RESEARCH ARTICLE

Solid-state fermentation: tool for bioremediation of adsorbed

textile dyestuff on distillery industry waste-yeast biomass
using isolated Bacillus cereus strain EBT1
Avinash A. Kadam & Jeevan D. Kamatkar &
Rahul V. Khandare & Jyoti P. Jadhav &
Sanjay P. Govindwar
Received: 8 March 2012 / Accepted: 10 April 2012 / Published online: 5 May 2012
#Springer-Verlag 2012
Abstract Bioremediation of textile dyestuffs under solid-
state fermentation (SSF) using industrial wastes as substrate
pose an economically feasible, promising, and eco-friendly
alternative. The purpose of this study was to adsorb Red M5B
dye, a sample of dyes mixture and a real textile effluent on
distillery industry waste-yeast biomass (DIW-YB) and its
further bioremediation using Bacillus cereus EBT1 under
SSF. Textile dyestuffs were allowed to adsorb on DIW-YB.
DIW-YB adsorbed dyestuffs were decolorized under SSF by
using B. cereus. Enzyme analysis was carried out to ensure
decolorization of Red M5B. Metabolites after dye degradation
were analyzed using UVVis spectroscopy, FTIR, HPLC, and
GC-MS. DIW-YB showed adsorption of Red M5B, dyes
mixture and a textile wastewater sample up to 87, 70, and
81 %, respectively. DIW-YB adsorbed Red M5B was decol-
orized up to 98 % by B. cereus in 36 h. Whereas B. cereus
could effectively reduce American Dye Manufacture Institute
value from DIW-YB adsorbed mixture of textile dyes and
textile wastewater up to 70 and 100 %, respectively. Induction
of extracellular enzymes such as laccase and azoreductase
suggests their involvement in dye degradation. Repeated uti-
lization of DIW-YB showed consistent adsorption and ADMI
removal from textile wastewater up to seven cycles. HPLC
and FTIR analysis confirms the biodegradation of Red M5B.
GC-MS analysis revealed the formation of new metabolites.
B. cereus has potential to bioremediate adsorbed textile dye-
stuffs on DIW-YB. B. cereus along with DIW-YB showed
enhanced decolorization performance in tray bioreactor which
suggests its potential for large-scale treatment procedures.
Keywords Decolorization
.
Distillery industry waste-yeast
biomass
.
Bacillus cereus
.
Red M5B
.
Solid-state
fermentation
Introduction
Water is a source of life and energy. Pollution of water due
to rapid pace of industrialization, pollution and unplanned
urbanization is causing millions of people worldwide to
suffer for storage of fresh and clean drinking water (Bhatnagar
and Sillanpaa 2010). Dyes are responsible for addition of
beautiful colors to the life and belongs to an important class
of chemicals in many industrial processes, but the presence of
very small amounts of dyes in water (less than 1 ppmfor some
dyes) is highly visible and undesirable (Robinson et al. 2001)
as color is the first contaminant to be recognized in wastewa-
ter. Nowadays, due to their extensive use, they have become
an integral part of industrial wastewater. Inefficiency of the
dyeing processes, poor handling of spent effluent, and insuf-
ficient treatment of wastes of the dyestuff industries lead to the
contamination of soil and natural water bodies (Nigam et al.
1996). The azo dyes are prominently used in leather, food, and
cosmetic industries as well as for tattooing, printing, and most
importantly, in textile dyeing because of their chemical stabil-
ity and versatility. It is now established that the azo dyes
causes skin and eye irritations (Mittal et al. 2012a). Many
synthetic azo dyes and their metabolites are toxic, carcinogen-
ic, and mutagenic leading to potential health hazards to
Responsible editor: Vinod Kumar Gupta
A. A. Kadam
:
J. D. Kamatkar
:
R. V. Khandare
:
J. P. Jadhav
Department of Biotechnology, Shivaji University,
Kolhapur 416004, India
S. P. Govindwar (*)
Department of Biochemistry, Shivaji University,
Kolhapur 416004, India
e-mail: spg_biochem@unishivaji.ac.in
Environ Sci Pollut Res (2013) 20:10091020
DOI 10.1007/s11356-012-0929-6
mankind (Nilsson et al. 1993). Therefore, the treatment of
industrial effluents containing azo dyes becomes necessary
prior to their final discharge to the wastewater.
Treatment of textile effluents is very important as they are
characterised by high BOD, COD, color, pH, and presence
of metals (Senan and Abraham 2004; Phugare et al. 2010).
Current technologies applied by textile industries to treat the
effluents mostly rely on physical and chemical methods.
However, the use of these methods have certain drawbacks
of being economically unfeasible, having more energy con-
sumption and involving the use of a large amount of chem-
icals and they still failed to remove recalcitrant azo dyes
completely, generating a significant amount of sludge that
may cause secondary pollution problems and may involve
complicated procedures (Zhang et al. 2004). Photochemical
and electrochemical methods have also been proposed for
dye removal (Gupta et al. 2007a, b). Among various textile
wastewater treatment technologies, adsorption is a fast, in-
expensive and universal method (Gupta et al. 2006a, 2007c;
Ali and Gupta 2007; Mittal et al. 2007). It is the most
economical method as it utilizes low-cost adsorption mate-
rials for removal of environmental pollutants like textile
dyes (Mittal 2006; Mittal and Kurup 2006; Gupta et al.
