59 biodegradation of leather

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


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


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


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)


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

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Fig-13. Decolorization of Acid blue 113 by Bacillus subtilis was confirmed by

TLC analysis.


Research Article

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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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Sharma, P., 2006. Use of Phenerochate

chrysosporium biomass for the removal

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Biotechnology 22: 835 839.

59 biodegradation of leather

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