Research Article
12 Advanced Biotech November | | 2008
Gurulakshmi. M, Sudarmani. D.N.P and Venba. R.
Biodegradation of Leather Acid dye by Bacillus subtilis
and degradation is an environmentally
friendly and cost competitive alternative to
chemical decomposition possess [4]
Unfortunately, most azo dyes are recalcitrant
to aerobic degradation by bacterial cells [8].
H o w e v e r , t h e r e a r e f e w k n o w n
microorganisms that have the ability to
reductively cleave azo bonds under aerobic
conditions [9,10,11,12].
Compared with chemical/Physical methods,
biological processes have received more
interest because of their cost effectiveness,
lower sludge production and environmental
friendliness. Various wood-rotting fungi were
able to decolorize azo dyes using peroxidases
or laccases. But fungal treatment of effluents
is usually time-consuming. Under static or
anaerobic conditions, bacterial decolorization
generally demonstrates good color removal
effects. However, aerobic treatment of azo
dyes with bacteria usually achieved low
efficiencies because oxygen is a more
efficient electron acceptor than azo dyes [13].
Although decolorization, under anaerobic
conditions generally cannot realize the
complete mineralization of azo dyes,
aromatic amines as decolorized products are
usually more susceptible to oxygenase attack.
Thus, bacterial mineralization of azo dyes
generally takes two steps:
Step 1: Two mechanisms for the
decolorization of azo dyes under anaerobic
conditions in bacterial systems have been
proposed [14]. The first one consists of direct
electron transfer to azo dyes as terminal
acceptors via enzymes during bacterial
catabolism, connected the ATP generation
(energy conservation). The second one
involves a free reduction of azo dyes by the
end products of bacterial catabolism, not
linked to ATP generation (eg., reduction of the
azo bond by reduced inorganic compounds,
such as Fe2+ or H2S, that are formed as the
end product of certain anaerobic bacterial
metabolic reactions). Figure 1 shows a
possible pathway for the degradation of azo
dyes under anaerobic conditions with whole
bacterial cells.
Step 2: During anaerobic degradation, a
reduction of the azo bond in the molecules is
observed. Then, aerobic conditions are
required for the complete mineralization of
the reactive azo dye molecule. The aromatic
compounds produced by the initial reduction
are degraded via hydroxylation and opening
in the process is necessary in which oxygen is
introduced after the initial anaerobic
reduction of the azo bond has taken place. The
optimum pH for colour removal is around pH
7-7.5. The rate of colour removal tends to
decrease rapidly under strongly acid or
strongly alkaline conditions. The optimum
cell culture growth temperature is between 35
and 45°C.
Introduction
Dyes are widely used in the Textile, rubber
product, paper, printing, color photography,
Pharmaceuticals, Cosetics and Many other
industries. [1] Amongst these, azo dyes
represent the largest and most versatile class
of synthetic dyes. [2] Approximately 10 - 15%
of the dyes are release into the environment
during manufacturing and usage. [3] Since
some of the dyes are harmful, dye-containing
wastes pore an important environmental
problem. [4] These dyes are poorly
biodegrabale because of their structures and
treatment of wastewater containg dyes
usually involves physical and / or chemical
methods [5] such as adsorption, Coagulation-
flocculation, Oxidation, filtration and
electrochemical methods [6]
Over the Past decades, Biological
decolorization has been investigated as a
method to transform, degrade or mineralize
azo dyes [7] Moreover, such decolorization
Abstract
The Bacillus subtilis was used to decolorize the Acidblue113. The bacterial culture exhibited
90% decolorization ability within 50 h. Maximum rate of decolorization was observed (90%)
when starch & peptone was supplemented in the medium. Decolorization of Acidblue113 was
monitored by TLC, which indicated that dye decolorization was due to its degradation into
unidentified intermediates. The optimum dye decolorizing activity of the culture was observed 0at pH 7.0 and incubation temperature of 37 C. Maximum, dye-decolorizing efficiency was
observed at 200 mg/l concentration of Acidblue113. A plate assay was performed for the
detection of decolorizing ability of bacteria. Clearing zone (decolorization) was formed
surrounding the bacterial culture. Decolorization was confirmed by UV-VIS
spectrophotometer. The initial dye solution showed high peak at the wavelength of 560nm.
The decolorized dye showed disappearance of peak, which indicated that the decolorization is
due to dye degradation. The dye decolorization was further confirmed by COD & BOD
Analysis.
Key words: Biodegradation, Acid Blue 113, Bushnell & Hass medium (BHM) and Bacillus
subtilis
13 Advanced Biotech November | | 2008
Research Article
Medium:
Measurement of dye concentration:
Study of Physico-Chemical Parameters
Plate Assay
Analysis of UV/ Visible
Spectrophotometer
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, MgSo 0.2, 4
CaCl 0.02, KH PO 1.0, K HPO 1.0, 2 2 4 2 4
NH No 1.0, FeCl 0.05, Glucose 0.9, Yeast 4 3 3
extract 0.9, Acid blue113-100mg
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:
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), oTemperature (20-50 C), and at different
culture conditions under Agitation and
stationary conditions.