2006b, c, 2007d; Mittal et al. 2009a, b, 2010a, b, c, d,
2012a, b; Mittal and Gupta 2010), toxic heavy metals
(Gupta et al. 1997, 1998, 2010; Gupta and Sharma 2003;
Gupta and Rastogi 2008a, b, 2009), fluorides (Gupta et al.
2007e), and organochlorine pestisides (Gupta and Ali 2008).
Membrane-based metal electrode ion sensors have also been
proposed for the detection of pharmaceuticals and metal
analytes from wastewater (Srivastava et al. 1995; Jain et
al. 1995, 1997; Gupta et al. 2009a). Wood saw dust has been
used as effective adsorbent for removal of Basic Red 46 and
Basic Yellow 28 from textile wastewater (Laasri et al. 2007).
Agricultural wastes like rice and wheat husk and de-oiled
soya have been used for removal of Safranin T, Reactofix
Golden Yellow 3 RFN, Brilliant Blue FCF and Acid Orange
7 dye from waste water (Gupta et al. 2006d, 2009b). Indus-
trial waste like bottom ash was used for the adsorption of
carmoisine A (Gupta et al. 2009c). Similarly, distillery in-
dustry waste-yeast biomass (DIW-YB) may serve as a low-
cost adsorbent for textile dyes and such adsorbed dyes can
be further remediated under SSF.
There are 600 sugarcane factories in India which gener-
ates 7.5 million tons of molasses as waste per year. This
molasses is further used in distillery industry as raw mate-
rial. There are 257 distillery industries in India which pro-
duce 1.5 billion liters of alcohol and generate 1011 billion
liters of wastewater annually (Kumar 2006). Large amounts
of raw yeast biomass are generated after distillation process
which remains as solid waste/sludge (Kumar et al. 2003).
Utilization of such waste-yeast biomass as solid-state sub-
strate for dye adsorption and decolorization is economical
and has engineering advantages over conventional sub-
merged fermentations (Phugare et al. 2010). Waste-yeast
biomass has been exploited for the biosorption of lead,
cadmium, and uranium (Riordan et al. 1997; Gksungur et
al. 2005).
Most of the studies on microbial dye decolorization were
carried out using the submerged culture conditions (Dawkar
et al. 2008; Telke et al. 2009; Kurade et al. 2011). Looking
at the huge volumes of textile wastewater generated per day,
it is not practically feasible to apply submerged culture
conditions. Hence, to overcome this problem solid-state
fermentation (SSF) was preferred (Murugesan et al. 2007).
In addition, this technique allows the utilization of diverse
agro-industrial wastes as support-substrate, making the pro-
cess more economical and eco-friendly. SSF, defined as the
fermentation of solids in the absence of free water, has the
advantage of supporting the growth and metabolism of
microorganisms under moist conditions (Pandey 2003).
SSF reflects the microbial growth in its natural condition,
while liquid cultures do not. Solid-state processes are be-
coming more popular due to their advantages over to the
submerged cultures, such as higher productivity, simpler
operation, and lower costs. Very few reports were available
for decolorization of dye under SSF (Papinutti et al. 2006;
Kadam et al. 2011).
This work reports the adsorption of textile dyestuff on
DIW-YB and the decolorization of adsorbed textile dyestuff
by B. cereus strain EBT1 under SSF conditions.
Materials and methods
Dyestuff and chemicals
All chemicals were of highest purity and of an analytical
grade. L-tyrosine, n-propanol, Methyl red and microbiological
media such as, nutrient broth were obtained from Hi-media
laboratory, India. 2,2-Azino-bis (3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS) was obtained from Sigma Aldrich (St.
Louis, MO, USA), and 2,6-dichlorophenolendophenol from
Sisco Research Laboratory, India. The textile dyes and highly
colored effluent of the textile processing industry were
obtained from local textile mill, Ichalkaranji, India. The mix-
ture of textile dyes was prepared by adding various textile
dyes (each 20 mg L
1
) such as Remazol Orange, Disperse
Brown, Solo Red 5B, Golden Yellow HE2R, Direct Red 5B,
Rubin GFL, Remazol Red R, Yellow HE4G, Solo Black BR
and Red M5B.
Preparation of DIW-YB substrate for SSF
DIW-YB was obtained from local distillery industry, Kolha-
pur, India. It was washed with tap water. pH was adjusted to
1010 Environ Sci Pollut Res (2013) 20:10091020
6.67.0 and then dried in sunlight. Dried DIW-YB grinded
in mortar and pestle to obtain dry uniform powder and
preserved in desiccators for further use.
