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

 Research Article

Medium: 10% (v/v) was 90% decolorized in 50 hours.

UV/Visible spectra of culture supernatants of
The Bacillus subtilis culture was routinely
0 hour and 50 hours were compared and
grown at 37°C in the basal culture medium,
possible degradation products were
Bushnell and Hass medium (BHM)
speculated.
containing the following in g/l, MgSo4 0.2,
CaCl2 0.02, KH2PO4 1.0, K2HPO4 1.0, Estimation Of Chemical Oxygen Demand
NH4No3 1.0, FeCl3 0.05, Glucose 0.9, Yeast (COD)
extract 0.9, Acid blue113-100mg Chemical oxygen demand was measured by
Measurement of dye concentration: the standard Potassium dichromate method.
Fig 1- A proposed redox reduction for the degradation 1ml of initial medium containing dye
of azo dye with whole bacterial cells
The dye concentrations were measured with a solution, decolorized medium, distilled water
Azoreductase is the key enzyme expressed in UV/VIS spectrophotometer (HITACHI- was added to COD Tube sample 1, sample 2,
azodye-degrading bacteria that catalyses the U.2000-Spectrophotometer) at regular Blank respectively. Then 1.5ml of distilled
reductive cleavage of the azo bond. intervals during the decolorisation process. water & reducing agent potassium
Azoreductase activity has been identified in The concentration of azo dye was detected dichromate and 3.5ml COD acid were added
several species of bacteria recently; such as spectrophotometrically by reading the culture to each tube. Duplicates were put up for all the
Caulobacter subvibrioides C7-D, Xenophilus supernatant at its specific max after tubes. All the tubes were kept in the COD
azovorans KF46F, Pigmentiphaga kullae centrifugation at 10,000 rpm for 10 min. incubator at 148oC for 2 hrs. After incubation
K24, Enterobacter agglomerans and (Superspin R-VIFm plasto crafts). The dye the entire content were transferred to a conical
Enterococcus faecalis [15, 16,17,18,19]. concentrations were determined from the flask. A drop of ferroin indicator was added to
attenuance (O.D) of the culture at 533 nm. it and was titrated against FAS in the burette.
Efforts to isolate bacterial cultures capable of
degrading azo dyes started in the 1970s with Decolorization activity was calculated as The readings were noted
reports of Bacillus subtilis [9], then follows:
Aeromonas hydrophila (20) followed by
Initial absorbance - Observed absorbance Volume of sample
Bacillus cereus [21]. Numerous bacteria Decolorization(%) =
Initial absorbance
capable of dye decolorization, either in pure A-volume of Ferrous Ammonium Sulphate
cultures or in consortia, have been reported Study of Physico-Chemical Parameters used for blank
[22, 7, 23,12,14,4].
Decolorization was studied using various Co- B-volume of ferrous Ammonium Sulphate
In the course of our study on the substrates (starch & peptone, sucrose, starch used for sample
biodegradation of Leather dye. We have & yeast extract, sucrose &yeast extract,
found that Bacillus subtilis are capable of Dextrose & yeast extract) and at different dye Equivalent weight of oxygen - 8
degrading C.I. Acid Blue 113 (C.I. No. concentrations (100-500 mg/l), inoculum size N-Normality of FAS - 0.1
26360). To the best of our knowledge, no (5, 10, 15, 20, 25, & 30% (v/v), pH (5-8),
other microorganism is reported to Temperature (20-50oC), and at different COD values were compared between the
biodegrade Acid Blue 113. This paper culture conditions under Agitation and initial medium containing dye solution and
describes the degradation of Acid Blue 113 by stationary conditions. decolorized medium.
Bacillus subtilis and shows a plausible initial
Plate Assay Estimation of Biological Oxygen Demand
pathway for the biodegradation of Acid Blue
(BOD)
113. We also report the optimization of Plate assay was performed for the detection of
parameters required for the dyes efficiently in decolourizing activity of bacteria. The 1ml of initial medium containing dye
a short period. nutrient agar and Acid blue 113 dye was solution, decolorized medium, distilled water
autoclaved at 121oC for 15 minutes. Bacillus was added to airtight BOD bottles sample 1,
Materials and Methods: subtilis culture was plated on nutrient agar sample 2, Blank respectively. Place desired
plates containing Acid blue (500mg/l). The volume of water in a suitable bottle and add
Chemicals: Acid blue 113 Dye (Figure 2) and 1ml of each of Phosphate buffer, MgSO4,
plates were wrapped with parafilm and were
the isolates (Bacillus subtilis) used in this FeCl3 and seeding/L of water. Before use
incubated at 37oC for 7days. The plates were
study were kindly provided by the Tannery
observed for clearance of the surrounding the bring dilution water temperature to 200C.
Division, CLRI, Chennai. All other reagents
colonies. Dilution water was aerated with organic free
used were of analytical grade.
filtered air. All the bottles are kept
Analysis of UV/ Visible in BOD incubator at 200C for 5 days.
Spectrophotometer After incubation 1ml of MnSO4, Alkali iodide
Under static conditions, the culture with an solution and sulphuric acid was added to form
initial dye (Acid blue 113) concentration of brown color solution. After color formation
Fig- 2 Structure of Acid blue 113

