Research Article

Biodegradation of Leather
Acid dye by Bacillus subtilis
Gurulakshmi. M, Sudarmani. D.N.P and Venba. R.

Although decolorization, under anaerobic

Abstract
conditions generally cannot realize the
The Bacillus subtilis was used to decolorize the Acidblue113. The bacterial culture exhibited complete mineralization of azo dyes,
90% decolorization ability within 50 h. Maximum rate of decolorization was observed (90%) aromatic amines as decolorized products are
when starch & peptone was supplemented in the medium. Decolorization of Acidblue113 was usually more susceptible to oxygenase attack.
monitored by TLC, which indicated that dye decolorization was due to its degradation into Thus, bacterial mineralization of azo dyes
unidentified intermediates. The optimum dye decolorizing activity of the culture was observed generally takes two steps:
at pH 7.0 and incubation temperature of 370C. Maximum, dye-decolorizing efficiency was
St e p 1 : Tw o m e c h a n i s m s f o r t h e
observed at 200 mg/l concentration of Acidblue113. A plate assay was performed for the
decolorization of azo dyes under anaerobic
detection of decolorizing ability of bacteria. Clearing zone (decolorization) was formed
conditions in bacterial systems have been
surrounding the bacterial culture. Decolorization was confirmed by UV-VIS
proposed [14]. The first one consists of direct
spectrophotometer. The initial dye solution showed high peak at the wavelength of 560nm.
electron transfer to azo dyes as terminal
The decolorized dye showed disappearance of peak, which indicated that the decolorization is
acceptors via enzymes during bacterial
due to dye degradation. The dye decolorization was further confirmed by COD & BOD
catabolism, connected the ATP generation
Analysis.
(energy conservation). The second one
Key words: Biodegradation, Acid Blue 113, Bushnell & Hass medium (BHM) and Bacillus involves a free reduction of azo dyes by the
subtilis end products of bacterial catabolism, not
linked to ATP generation (eg., reduction of the
azo bond by reduced inorganic compounds,
Introduction and degradation is an environmentally
such as Fe2+ or H2S, that are formed as the
friendly and cost competitive alternative to
Dyes are widely used in the Textile, rubber end product of certain anaerobic bacterial
chemical decomposition possess [4]
product, paper, printing, color photography, metabolic reactions). Figure 1 shows a
Unfortunately, most azo dyes are recalcitrant
Pharmaceuticals, Cosetics and Many other possible pathway for the degradation of azo
to aerobic degradation by bacterial cells [8].
industries. [1] Amongst these, azo dyes dyes under anaerobic conditions with whole
H o w e v e r, t h e r e a r e f e w k n o w n
represent the largest and most versatile class bacterial cells.
microorganisms that have the ability to
of synthetic dyes. [2] Approximately 10 - 15% reductively cleave azo bonds under aerobic Step 2: During anaerobic degradation, a
of the dyes are release into the environment conditions [9,10,11,12]. reduction of the azo bond in the molecules is
during manufacturing and usage. [3] Since observed. Then, aerobic conditions are
Compared with chemical/Physical methods,
some of the dyes are harmful, dye-containing required for the complete mineralization of
biological processes have received more
wastes pore an important environmental the reactive azo dye molecule. The aromatic
interest because of their cost effectiveness,
problem. [4] These dyes are poorly compounds produced by the initial reduction
lower sludge production and environmental
biodegrabale because of their structures and are degraded via hydroxylation and opening
friendliness. Various wood-rotting fungi were
treatment of wastewater containg dyes in the process is necessary in which oxygen is
able to decolorize azo dyes using peroxidases
usually involves physical and / or chemical introduced after the initial anaerobic
or laccases. But fungal treatment of effluents
methods [5] such as adsorption, Coagulation- reduction of the azo bond has taken place. The
is usually time-consuming. Under static or
flocculation, Oxidation, filtration and optimum pH for colour removal is around pH
anaerobic conditions, bacterial decolorization
electrochemical methods [6] 7-7.5. The rate of colour removal tends to
generally demonstrates good color removal
Over the Past decades, Biological decrease rapidly under strongly acid or
effects. However, aerobic treatment of azo
decolorization has been investigated as a strongly alkaline conditions. The optimum
dyes with bacteria usually achieved low
method to transform, degrade or mineralize cell culture growth temperature is between 35
efficiencies because oxygen is a more
azo dyes [7] Moreover, such decolorization and 45°C.
efficient electron acceptor than azo dyes [13].