Isolation, screening, and identification of DIW-YB adsorbed
textile dye degrading microorganism
Five grams of DIW-YB was taken in 250-mL Erlenmeyer
flasks and 50 mL solution of the dye Red M5B
(100 mg L
1
) was added. The supernatant was separated
by centrifugation at 2,851g for 15 min. The flasks contain-
ing dye-adsorbed DIW-YB were autoclaved and inoculated
with 0.5 g of soil sample collected from the waste disposal
site of textile processing and dye manufacturing units in and
around Ichalkaranji (India). These flasks were incubated at
302 C at static and shaking condition. From the flask
showing decolorization at static condition, 0.5 g of decolor-
ized medium was serially diluted and poured on nutrient
agar plates by four quadrant spread plate technique. The
isolated pure colonies were screened for decolorization of
DIW-YB adsorbed Red M5B. The colony showing decolor-
ization consistently was selected and transferred on nutrient
agar (in grams per liter; peptone 10, sodium chloride 10 and
beef extract 2) slants and stored at 4 C for further use.
The isolate was identified using 16S rRNA sequence
analysis. 16S rRNA gene sequencing of isolated bacteria
was carried out at Bangalore Genei, Bangalore, India. The
nucleotide sequence alignment of the sequence was done at
Blast-n site at NCBI server (http://www.ncbi.nlm.nih.gov/
BLAST). The alignment of the sequences was done by
using CLUSTALW program V1.82 at European bioinfor-
matics site (http://www.ebi.ac.uk/clustalw). The Phyloge-
netic tree was constructed using the aligned sequences by
the neighbor-joining method using Kimura-2-parameter dis-
tances in MEGA 4 software. The 16 rRNA sequence was
deposited in Genbank databases.
Adsorption of textile dyestuff
Adsorption of Red M5B on DIW-YB was performed in a
batch technique. Five grams of DIW-YB was taken in 250-
mL Erlenmeyer flasks and 50 mL solution of the dye Red
M5B (100 mg L
1
) was added. This mixture was kept at
shaking condition at 120 rpm for 20 min. The clear super-
natant was collected by centrifuging the medium at 2,851g
for 15 min. The intensity of color was measured at maxi-
mum absorbance wavelength (520 nm) of Red M5B using
spectrophotometer (Hitachi U-2800 Japan). A similar pro-
cess was used for various textile dyes, mixture of textile
dyes and textile effluent. The percentage of dye adsorbed
was calculated from the following equation.
%Adsorbed dye A
0
A =A
0
100
Where, A
0
is the absorbance of sample before addition of
the DIW-YB and A is the absorbance of sample after ad-
sorption on DIW-YB. The characteristics of the textile ef-
fluent such as biological oxygen demand (BOD), chemical
oxygen demand (COD), alkalinity, hardness, total dissolved
solids (TDS) and total suspended solids (TSS) were carried
out at before and after the adsorption in accordance with
(APHA 1998). The dye-adsorbed DIW-YB was used further
for decolorization study.
Inoculums development and decolorization experiment
One loop full of bacterial culture was inoculated into 1.5 mL
nutrient broth (in grams per liter; peptone 10, sodium chlo-
ride 10, and beef extract 2) and incubated at 30 C under
static condition. Textile dye Red M5B adsorbed DIW-YB
(5 g) was added into 250-mL Erlenmeyer flasks. These
flasks were sterilized after pH adjustment (7.5 to 8). The
flasks were inoculated with 1.5 mL of log phase culture of
B. cereus for decolorization study. The moisture content of
the decolorization medium was maintained between 65 and
70 %. All flasks were incubated at 30 C under static and
shaking (120 rpm) conditions. A similar process was used
for various textile dyes, mixture of textile dyes, and textile
effluent. In order to measure the decolorization, textile dyes
adsorbed on DIW-YB were desorbed by dimethyl sulfoxide
(DMSO; Kadam et al. 2011). DMSO (50 mL) was added
into a 250-mL Erlenmeyer flask containing 5 g of dye-
adsorbed DIW-YB and kept under shaking condition
(120 rpm) for 20 min. The solution was filtered through
Whatman filter paper no. 42 and filtered solution was then
centrifuged at 2,851g for 10 min. The clear supernatant
was used for color measurement and the intensity of color was
measured at maximum absorbance wavelength of respective
dyes using spectrophotometer (Hitachi U-2800 Japan). Decol-
orization Percentage was calculated using initial and final
absorbance measerments.
To measure the decolorization of mixture of textile dyes
and textile effluent the % ADMI removal values were mea-
sured. The characteristics of mixture of dyes and textile
effluent are highly erratic in both, the hues and the concen-
tration of color. Being a complex mixture of dyes, it did
not show well-defined peaks in the visible region hence
the decolorization of the mixture of dyes was determined in
terms of the ADMI value (Kurade et al. 2011). The ADMI
removal ratio was calculated initial and final ADMI
values (APHA 1998). The ADMI [three wavelengths
(590, 540, and 438 nm); NY, USA] tristimulus filter method
has been used to measure decolorization of mixture of
dyes and textile effluent (Kurade et al. 2011). All de-
colorization experiments were performed in three sets.
Abiotic (without microorganism) controls were always
included.
