6. Percentage of decolorization of textile dyes by green alga.
rates 32, 33. Microalga could contribute to a valuable procedure
that might be associated with pre-existing treatment methods23. Conclusions
Thus, Peralta-Zamora et al. 34 showed that aerobic and/or The present study showed that green microalga may grow in textile
anaerobic procedures offer an extensive range of valid choices wastewater plant in spite of the high polluted charge of dyes and
for improving industrial wastewater. can be cultivated in media like bacteria. The results of optimal
conditions indicate that microalga was naturally acclimated and
contribute to reduce dyes pollution with efficiency values. Indeed,
biological treatment by activated sludge is better to reduce
chemical pollution from textile industry because it is inspired from
0.30 natural system of purification. Photosynthetic microalgae may be
0.25
introduced as a secondary stage for industrial textile effluent
Absorbance at 665 nm

treatment. Finally, for our study it is very interesting to realize


0.20 molecular and cytological analysis in order to determine exactly
the kind and the species of the isolated alga.
0.15

0.10 Acknowledgements
This study was supported by operating grants from “SITEX”
0.05
society. Many thanks to Professor Mahmoud Rouabhia, Faculté
0 de Médecine Dentaire, Bureau 1728, Université Laval
1 2 3 4 5 6 7 8
Incubation period (days) Québec, Qc, Canada, G1K 7P4.
Figure 4. Effect of dyes concentration (10 mg L-1) on green alga growth
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704 Journal of Food, Agriculture & Environment, Vol.7 (3&4), July-October 2009

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