12 | Advanced Biotech | November 2008


Fig 1- A proposed redox reduction for the degradation
of azo dye with whole bacterial cells
Azoreductase is the key enzyme expressed in
azodye-degrading bacteria that catalyses the
reductive cleavage of the azo bond.
Azoreductase activity has been identified in
several species of bacteria recently; such as
Caulobacter subvibrioides C7-D, Xenophilus
azovorans KF46F, Pigmentiphaga kullae
K24, Enterobacter agglomerans and
Enterococcus faecalis [15, 16,17,18,19].
Efforts to isolate bacterial cultures capable of
degrading azo dyes started in the 1970s with
reports of Bacillus subtilis [9], then
Aeromonas hydrophila (20) followed by
Bacillus cereus [21]. Numerous bacteria
capable of dye decolorization, either in pure
cultures or in consortia, have been reported
[22, 7, 23,12,14,4].
In the course of our study on the
biodegradation of Leather dye. We have
found that Bacillus subtilis are capable of
degrading C.I. Acid Blue 113 (C.I. No.
26360). To the best of our knowledge, no
other microorganism is reported to
biodegrade Acid Blue 113. This paper
describes the degradation of Acid Blue 113 by
Bacillus subtilis and shows a plausible initial
pathway for the biodegradation of Acid Blue
113. We also report the optimization of
parameters required for the dyes efficiently in
a short period.
Materials and Methods:
Chemicals: Acid blue 113 Dye (Figure 2) and
the isolates (Bacillus subtilis) used in this
study were kindly provided by the Tannery
Division, CLRI, Chennai. All other reagents
used were of analytical grade.
Fig- 2 Structure of Acid blue 113
13 | Advanced Biotech | November 2008
Medium:
The Bacillus subtilis culture was routinely
grown at 37°C in the basal culture medium,
Bushnell and Hass medium (BHM)
containing the following in g/l, MgSo4 0.2,
CaCl2 0.02, KH2PO4 1.0, K2HPO4 1.0,
NH4No3 1.0, FeCl3 0.05, Glucose 0.9, Yeast
extract 0.9, Acid blue113-100mg
Measurement of dye concentration:
The dye concentrations were measured with a
UV/VIS spectrophotometer (HITACHI-
U.2000-Spectrophotometer) at regular
intervals during the decolorisation process.
The concentration of azo dye was detected
spectrophotometrically by reading the culture
supernatant at its specific max after
centrifugation at 10,000 rpm for 10 min.
(Superspin R-VIFm plasto crafts). The dye
concentrations were determined from the
attenuance (O.D) of the culture at 533 nm.
Decolorization activity was calculated as
follows:
Initial absorbance - Observed absorbance
Decolorization(%) =
Initial absorbance
Study of Physico-Chemical Parameters
Decolorization was studied using various Co-
substrates (starch & peptone, sucrose, starch
& yeast extract, sucrose &yeast extract,
Dextrose & yeast extract) and at different dye
concentrations (100-500 mg/l), inoculum size
(5, 10, 15, 20, 25, & 30% (v/v), pH (5-8),
Temperature (20-50oC), and at different
culture conditions under Agitation and
stationary conditions.