Environ Sci Pollut Res (2013) 20:10091020 1011
Optimization of pH, temperature, and various carbon
and nitrogen sources
To optimize the pH for decolorization of adsorbed Red M5B
by B. cereus, the pH of the medium was adjusted to 2, 4, 6,
8, and 10 then the flasks were inoculated with B. cereus and
incubated at 30 C at static condition. pH measurement was
done with a Horiba pH meter (M13, Japan). The optimum
temperature was studied at different temperatures such as
20, 30, 40, and 50 C at static condition by keeping the
pH 8. The effect of various carbon and nitrogen sources on
decolorization of adsorbed Red M5B was studied as the
DIW-YB was supplemented with 3 mL of 1 % glucose,
starch, beef extract, ammonium chloride (NH
4
Cl), urea,
peptone, maize bran, bagasse, and lactose.
Repeated utilization of DIW-YB for textile dyes adsorption
and decolorization
After complete decolorization of dyes adsorbed on DIW-YB
by B. cereus strain EBT1, sterilized 50 mL solution of
textile effluent was aseptically added to decolorized medium
and kept for shaking at 120 rpm for 20 min. Then, it was
centrifuged at 5,587g for 15 min at 4 C. The intensity of
color of clear supernatant was measured at maximum ab-
sorbance wavelength of textile effluent to determine adsorp-
tion. The pellets containing dyes adsorbed on DIW-YB in
which B. cereus was grown on DIW-YB during first decolor-
ization cycle was taken as inoculum for next decolorization
cycle. The pellet was then taken aseptically into 250-mL
Erlenmeyer flask and incubated at 30 C at static condition
to study decolorization and the time required for decoloriza-
tion was measured. Only a 0.1 g of sample was removed
aseptically from decolorization medium and abiotic control
and 2 mL of DMSO was added. The solution was filtered
through Whatman filter paper No. 42 and then centri-
fuged at 2,851g for 10 min. The clear supernatant was
used for measurement of decolorization. This process was
repeated for number of cycles up to which it is capable of
decolorization.
Enzyme studies during decolorization
The culture medium were added with 20 mL of sodium
phosphate buffer (20 mM, pH 7.0) and then kept under shak-
ing condition (120 rpm) for half an hour at 30 C. The medium
was filtered using muslin cloth and then centrifuged at
5,587g for 10 min at 30 C. The clear supernatant was used
as source of extracellular enzymes (Kadam et al. 2011).
Extracellular supernatant was used to determine all en-
zyme activities at room temperature (25 C). Laccase activ-
ity was determined in a reaction mixture of 2 mL containing
10 % ABTS in 20 mM potassium phosphate buffer (pH 4.0)
and increase in the optical density was measured at 420 nm.
Molar extinction coefficient of ABTS was 0.036 M
1
cm
1
at 420 nm (Telke et al. 2010). Lignin peroxidase activity
was determined by monitoring the formation of propanalde-
hyde at 300 nm in a 2.5-mL reaction mixture containing
100 mM n-propanol, 250 mM tartaric acid, 10 mM H
2
O
2
(Jadhav and Govindwar 2006). Tyrosinase activity was de-
termined in a reaction mixture (3.0 mL) containing 2.5 mL
of sodium acetate buffer (20 mM, pH 4.0) and 100 M of L-
tyrosine. The reaction was started by adding 0.2 mL of en-
zyme solution and increase in absorbance was measured at
280 nm (Duckworth and Coleman 1970). One unit of enzyme
activity was defined as the amount of enzyme required for an
increase in 1.0 ABS unit min
1
under assay condition.
Azoreductase assay mixture (2.0 mL) contained 4.45 M
of Methyl red, 50 mM potassium phosphate buffer (pH 7.5)
and 0.2 mL of enzyme solution. The reaction was started by
adding 100 M of NADH and then monitored for the
decrease in the color absorbance at 430 nm. The molar
extinction coefficient of Methyl red was 0.023 M
1
cm
1
at 430 nm (Chen et al. 2005). NADH-DCIP reductase assay
mixture contained 25 M DCIP, 50 mM potassium phos-
phate buffer (pH 7.5) and 0.2 mL of enzyme solution in a
total volume of 5.0 mL. The reaction was started by adding
100 M of NADH. The decrease in color intensity was
measured at 595 nm. The molar extinction coefficient of
DCIP was 0.021 M
1
cm
1
at 595 nm (Salokhe and
Govindwar 1999).
Tray bioreactor study for textile wastewater decolorization
DIW-YB (500 g) was added in 5 L of the textile effluent for
adsorption of dyes with continuous stirring. The solid slurry
of dyes adsorbed DIW-YB was separated by allowing it to
settle for 23 h. The unautoclaved DIW-YB spread uniform-
ly in the tray, having dimensions of 48 cm33 cm (length
width) and thickness 3.5 mm. The above procedure was
used for preparation of control and test trays. The test tray
was inoculated with 50 mL of 24 h grown B. cereus culture
and incubated at room temperature and non-sterile condi-
tions. A 2-g sample was removed after 4 h interval from
inoculated test tray and control tray (without inoculation)
and extracted with 20 ml DMSO for color measurement. All
experiments were carried out at non-sterile conditions and at
room temperature. Design of continuous flow packed-bed
bioreactor for acid wastewater decolorization by fungus
under SSF on agro-industrial waste was reported earlier by
(Landolo et al. 2011).