Plate assay was performed for the detection of
decolourizing activity of bacteria. The
nutrient agar and Acid blue 113 dye was oautoclaved at 121 C 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 oincubated at 37 C for 7days. The plates were
observed for clearance of the surrounding the
colonies.
Under static conditions, the culture with an
initial dye (Acid blue 113) concentration of
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.
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.
Materials and Methods:
Chemicals:
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.
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 oincubator at 148 C 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
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.
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, MgSO , 4
FeCl and seeding/L of water. Before use 3
0bring dilution water temperature to 20 C.
Dilution water was aerated with organic free
filtered air. All the bottles are kept 0in BOD incubator at 20 C for 5 days.
After incubation 1ml of MnSO , Alkali iodide 4
solution and sulphuric acid was added to form
brown color solution. After color formation
Estimation Of Chemical Oxygen Demand
(COD)
Estimation of Biological Oxygen Demand
(BOD)
Fig 1- A proposed redox reduction for the degradation of azo dye with whole bacterial cells
Fig- 2 Structure of Acid blue 113
Decolorization(%) = Initial absorbance - Observed absorbance
Initial absorbance
Volume of sample
14 Advanced Biotech November | | 2008
Research Article
were similar to those studies on E. coli NO 3
and Pseudomonas luteola [27].
Bacterial culture generally exhibited
maximum decolorization rate at pH values
near 7. Decolorization of CI Acid blue 113 at
various pH value by the Bacillus subtilis is in
Fig. 5. It shows that an increase in pH from 5.0
(0.764) to 7 (1.244 mg/l/h) (Table 2) while the
decolorization rate value decreased as pH was
Effect of pH on dye decolorization
they were titrated against their Na SO for 2 4
their BOD values. The readings were noted.
B-volume of Na SO used for blank 2 3
T (v)-volume of Na So used for sample2 3
S (v)-volume of sample
BOD values were compared between the
initial medium containing dye solution and
decolourised medium.
Degradation of dye, Acid blue 113, was
analysed by TLC using silica gel plates. 5ml
of the sample was extracted with equal
volume of ethyl acetate and then evaporated
under vacuum. The gel plates supplied by the
residue was spotted on TLC plates in which
micro syringe was used. The solvent system
used was isopropanol: acidic acid: water, in
the ratio of 19:9:1 respectively.
The culture under agitation conditions
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
azo dyes are generally resistant to attack by
bacteria [25]. Azo dye decolorization by
bacterial species if often initiated by
enzymatic reduction of azo bonds, the
presence of oxygen normally inhibits the azo
bond reduction activity since aerobic
respiration may dominate utilization of
NADH; thus impeding the electron transfer
from NADH to azo bonds. [26]. The results
Thin Layer Chromatography (TLC)
Effect of culture conditions on dye
decolorization
Results and Discussion:
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 reduct ion in
decolorization activity at pH 5.0. Both E. coli
and Pseudomonas luteola exhibited best
decolorization rate at pH 7 with constant
decolorization rates upto pH
9 . 5 ( 2 6 . K l e b s i e l l a
p n e u m o n i a e R S . 1 3
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
w a s o p t i m u m f o r
d e c o l o r i z a t i o n o f
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].
The dye decolorization activity of our culture
was found to increase with increase in
incubation temperature (Figure-6) from 25 to
E f f e c t o f Te m p e r a t u r e o n d y e
decolorization
Fig-3 Showing the decolorization of Acid blue113 by Bacillus subtilis
Fig 4- Decolorization of Acid blue113 by Bacillus subtilis under different culture conditions.
Fig 5- Decolorization rate of Acid blue 113 by Bacillus subtilis
at different initial pH.
S. No pH Decolorization rate mg/l/h
1 5.0 0.7642 5.5 0.9883 6.0 1.0224 6.5 1.0995 7.0 1.2446 7.5 1.1987 8.0 1.129
Table 2 - Decolorization rate at different initial pH of medium.
Table 1. Decolorization Activity of B.subtilis under different culture conditions
1234567891011
05101520253035404550
---0.641116202325272830
---19285468828587888990
S. No Incubation Period (h)
% of DecolorizationUnder Agitated Conditions
Under Stationary Conditions
15 Advanced Biotech November| | 2008
Research Article
S. No Temperature Decolorization rate mg/l/h
1 20ºC 0.6482 30ºC 1.0873 37ºC 1.2964 40ºC 1.0325 50ºC 0.536
Table 3 - Decolorization rate at different incubation temperatures
o o37 C with maximum activity attained at 37 C
(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
decolorization activity of the bacterial oconsortium NBNJ6 was noticed at 37 C [31].