Plate Assay
Plate assay was performed for the detection of
decolourizing activity of bacteria. The
nutrient agar and Acid blue 113 dye was
autoclaved at 121oC for 15 minutes. Bacillus
subtilis culture was plated on nutrient agar
plates containing Acid blue (500mg/l). The
plates were wrapped with parafilm and were
incubated at 37oC for 7days. The plates were
observed for clearance of the surrounding the
colonies.
Analysis of UV/ Visible
Spectrophotometer
Under static conditions, the culture with an
initial dye (Acid blue 113) concentration of
Research Article
10% (v/v) was 90% decolorized in 50 hours.
UV/Visible spectra of culture supernatants of
0 hour and 50 hours were compared and
possible degradation products were
speculated.
Estimation Of Chemical Oxygen Demand
(COD)
Chemical oxygen demand was measured by
the standard Potassium dichromate method.
1ml of initial medium containing dye
solution, decolorized medium, distilled water
was added to COD Tube sample 1, sample 2,
Blank respectively. Then 1.5ml of distilled
water & reducing agent potassium
dichromate and 3.5ml COD acid were added
to each tube. Duplicates were put up for all the
tubes. All the tubes were kept in the COD
incubator at 148oC for 2 hrs. After incubation
the entire content were transferred to a conical
flask. A drop of ferroin indicator was added to
it and was titrated against FAS in the burette.
The readings were noted
Volume of sample
A-volume of Ferrous Ammonium Sulphate
used for blank
B-volume of ferrous Ammonium Sulphate
used for sample
Equivalent weight of oxygen - 8
N-Normality of FAS - 0.1
COD values were compared between the
initial medium containing dye solution and
decolorized medium.
Estimation of Biological Oxygen Demand
(BOD)
1ml of initial medium containing dye
solution, decolorized medium, distilled water
was added to airtight BOD bottles sample 1,
sample 2, Blank respectively. Place desired
volume of water in a suitable bottle and add
1ml of each of Phosphate buffer, MgSO4,
FeCl3 and seeding/L of water. Before use
bring dilution water temperature to 200C.
Dilution water was aerated with organic free
filtered air. All the bottles are kept
in BOD incubator at 200C for 5 days.
After incubation 1ml of MnSO4, Alkali iodide
solution and sulphuric acid was added to form
brown color solution. After color formation
 Research Article
they were titrated against their Na2SO4for were similar to those studies on E. coli NO3
their BOD values. The readings were noted. and Pseudomonas luteola [27].
B-volume of Na2SO3 used for blank
T (v)-volume of Na2So3used for sample
S (v)-volume of sample
BOD values were compared between the
initial medium containing dye solution and
decolourised medium.
Fig-3 Showing the decolorization of
Thin Layer Chromatography (TLC)
Acid blue113 by Bacillus subtilis
Degradation of dye, Acid blue 113, was
Table 1. Decolorization Activity of
analysed by TLC using silica gel plates. 5ml B.subtilis under different culture conditions
of the sample was extracted with equal S. No Incubation % of Decolorization
volume of ethyl acetate and then evaporated Period (h) Under Under
under vacuum. The gel plates supplied by the Agitated Stationary
residue was spotted on TLC plates in which Conditions Conditions
micro syringe was used. The solvent system 1 0 --- ---
used was isopropanol: acidic acid: water, in 2 5 0.6 19
3 10 4 28
the ratio of 19:9:1 respectively.
4 15 11 54
5 20 16 68
Results and Discussion: 6 25 20 82
7 30 23 85
Effect of culture conditions on dye 25 87
8 35
decolorization 27 88
9 40
10 45 28 89
The culture under agitation conditions
11 50 30 90
demonstrated a better growth than that under
static conditions. But the bacterial species
Bacillus subtilis exhibited dye decolorizing
activity only when incubated under the
stationary conditions, where as, negligible
decolorization (30%) was noticed under the
agitating conditions. Stationary cultures
exhibited apparently complete decolorization
(90%) of Acid blue 113 (Fig 3) with in 50 hrs
of incubation (Fig 4) (Table 1) and further
incubation did not improve decolorization.