Metabolites analysis after degradation
After desorption of the samples which were obtained after
the complete decolorization of adsorbed textile dyestuff,
1012 Environ Sci Pollut Res (2013) 20:10091020
they were scanned between 400 and 800 nm using spectro-
photometer (Hitachi U-2800 Japan), for qualitative analysis
of decolorization. The decolorized medium was added with
100 mL of distilled water and kept at shaking condition
120 rpm for 1 h. The medium was centrifuged at 11,404g
for 20 min. The supernatant obtained was used to extract
metabolites with an equal volume of ethyl acetate and ex-
tract was then evaporated in vacuum over anhydrous
Na
2
SO
4
and dried (Kadam et al. 2011). High-performance
liquid chromatography (HPLC) analysis (Waters model no.
2690, USA) was carried out with C
18
column (symmetry,
4.6250 mm) using isocratic method with 10 min run time.
The mobile phase used was HPLC-grade methanol with a
flow rate 0.50 mL min
.
1
The Fourier-transform infrared
spectroscopy (FTIR; Perkin Elmer Spectrum one, USA)
analysis was carried out in the mid IR region of 450
4,000 cm
1
with 16 scan speed. The GC-MS analysis of
metabolites was carried out using MS Engine (Shimadzu
QP2010 Japan) equipped with integrated gas chromatograph
with a HP1 column (60 m long, 0.25 mm id, nonpolar).
Helium was used as carrier gas at a flow rate of 1 mL min
1
.
The temperature was maintained at 280 C with oven con-
ditions as: 80 C kept constant for 2 minincreased up to
200 Cwith 10 Cmin
1
raised up to 280 Cwith 20 Cmin
1
rate. The compounds were identified on the basis of mass
spectra and using the NIST library.
Phytotoxicity study
Phytotoxicity tests were performed in order to study the
toxicity effects of Red M5B and its degradation products
at the concentration of 500 ppm (Telke et al. 2009). Sorghum
vulgare (monocot) and Phaseolus mungo (dicot) were select-
ed for toxicological analysis. The phytotoxicity study was
carried out at room temperature (ten seeds of each) by water-
ing separately 5 mL sample of control Red M5B and its
degradation product per day. Control set was carried out using
water at the same time. Lengths of plumules (shoot) and
radicles (root) were recorded on the 7th day and germination
percentage was calculated.
Statistical analysis
Data were analyzed by one-way analysis of variance
(ANOVA) with TukeyKramer multiple comparison test.
Results and discussions
Isolation and identification of microorganism
The isolated bacterium was able to decolorize the textile dye
Red M5B. 16S rRNA sequencing (1,355 bp) from the
isolated bacterial strain and phylogenic analysis (Fig. 1)
showed its close relationship to Bacillus cereus hence, it
was named as Bacillus cereus strain EBT1. 16S rRNA
sequence was deposited in GenBank database with the ac-
cession number JF750734.
Adsorption of dyes on DIW-YB
Adsorption of textile dyestuff on various agricultural wastes
and domestic wastes has been extensively studied (Bhatnagar
and Sillanpaa 2010). Saccharomyces cerevisiae cells were
used for the recovery of textile dyes by adsorption from
wastewater effluent (Polman and Breckenridge 1996). S. cer-
evisiae cells also showed bioaccumulation of reactive textile
dyes namely Remazol Blue, Remazol Black B and Remazol
Red RB during growth in molasses (Aksu 2003). Yeast cells
have also been reported for dye adsorption and decolorization
(Jadhav and Govindwar 2006) but no reports are available for
the decolorization of adsorbed textile dyes on distillery waste-
yeast biomass using bacteria. DIW-YB showed 87, 84 and
81 % adsorption of the textile dye Red M5B, a mixture of
textile dyes and a textile industry wastewater, respectively
(Table 1). The BOD, COD, alkalinity, hardness, TDS and
TSS of the textile effluent before adsorption were found to
be 90, 6,160, 100, 100, 560, and 150 mg L
1
, respectively.
After adsorption all these characteristics of effluent showed
Fig. 1 Phylogenetic tree for isolated B. cereus strain EBT1 (neighbor-
joining tree). Scale bar number of nucleotide changes per sequence
position. The number at nodes shows the bootstrap values obtained
with 1,000 resampling analysis
Environ Sci Pollut Res (2013) 20:10091020 1013
54, 54, 75, 44, 44, and 80 % reduction, respectively. The
concurrent reduction of COD of the textile effluent while
using corn pith activated carbon as adsorbent was reported
earlier (Santhy and Selvapathy 2006). DIW-YB showed dif-
ferent adsorption capacities for various textile dyes namely
Remazol Orange, Disperse Brown, Solo Red 5B, Golden
yellow HE2R, Direct red 5B, Rubin GFL, Remazol Red R,
Yellow HE4G, and Solo Black BR which showed 20, 24, 80,
15, 77, 60, 46, 10, and 78 %adsorption, respectively (Table 1).
A large volume of yeast biomass is generated as a waste from
distillery industry, making it an advisable tool to be used as an
adsorbent for textile wastewater. The dye-adsorbed DIW-YB
was used for further decolorization study. Adsorption of dyes
has been known to be the most efficient method of dye
removal from wastewaters (Gupta et al. 2009a, b, c); but the
problem of persistence of dyes in the environment remains
unsolved. SSF could prove to be a potential tool to bioremedi-
ate these adsorbed dyes.