Decolorization activity of the bacterial
culture Bacillus subtilis was studied using
Acid blue 113 at different ini t ial
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
E f f e c t o f d y e & i n o c u l u m s i z e
concentrations on dye decolorization
Table 6 - The effect of various co-substrates on decolorization of dye
reported that the Direct red 81 decolorization
rate was increased with increase in initial dye
concentration upto 200 ppm (2.29 mg/l/h) by
using bacterial consortium NBNJ6. Bacillus
subtilis could decolorize the dye at
concentrations much above those reported in
waste waters and thus it can be successfully
explosed for treatment of dye bearing
industrial waste waters.
In order to find out the optimum Bacillus
subtilis inoculum needed for faster and higher
percentage decolorization by decolorizing
ability was tested at different inoculum
concentrations starting from 5 to 30% (v/v)
(Fig 8). The decolorization rate increased
with increase in the inoculum size, reaching
maximum (1.984 mg/l/h) (Table 5) at
20% (v/v) inoculum size. However, beyond
20% (v /v) inoculum s ize ra te of
decolorization did not vary significantly.
There was no proportionate increase in the
percentage of decolorization with increase in
the inoculum size of Kurthia sp. When
inoculated in textile effluent (34). [31] They
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.
Bacterial culture Bacillus subtilis exhibited
maximum decolorization of Acid blue 113
dye when starch & peptone were
supplemented in the medium (Table 6). In
absence of co-substrate the bacterial culture
was unable to decolorize the dye, with
indicates the availability of supplementary
carbon source seems to be necessary for
growth and decolorization of dyes [35]. The
ability of our culture to use starch & peptone
as co-substrates was encouraging from a
commercial point of view. Other combination
of two carbon sources also seemed to be
reasonably effective. In order to optimize the
concentration of starch on the medium for
maximum decolorization 89% of Acid blue
113 with in 50 hour of incubation. [36] They
reported lactose (5g/l) and yeast extract (50
E f f e c t o f c o - s u b s t r a t e o n d y e
decolorization
Table 5 Decolorization rate at different inoculum size in medium
S. No Different Decolorizationinoculum rate mg/l/hSize % (v/v)
1 5 1.2462 10 1.4923 15 1.7654 20 1.9845 25 1.6536 30 1.371
Table 4 - Decolorization rate at different initial dye concentration
in medium.
S. No Concentration Decolorizationof Acid blue rate mg/l/h113 (mg/l)
1 50 1.2372 100 1.3843 150 1.5374 200 1.7465 250 0.8646 300 0.725
Fig 6 - Decolorization rate of Acid blue 113 by Bacillus subtilis at
different temperatures (ºC)
Fig 8-Decolorization rate of Acid blue 113 by Bacillus subtilis at different
inoculum size (v/v)
Fig 7- Decolorization rate of Acid blue 113 by Bacillus subtilis
Analysis of UV/VIS-spectra
COD Determination
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.
The Chemical oxygen demand was measured
by calculating the amount of oxidizing agent
Fig 12-Biodegradation rate was measured by COD & BOD Determination.
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 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.
Decolorizing Bacteria
16 Advanced Biotech November | | 2008
Research Article
i.e., K Cr O consumed during oxidation of 2 2 7
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 oadsorption at pH 2, 40 C and 150 rev/min to
3.54 g mycelium of P.chrysosporium for
24 hrs.
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
s o l u t i o n g e t s c o n s i d e r a b l y a f t e r
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
and ferrous iron. It also may measure the
oxygen used to oxidize reduce forms of
Nitrogen (Nitrogenous demand).
The dye decolorization study of Bacillus
subtilis was further supported by TLC
BOD Determination
TLC Analysis
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)
17 Advanced Biotech November | | 2008
Research Article
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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0.81) and no spot was observed in the
decolorized medium (Fig 13), indicating that
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decolorize the leather dye Acid Blue 113 with
decolorization efficiency of 90%, thus
suggesting its application for decolorization
of dye bearing of industrial wastewaters.
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Peptone) is the essential conditions for
attaining maximum decolorization efficiency.
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Fig-13. Decolorization of Acid blue 113 by Bacillus subtilis was confirmed by
TLC analysis.
18 Advanced Biotech November | | 2008
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About the Authors
M.Gurulakshmi
M.Sc., Biotechnology
Dr. D.N.P. Sudarmani Ph.D.,
Lecturer,
P.G. Department of Biotechnology,
Ayya Nadar Janaki Ammal College,
(Autonomous),
Sivakasi 626 123, Virudhunagar Dist., T.N.
Mrs. R. Venba,
Sr. Assistant Director,
Tannery Division,
Central Leather Research Institute,
Adyar, Chennai - 600 020.
For Correspondence:
M.Gurulakshmi,
E-mail: [email protected]
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