Anaerobic or static conditions were necessary
for bacterial decolorization through the cell
growth was poorer than that under aerobic
conditions. [24]. Under aerobic conditions Fig 4- Decolorization of Acid blue113 by
Bacillus subtilis under different culture conditions.
azo dyes are generally resistant to attack by
bacteria [25]. Azo dye decolorization by Effect of pH on dye decolorization
bacterial species if often initiated by
Bacterial culture generally exhibited
enzymatic reduction of azo bonds, the
maximum decolorization rate at pH values
presence of oxygen normally inhibits the azo
near 7. Decolorization of CI Acid blue 113 at
bond reduction activity since aerobic
various pH value by the Bacillus subtilis is in
respiration may dominate utilization of
Fig. 5. It shows that an increase in pH from 5.0
NADH; thus impeding the electron transfer
(0.764) to 7 (1.244 mg/l/h) (Table 2) while the
from NADH to azo bonds. [26]. The results
decolorization rate value decreased as pH was
14 | Advanced Biotech | November 2008
increased further from 7.0 (1.244 mg/l/h) to
8.0 (1.129 mg/l/h). The rate of decolorization
for B. subtilis was optimum in the narrow pH
range from 7.0 (1.244 mg/l/h) to 8.0 (1.129
mg/l/h) with marked reduction in
decolorization activity at pH 5.0. Both E. coli
and Pseudomonas luteola exhibited best
decolorization rate at pH 7 with constant
Table 2 - Decolorization rate at
different initial pH of medium.
S. No pH Decolorization
rate mg/l/h
1 5.0 0.764
2 5.5 0.988
3 6.0 1.022
4 6.5 1.099
5 7.0 1.244
6 7.5 1.198
7 8.0 1.129
Fig 5- Decolorization rate of Acid
blue 113 by Bacillus subtilis
at different initial pH.
decolorization rates upto pH
9.5 (26. Klebsiella
pneumoniae RS.13
completely degraded methyl
red in pH range from 6.0 to
8.0 [28]. [29] They found that
a pH value between 6 and 9
was optimum for
decolorization of
triphenylmethanes and azo
dyes by Pseudomonas sp.
Moreover, it has been
reported that generally azo
dye reduction cultures to
more basic aromatic amines
leads to a rise in pH of the medium by about
0.8-1.0 values [25, 30].
E f f e c t o f Te m p e r a t u r e o n d y e
decolorization
The dye decolorization activity of our culture
was found to increase with increase in
incubation temperature (Figure-6) from 25 to

37oC with maximum activity attained at 37oC
(1.296 mg/l/h). Further increase in
temperature resulted in maginal reduction in
decolorization activity of the bacterial culture
Bacillus subtilis (Table-3) so the bacterial
culture B.subtilis was more sensitive to
temperature.
Decline in decolorization activity at higher
temperature can be attributed to the loss of
cell viability or to the denaturation of the azo-
reductase enzyme (14). Maximum dye
Table 3 - Decolorization rate at different
incubation temperatures
S. No Temperature Decolorization
rate mg/l/h
1 20ºC 0.648
2 30ºC 1.087
3 37ºC 1.296
4 40ºC 1.032
5 50ºC 0.536
Fig 6 - Decolorization rate of Acid
blue 113 by Bacillus subtilis at
different temperatures (ºC)
decolorization activity of the bacterial
consortium NBNJ6 was noticed at 37o C [31].
Effect of dye & inoculum size
concentrations on dye decolorization
Decolorization activity of the bacterial
culture Bacillus subtilis was studied using
Acid blue 113 at different initial
concentrations varying from 50 to 300 mg/l
(Fig.7). Rate of decolorization increased with
increase in initial dye concentration up to
200 mg/l (1.746 mg/l/h) Table 4. Further
increase in dye concentration resulted in
reduction in decolorization rates. Lower
decolorization efficiency is due to higher
inhibition at high dyestuff concentration [32].