13 | Advanced Biotech | November 2008

 Research Article

they were titrated against their Na2SO4for were similar to those studies on E. coli NO3 increased further from 7.0 (1.244 mg/l/h) to
their BOD values. The readings were noted. and Pseudomonas luteola [27]. 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
B-volume of Na2SO3 used for blank and Pseudomonas luteola exhibited best
T (v)-volume of Na2So3used for sample decolorization rate at pH 7 with constant

S (v)-volume of sample Table 2 - Decolorization rate at

BOD values were compared between the different initial pH of medium.
initial medium containing dye solution and S. No pH Decolorization
decolourised medium. rate mg/l/h
Fig-3 Showing the decolorization of 1 5.0 0.764
Thin Layer Chromatography (TLC) 2 5.5 0.988
Acid blue113 by Bacillus subtilis
3 6.0 1.022
Degradation of dye, Acid blue 113, was
Table 1. Decolorization Activity of 4 6.5 1.099
analysed by TLC using silica gel plates. 5ml B.subtilis under different culture conditions 5 7.0 1.244
of the sample was extracted with equal S. No Incubation % of Decolorization 6 7.5 1.198
volume of ethyl acetate and then evaporated Period (h) Under Under 7 8.0 1.129
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 Fig 5- Decolorization rate of Acid
10 45 28 89
The culture under agitation conditions blue 113 by Bacillus subtilis
11 50 30 90
demonstrated a better growth than that under at different initial pH.
static conditions. But the bacterial species decolorization rates upto pH
Bacillus subtilis exhibited dye decolorizing 9.5 (26. Klebsiella
activity only when incubated under the pneumoniae RS.13
stationary conditions, where as, negligible completely degraded methyl
decolorization (30%) was noticed under the red in pH range from 6.0 to
agitating conditions. Stationary cultures 8.0 [28]. [29] They found that
exhibited apparently complete decolorization a pH value between 6 and 9
(90%) of Acid blue 113 (Fig 3) with in 50 hrs was optimum for
of incubation (Fig 4) (Table 1) and further decolorization of
incubation did not improve decolorization. triphenylmethanes and azo
Anaerobic or static conditions were necessary dyes by Pseudomonas sp.
for bacterial decolorization through the cell Moreover, it has been
growth was poorer than that under aerobic reported that generally azo
conditions. [24]. Under aerobic conditions Fig 4- Decolorization of Acid blue113 by
Bacillus subtilis under different culture conditions. dye reduction cultures to
azo dyes are generally resistant to attack by more basic aromatic amines
bacteria [25]. Azo dye decolorization by Effect of pH on dye decolorization leads to a rise in pH of the medium by about
bacterial species if often initiated by 0.8-1.0 values [25, 30].
Bacterial culture generally exhibited
enzymatic reduction of azo bonds, the
maximum decolorization rate at pH values E f f e c t o f Te m p e r a t u r e o n d y e
presence of oxygen normally inhibits the azo
near 7. Decolorization of CI Acid blue 113 at decolorization
bond reduction activity since aerobic
various pH value by the Bacillus subtilis is in
respiration may dominate utilization of The dye decolorization activity of our culture
Fig. 5. It shows that an increase in pH from 5.0
NADH; thus impeding the electron transfer was found to increase with increase in
(0.764) to 7 (1.244 mg/l/h) (Table 2) while the
from NADH to azo bonds. [26]. The results incubation temperature (Figure-6) from 25 to
decolorization rate value decreased as pH was