Decolorization of adsorbed textile dyestuff under SSF
B. cereus showed 98 % decolorization of textile dye Red
M5B within 36 h under SSF (Table 1). Textile industry
effluent generally contains the mixture of highly complex
dyes. Therefore, it is necessary to study the capacity of
microorganism for decolorization of mixture of dyes and
effluents. We evaluated the decolorization ability of mixture
of various industrial dyes such as; Remazol Orange, Dis-
perse Brown, Solo Red 5B, Golden Yellow HE2R, Direct
Red 5B, Rubin GFL, Remazol Red R, Yellow HE4G, Solo
Black BR and Red M5B at a concentration of 20 mg L
1
by
using B. cereus. The mixture of textile dyes and effluent had
initial ADMI values 8,725 and 1,886 which were reduced to
605 and 9.3, respectively, after the treatment by B. cereus
(Table 1). Bjerkandera adusta showed 53 % decolorization
of barley husk adsorbed textile effluent under SSF within
21 days (Robinson and Nigam 2008), whereas B. cereus
could decolorize textile effluent sample up to 99 % within
36 h. B. cereus also decolorized DIW-YB adsorbed dyes
such as Remazol Orange, Disperse Brown, Solo Red 5B,
Golden Yellow HE2R, Direct Red 5B, Rubin GFL, Remazol
Red R, Yellow HE4G and Solo Black BR up to 90, 92, 91,
83, 83, 91, 93, 78 and 82 %, respectively (Table 1). These
results suggest the bioremediation potential of B. cereus to
decolorize the adsorbed textile dyestuff from mixture of
textile dyes and textile industry wastewater under SSF.
SSF conditions do not only degrade the dyes but also keeps
the nutritional value of the dye-adsorbed waste enriched.
Hence, it can be used as manure in agricultural fields
(Robinson et al. 2001).
Optimization of pH, temperature, and various carbon
and nitrogen sources
Decolorization of Red M5B by B. cereus was found to be
maximum (98 %) at pH 8 with in 36 h. However, the
decolorization of Red M5B observed at pH 2, 4, 6, 10 were
22, 47, 60 and 97 % respectively, within 36 h (Fig. 2).
Maximum decolorization was found to be in alkaline con-
ditions. Textile effluents mostly have alkaline pH (Kumar
2006), therefore this study provides a direct protocol for
textile wastewater treatment. The optimum temperature for
decolorization of adsorbed textile dye Red M5B by B.
cereus was 30 C showing 98 % decolorization in 36 h.
However, decolorization at 20, 40 and 50 C was found to
be 33, 65 and 43 %, respectively (Fig. 2). Similar
Table 1 Decolorization of
DIW-YB adsorbed textile
dyestuffs by B. cereus
in 36 h
Values are mean of three
experiments
ND No decolorization
a
% ADMI removal ratio
Dyes max (nm) Adsorption (%) Decolorization (%)
Static Shaking
Red M5B (100 mg L
1
) 520 872.3 980.5 ND
Mixture of textile dyes
a
(20 mg L
1
each dye) 520 841.4 932.5 ND
Textile wastewater
a
540 812.7 990.1 ND
Various textile dyes (100 mg L
1
each dye)
Remazol Orange 500 201.5 902.0 ND
Disperse Brown 440 241.5 920.9 ND
Solo Red 5B 530 801.7 911.5 ND
Golden Yellow HE2R 440 151.2 831.2 ND
Direct Red 5B 500 771.5 831.2 ND
Rubin GFL 560 601.2 911.5 ND
Remazol Red R 500 461.2 930.9 ND
Yellow HE4G 440 101.2 781.6 ND
Solo Black BR 620 780.6 821.5 ND
1014 Environ Sci Pollut Res (2013) 20:10091020
observations were made during the decolorization of Scarlet
R (Saratale et al. 2009).
B. cereus showed 54 % decolorization of adsorbed Red
M5B in presence of control DIW-YB with in 18 h (Table 2)
at static conditions. Decolorization percentage was found to
be higher with nitrogen sources such as peptone (65 %) and
beef extract (62 %). However, using agricultural waste like
bagasse, carbon source as glucose, and nitrogen source as
ammonium chloride consistent decolorization (52 %) with
DIW-YB was observed. The decolorization was lowered up
to 35 % when carbon sources were lactose and starch
(Table 2). The organic nitrogen sources can regenerate
NADH, which acts as an electron donor for the reduction
of azo dyes (Hu 1994). These observations might help to
scale up the protocol for treatment of larger volumes of
textile effluents.
Repeated utilization of DIW-YB for textile dyes adsorption
and decolorization
The repeated use of dye degrading microorganism and sub-
strate DIW-YB for decolorization of dyes from textile
wastewater is important to enhance its commercial value.
Repeated addition of dye aliquots to access ability of mi-
croorganism for dye decolorization was studied earlier in
submerged fermentation conditions (Saratale et al. 2009).