[33] They reported that the dye concentration
in the reactive dye bath effluent was observed
with in narrow range of 0.1-0.2 g/l. [31] They
15 | Advanced Biotech | November 2008
Research Article
reported that the Direct red 81 decolorization decolorization did not vary significantly.
rate was increased with increase in initial dye There was no proportionate increase in the
concentration upto 200 ppm (2.29 mg/l/h) by percentage of decolorization with increase in
using bacterial consortium NBNJ6. Bacillus the inoculum size of Kurthia sp. When
subtilis could decolorize the dye at inoculated in textile effluent (34). [31] They
concentrations much above those reported in
Table 4 - Decolorization rate at
different initial dye concentration
in medium.
S. No Concentration Decolorization
of Acid blue rate mg/l/h
113 (mg/l)
1 50 1.237
2 100 1.384
3 150 1.537
4 200 1.746 Fig 8-Decolorization rate of Acid blue
5 250 0.864 113 by Bacillus subtilis at different
6 300 0.725 inoculum size (v/v)
reported that the Direct Red 81 decolorization
rate was increased with increase in the
inoculum size, reaching maximum (2.53
mg/l/h) at 20% (v/v) inoculum size.
Effect of co-substrate on dye
decolorization
Bacterial culture Bacillus subtilis exhibited
maximum decolorization of Acid blue 113
dye when starch & peptone were
Fig 7- Decolorization rate of supplemented in the medium (Table 6). In
Acid blue 113 by Bacillus subtilis absence of co-substrate the bacterial culture
waste waters and thus it can be successfully was unable to decolorize the dye, with
explosed for treatment of dye bearing indicates the availability of supplementary
carbon source seems to be necessary for
industrial waste waters.
growth and decolorization of dyes [35]. The
In order to find out the optimum Bacillus ability of our culture to use starch & peptone
subtilis inoculum needed for faster and higher as co-substrates was encouraging from a
percentage decolorization by decolorizing commercial point of view. Other combination
ability was tested at different inoculum of two carbon sources also seemed to be
concentrations starting from 5 to 30% (v/v) reasonably effective. In order to optimize the
(Fig 8). The decolorization rate increased concentration of starch on the medium for
with increase in the inoculum size, reaching maximum decolorization 89% of Acid blue
maximum (1.984 mg/l/h) (Table 5) at 113 with in 50 hour of incubation. [36] They
20% (v/v) inoculum size. However, beyond reported lactose (5g/l) and yeast extract (50
20% (v/v) inoculum size rate of
Table 6 - The effect of various
co-substrates on decolorization of dye
Table 5 Decolorization rate at
different inoculum size in medium
S. No Different Decolorization
inoculum rate mg/l/h
Size % (v/v)
1 5 1.246
2 10 1.492
3 15 1.765
4 20 1.984
5 25 1.653
6 30 1.371
 Research Article
mg/l) to be the most effective carbon-nitrogen
source in decolorization of Everzol Red RBN
by bacterial-consortium PDW. [31] They
reported that starch and casein to be the most6
effective carbon-nitrogen source in
decolorization of Direct Red 81 by bacterial
consortium NBNJ6.
Decolorizing Bacteria
Decolorizing activity of bacteria was detected
by plate assay. Clearing zone was formed
surrounding the bacterial culture which
grown on Nutrient agar plate containing Acid
blue 113 dye. The decolorization ability of
Bacillus subtilis was shown in Fig 9.
Fig-9. Decolorization activity of Bacillus subtilis was detected by plate assay
A) Initial period of incubation. B) After incubation, clearance of zone
(decolorization Zone) was observed surrounding the culture
Fig 10-UV/VIS spectral analysis of initial period of incubation of inoculated medium
Fig 11-UV/VIS spectral analysis of decolorized medium (after 50hrs incubation)
Analysis of UV/VIS-spectra
The UV-VIS spectra corresponding to initial
(Fig 10) & final samples of decolorization
experiments for Acid blue 113 are shown in
Fig 11. The absorbance analysed from 400 to
700nm. The initial dye solution showed high
peak at the wavelength of 533 nm. The
decolorized dye showed disappearance of
peak, which indicates that the decolorization
is due to dye degradation.