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37oC with maximum activity attained at 37oC reported that the Direct red 81 decolorization decolorization did not vary significantly.
(1.296 mg/l/h). Further increase in rate was increased with increase in initial dye There was no proportionate increase in the
temperature resulted in maginal reduction in concentration upto 200 ppm (2.29 mg/l/h) by percentage of decolorization with increase in
decolorization activity of the bacterial culture using bacterial consortium NBNJ6. Bacillus the inoculum size of Kurthia sp. When
Bacillus subtilis (Table-3) so the bacterial subtilis could decolorize the dye at inoculated in textile effluent (34). [31] They
culture B.subtilis was more sensitive to concentrations much above those reported in
temperature.
Table 4 - Decolorization rate at
Decline in decolorization activity at higher different initial dye concentration
temperature can be attributed to the loss of in medium.
cell viability or to the denaturation of the azo- S. No Concentration Decolorization
reductase enzyme (14). Maximum dye of Acid blue rate mg/l/h
113 (mg/l)
Table 3 - Decolorization rate at different 1 50 1.237
incubation temperatures 2 100 1.384
3 150 1.537
S. No Temperature Decolorization 4 200 1.746 Fig 8-Decolorization rate of Acid blue
rate mg/l/h 5 250 0.864 113 by Bacillus subtilis at different
6 300 0.725 inoculum size (v/v)
1 20ºC 0.648
2 30ºC 1.087 reported that the Direct Red 81 decolorization
3 37ºC 1.296 rate was increased with increase in the
4 40ºC 1.032 inoculum size, reaching maximum (2.53
5 50ºC 0.536 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
Fig 6 - Decolorization rate of Acid carbon source seems to be necessary for
industrial waste waters.
blue 113 by Bacillus subtilis at growth and decolorization of dyes [35]. The
different temperatures (ºC) 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
decolorization activity of the bacterial
percentage decolorization by decolorizing commercial point of view. Other combination
consortium NBNJ6 was noticed at 37o C [31].
ability was tested at different inoculum of two carbon sources also seemed to be
Effect of dye & inoculum size concentrations starting from 5 to 30% (v/v) reasonably effective. In order to optimize the
concentrations on dye decolorization (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
Decolorization activity of the bacterial
maximum (1.984 mg/l/h) (Table 5) at 113 with in 50 hour of incubation. [36] They
culture Bacillus subtilis was studied using
20% (v/v) inoculum size. However, beyond reported lactose (5g/l) and yeast extract (50
Acid blue 113 at different initial
20% (v/v) inoculum size rate of
concentrations varying from 50 to 300 mg/l Table 6 - The effect of various
(Fig.7). Rate of decolorization increased with co-substrates on decolorization of dye
increase in initial dye concentration up to Table 5 Decolorization rate at
different inoculum size in medium
200 mg/l (1.746 mg/l/h) Table 4. Further
increase in dye concentration resulted in S. No Different Decolorization
reduction in decolorization rates. Lower inoculum rate mg/l/h
Size % (v/v)
decolorization efficiency is due to higher
1 5 1.246
inhibition at high dyestuff concentration [32].
2 10 1.492
[33] They reported that the dye concentration 3 15 1.765
in the reactive dye bath effluent was observed 4 20 1.984
with in narrow range of 0.1-0.2 g/l. [31] They 5 25 1.653
6 30 1.371

15 | Advanced Biotech | November 2008

 Research Article

mg/l) to be the most effective carbon-nitrogen Analysis of UV/VIS-spectra i.e., K2Cr2O7 consumed during oxidation of
source in decolorization of Everzol Red RBN organic matter (biodegradable and non-
The UV-VIS spectra corresponding to initial
by bacterial-consortium PDW. [31] They biodegradable) under acidic conditions.
(Fig 10) & final samples of decolorization
reported that starch and casein to be the most6 Chemical oxygen demand of degraded dye
experiments for Acid blue 113 are shown in
effective carbon-nitrogen source in solution gets considerably reduced after
Fig 11. The absorbance analysed from 400 to
decolorization of Direct Red 81 by bacterial degradation by Bacillus subtilis. COD of the
700nm. The initial dye solution showed high
consortium NBNJ6. solutions after degradation shows significant
peak at the wavelength of 533 nm. The
Decolorizing Bacteria decolorized dye showed disappearance of decrease from 13600 mg/l to 3200 mg/l.
peak, which indicates that the decolorization Similarly [37] They reported that the COD of
Decolorizing activity of bacteria was detected the synthetic effluent (5200 mg/l) and
is due to dye degradation.
by plate assay. Clearing zone was formed Reactofix Golden Yellow (4000 mg/l)
surrounding the bacterial culture which COD Determination decreased by 57% and 54% respectively after
grown on Nutrient agar plate containing Acid adsorption at pH 2, 40o C and 150 rev/min to
The Chemical oxygen demand was measured
blue 113 dye. The decolorization ability of 3.54 g mycelium of P.chrysosporium for
by calculating the amount of oxidizing agent
Bacillus subtilis was shown in Fig 9. 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
Fig-9. Decolorization activity of Bacillus subtilis was detected by plate assay
A) Initial period of incubation. B) After incubation, clearance of zone during a specified incubation period for the
(decolorization Zone) was observed surrounding the culture biochemical degradation of organic matter
(Carbonaceous demand) and the oxygen used
to utilize in organic material such as sulfides

Fig 10-UV/VIS spectral analysis of initial period of incubation of inoculated medium

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
Fig 11-UV/VIS spectral analysis of decolorized medium (after 50hrs incubation)

16 | Advanced Biotech | November 2008

 Research Article

analysis. The spot was observed in the initial aromatic amines The amine intermediates E u ro p e a n J o u r n a l o f Applied
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About the Authors
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and Elichi ldaka, 1981 Biodegradability azo dyes by different white rot fungai. P.G. Department of Biotechnology,
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Mrs. R. Venba,
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Biotechnology 54, 268 273. Decolorization of triphenyl methanes
24. Guang-fei Liu, Ji-ti Zhou, Jing Wang, Zhi- dyes and textile and dye stuff effluent by For Correspondence:
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18 | Advanced Biotech | November 2008