This study was carried out to examine the ability of B.
cereus to decolorize repeatedly adsorbed dyes from textile
effluent at static conditions. In the first cycle, the DIW-YB
showed 81 % adsorption of dyes from textile effluent and
99.5 % decolorization of adsorbed textile dyes by B. cereus
in 36 h. In second and third cycle there was 77 and 75 %
adsorption and decolorization was 82 and 94 % in 36 and
24 h, respectively (Table 3). Further, the fourth, fifth, and
sixth cycles showed 73, 77, and 76 % adsorption and 83, 82,
Fig. 2 Effect of pH and temperature on decolorization of Red M5B at
static condition under SSF
Table 2 Effect of various carbon and nitrogen sources on the decol-
orization of adsorbed Red M5B by B. cereus strain EBT1 in 18 h
Various carbon and nitrogen sources Decolorization (%)
Control DIW-YB 540.66
DIW-YB + glucose 500.57
DIW-YB + starch 384.00
DIW-YB + NH
4
Cl 523.84
DIW-YB + urea 611.85
DIW-YB + lactose 320.88
DIW-YB + peptone 650.57
DIW-YB + beef extract 621.76
DIW-YB + rice bran 311.67
DIW-YB + bagasse 530.33
Values are mean of three experimentsSEM
Table 3 Repeated use of DIW-YB for adsorption of dyes from textile
effluent and then decolorization by B. cereus at static condition
Repeated
cycles
Adsorption
(%)
ADMI removal
ratio (%)
Time required for
decolorization in hours
Cycle 1 811.2 990.1 36
Cycle 2 770.9 820.3 36
Cycle 3 750.8 941.5 24
Cycle 4 731.5 830.6 20
Cycle 5 770.9 821.2 24
Cycle 6 761.2 831.5 24
Cycle 7 760.6 391.7 48
Values are mean of three experimentsSEM
Table 4 Extracellular enzyme status of medium in presence and
absence of adsorbed Red M5B during SSF using B. cereus
Enzymes Control Test
Laccase
a
ND 130.3
Tyrosinase
b
ND 0.10.1
Lignin peroxidase
b
4.60.1 0.10.1*
Azoreductase
c
4.40.1 110.2*
NADH-DCIP reductase
d
18.70.4 5.70.1*
Values are mean of three experimentsSEM
ND Not detected
*P<0.001; significantly different fromcontrol cells (by one-way analysis
of variance (ANOVA) with TukeyKramer multiple comparison test)
a
M of ABTS oxidized mL
1
min
1
b
Activity in U mL
1
min
1
c
M of Methyl red reduced mL
1
min
1
d
M of DCIP reduced mL
1
min
1
Environ Sci Pollut Res (2013) 20:10091020 1015
and 83 % decolorization in 20, 24, and 24 h, respectively
(Table 2). In the seventh cycle, the decolorization decreased
to 39 % which took 48 h (Table 3). This indicates cessation
of decolorization rate, which is likely to be due to nutrient
depletion and accumulation of toxicants in DIW-YB. Simi-
lar results were observed during decolorization of Scarlet R
(Saratale et al. 2009) in submerged fermentation conditions.
The use of same DIW-YB medium for adsorption and
decolorization repeatedly has given insights to develop
large-scale treatment protocols.
Enzyme activities
B. cereus, when grown in absence of Red M5B, did not
show laccase and tyrosinase activities; while in the presence
of Red M5B, these enzymes were found to be active (Table 4).
Induction in the enzyme activities in presence of the dye was
also reported earlier (Dawkar et al. 2008). In this study,
significant inductions in the activities of laccase, azo reduc-
tase, tyrosinase, and presence of enzyme activities of lignin
peroxidase and NADH-DCIP reductase suggest their role in
decolorization of Red M5B (Table 4). The role of oxidore-
ductases in decolorization of textile dyes has been reported
earlier (Jadhav and Govindwar 2006); all the enzymes assayed
are well known to have important role in the decolorization of
the textile dyes.
Tray bioreactor study
The DIW-YB showed 81 % adsorption of dyes from textile
effluent. The test bioreactor tray showed 70 and 91 % de-
colorization of adsorbed dye in 4 and 8 h respectively, while
control trays did not show any decolorization within this
time. After 12 h, control trays showed 42 % decolorization
Fig. 3 UVVisible spectrophotometric analysis of control dye Red
M5B (filled diamond) and after decolorization (filled upright triangle)
by B. cereus
Fig. 4 Biodegradation analysis as a HPLC pattern of Red M5B; b HPLC pattern of metabolites; c FTIR pattern of Red M5B; d FTIR pattern of
metabolites
1016 Environ Sci Pollut Res (2013) 20:10091020
and test tray showed 100 % decolorization. Complete de-
colorization in control trays required 20 h which suggests
that DIW-YB also has the decolorization ability similar to
earlier yeast decolorization reports (Jadhav and Govindwar
2006; Phugare et al. 2010). DIW-YB in submerged fermen-
tation conditions took 48 h for 69 % decolorization of textile
effluent as reported by Phugare et al. (2010). While, at SSF
conditions it showed 100 % decolorization within 12 h,
ensuring its economical feasibility with better decolorization
performance. The presence of B. cereus significantly re-
duced the time required for decolorization. Synergic action
of yeast and bacteria showed enhanced degradation of tex-
tile wastewater (Phugare et al. 2011). B. cereus along with
DIW-YB showed faster, efficient and effective decoloriza-
tion performance in bioreactor which suggests its potential
for large scale treatment procedures.