COD Determination
The Chemical oxygen demand was measured
by calculating the amount of oxidizing agent
16 | Advanced Biotech | November 2008
i.e., K2Cr2O7 consumed during oxidation of
organic matter (biodegradable and non-
biodegradable) under acidic conditions.
Chemical oxygen demand of degraded dye
solution gets considerably reduced after
degradation by Bacillus subtilis. COD of the
solutions after degradation shows significant
decrease from 13600 mg/l to 3200 mg/l.
Similarly [37] They reported that the COD of
the synthetic effluent (5200 mg/l) and
Reactofix Golden Yellow (4000 mg/l)
decreased by 57% and 54% respectively after
adsorption at pH 2, 40o C and 150 rev/min to
3.54 g mycelium of P.chrysosporium for
24 hrs.
BOD Determination
The rate of removal (that is Consumption) of
Oxygen by microorganism in aerobic
degradation of the dissolved or even
particulate organic matter in water that is
called Biological Oxygen Demand (BOD).
The BOD determination was used to
determine the relative oxygen requirements
of dye solution. The BOD of degraded dye
solution gets considerably after
biodegradation by Bacillus subtilis. BOD of
the solution shows significant decrease from
3625 mg/l to 1375 mg/l after degradation at
pH 7. The test measures the Oxygen utilized
during a specified incubation period for the
biochemical degradation of organic matter
(Carbonaceous demand) and the oxygen used
to utilize in organic material such as sulfides
Fig 12-Biodegradation rate was
measured by COD & BOD Determination.
and ferrous iron. It also may measure the
oxygen used to oxidize reduce forms of
Nitrogen (Nitrogenous demand).
TLC Analysis
The dye decolorization study of Bacillus
subtilis was further supported by TLC

analysis. The spot was observed in the initial
dye solution (Rf value of Acid blue 113 =
0.81) and no spot was observed in the
decolorized medium (Fig 13), indicating that
decolorization was due to its degradation into
unidentified intermediates. The initial step in
bacterial degradation of dye is due to the
reduction of azo bonds leading to the
formation of aromatic amines. These
aromatic amines are likely to be formed
during reductive cleavage of are bonds
through which the amines are linked in the
dye.
Fig-13. Decolorization of Acid blue 113
by Bacillus subtilis was confirmed by
TLC analysis.
(31) They reported that the preliminary result,
TLC indicated that the spots of decolorized
medium (Rf value of DR 81 = 0.48). When the
dye chromatogram was observed in UV light,
fluorescent bands with Rf value (0.40, 0.51,
0.60 and 0.62) different from that of dye were
detected in the lanes corresponding to the
spots of decolorized medium no such bands
were observed for spots of uninoculated
medium. Different bands, which were formed
in the critical period of incubation of
inoculated medium, disappeared upon
extracted incubation supporting degradation.
Conclusion
The present study confirms the ability of
bacterial culture Bacillus subtilis to
decolorize the leather dye Acid Blue 113 with
decolorization efficiency of 90%, thus
suggesting its application for decolorization
of dye bearing of industrial wastewaters.
Presence of a Co-Substrate (Starch &
Peptone) is the essential conditions for
attaining maximum decolorization efficiency.
The anaerobic decolorization of Acid Blue
113 dye occurs as a result of reduction of
N=N- bond accompanied by the formation of
aromatic amines The amine intermediates
formed in static conditions treatment can be
removed by agitating conditions &
approximately 30% decolorization under
agitating conditions after a reaction period of
50 hrs.
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About the Authors
21. Wuhrmann, K., Mechsner, K.L. & diazo dye Direct Red 81 by a novel
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18 | Advanced Biotech | November 2008