Fig. 5 Proposed pathway for biodegradation of textile dye Red M5B by B. cereus
Table 5 The phytotoxicity study of the dye Red M5B and metabolite obtained after its degradation
Parameters Phaseolus mungo Sorghum vulgare
Water Red M5B Product Water Red M5B Product
Germination (%) 100 30 80 100 40 90
Plumule (cm) 100.3 5.40.2** 8.30.2* 9.20.3 4.30.3** 100.1
Radical (cm) 6.50.2 3.50.3** 5.90.1 8.70.1 5.10.1** 100.1**
*P<0.01; **P<0.001; the values are significantly different from control (by one-way analysis of variance (ANOVA) with TukeyKramer
comparison test)
Values are mean of three experimentsSEM
Environ Sci Pollut Res (2013) 20:10091020 1017
Metabolite analysis after degradation
Red M5B showed maximum absorbance at 520 nm. The
sample obtained after decolorization showed less absor-
bance which suggests its 98 % decolorization (Fig. 3).
HPLC spectrum of Red M5B showed the peaks at retention
time 2.54 and 2.88 min (Fig. 4a). The metabolites obtained
after its degradation by B. cereus showed the peaks at
retention time 3.23, 3.48, 3.73, 4.26, and 4.60 min
(Fig. 4b). Thus, the difference in the retention times of
control dye Red M5B and metabolites formed after its
degradation by B. cereus confirmed the biodegradation of
Red M5B. Comparison of FTIR spectra of control dye and
the products formed after complete degradation revealed the
biodegradation of Red M5B by B. cereus. The spectrum of
control Red M5B showed the peaks at 3,430.5, 1,623.8,
1,533.9, 1,393.0, 1,114.8, 1,048.6, 847.4, 753.4, and
619.8 cm
1
which represents NH stretching, NN stretch-
ing as in azo compounds, NH deformation, CN stretch-
ing, OH deformation, COH stretching, SO stretching as
in sulfonic acid, CH stretching and CH deformation as in
benzene ring and CCl stretching, respectively (Fig. 4c).
Thus, differential spectra of the untreated and treated dye
confirmed its degradation (Fig. 4d). Absence of peak at
1,623.8 cm
1
in product spectra confirms removal of azo
bond during degradation which also supports the enzymatic
pattern of B. cereus. Removal of the peak at 1,048.6 and
619.8 cm
1
for SO stretching and CCl stretching in
product spectra confirmed desulfonation and dechlorination
of Red M5B.
A pathway of degradation of Red M5B by B. cereus was
proposed based on the basis of GC-MS analysis (Fig. 5). B.
cereus cleaved Red M5B asymmetrically and desulfonated
it to form [A] 8-amino-2-diazenylnaphthalen-1-ol [R
t
17.88 min, Mw (m/z), 187 (M-2)] and [I] which was an
unidentified compound which further undergoes dechlorina-
tion and hydroxylation to give [B] 1,3,5-triazine-2,4-diole
[R
t
20.76 min, Mw (m/z), 113 (M-3)] (Fig. 5). 8-amino-2-
diazenylnaphthalen-1-ol [A] further degraded to form [C]
naphthalene-2-amine [R
t
21.29 min, Mw (m/z), 143].
Phytotoxicity study
The textile dyeing effluents may cause serious environmen-
tal and health hazards if used for agriculture or disposed in
water bodies. Thus, phytotoxicity studies of dye Red M5B
were carried out before and after degradation to assess its
toxic nature. The germination percentage of seeds of the
both S. vulgare and P. mungo was 40 and 30 % in presence
of the Red M5B where as the germination percentage was
increased up to 90 and 80 %, respectively, in the presence of
metabolites. The shoot and root lengths of S. vulgare in
presence of Red M5B were significantly reduced as
compared to control plants in distilled water (Table 5). The
shoot and root lengths of S. vulgare and P. mungo in pres-
ence of degraded products of Red M5B were similar to
control plants indicating detoxification of textile sulfonated
azo dye Red M5B. These results confirm the significant
reduction in toxicity of Red M5B.
Conclusion
B. cereus has potential to decolorize adsorbed textile dyestuff
on DIW-YB under SSF. This developed system showed con-
sistent adsorption and decolorization of textile effluent up to
seven cycles. The extracellular oxidoreductase enzymes sug-
gest their role in decolorization of Red M5B. B. cereus along
with DIW-YB also showed improved decolorization in tray
bioreactors that gave an additional insight to treat textile
wastewater under SSF, which offers an economically feasible
and eco-friendly approach. SSF using DIW-YB gives a dual
solution for solid waste management of distillery industry as
well as for the treatment of textile wastewaters.
Recommendations and perspectives
The DIW-YB serves as a low-cost adsorbent for removal of
dyes from textile wastewater. B. cereus has potential bio-
remediator for adsorbed dyes under SSF. Tray bioreactor
study provides a tool for textile wastewater treatment at
large scale in SSF conditions.
Acknowledgments The author Avinash A. Kadam is thankful to
Department of Science and Technology, New Delhi for providing the
DST-PURSE fellowship.
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