Illanjiam S et al.

/ Journal of Pharmacy Research 2011,4(9),2969-2974

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Biodegradation of Azodye Direct Brown II by Pseudomonas aeruginosa, Bacillus subtilis & Pseudomonas putida
Illanjiam S and Kantha D Arunachalam Department of Microbiology, Hindustan Colllege of Arts and Science, Padur, Chennai, Tamilnadu,India. Department of Interdiscipilinary Research SRM university, Kattankulathur, Chennai, Tamilnadu,India.

Received on: 19-05-2011; Revised on: 08-06-2011; Accepted on:01-07-2011 ABSTRACT

The Pseudomonas aeruginosa Pseudomonas putida & Bacillus subtilis was used to decolorize the Direct Brown II. The bacterial culture exhibited 93% decolorization ability within 72 h. Maximum rate of decolorization was observed (93%) when yeast extract was supplemented in the medium. Decolorization of Direct Brown II 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 at pH 7.0 and incubation temperature of 37C. Maximum, dye-decolorizing efficiency was observed at 25 ppm concentration of Diredct Brown II. 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 438nm. 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 , Azodye Direct Brown II by Pseudomonas aeruginosa. Bushnell and Hass medium (BHM)

INTRODUCTION
Dyes are widely used in the Textile, rubber product, paper, printing, color photography, Pharmaceuticals, Cosmetics 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 biodegradable because of their structures and treatment of wastewater containing 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 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]. However, there are few known 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 peroxides or lactases. 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 azodyes 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
Fig 1 . A proposed redox reduction for the degradation of azo dye with whole bacterial cell.

end products of bacterial catabolism, not linked to ATP generation (e.g., 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 isObserved. 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 7-9The rate of colour removal tends to decrease rapidly under strongly acid or strongly alkaline conditions. The optimum cell culture growth temperature is between 37 and 45C.

*Corresponding author.
S.Illanjiam, Department of Microbiology, Hindustan College of Arts and Science, Padur, Chennai,Tamilnadu,India. Tel.:9840930924 E-mail:illanjiam@gmail.com

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)

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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 textile dye. We have found t h a t Bacillus subtilis are capable of degrading C.I. Direct brown 2(C.I. No.17065.) To the best of our knowledge, no other microorganism is reported to biodegrade Direct brown2.This paper describes the degradation of direct brown 2by Bacillus subtilis and shows a plausible initial pathway for the biodegradation of Directbrown2. We also report the optimization of parameters required for the dyes efficiently in a short period. MATERIALS AND METHODS: Chemicals Direct brown 2 Dye (Figure 2) provided by the tannary division and the isolates (Bacillus subtilis Pseudomonas aeruginosa, Psedomonas putida) used in these studies were isolated from textile effluent collected from Tirupur, Tannery effluent Pallavaram, Chennai. All other reagents used were of analytical grade .
Control 4 Photo plate 4 Day 1 Day 2 Day 3 Day Photo plate 3 - Decolorization of Azo dye Direct Brown II by Azo Reductase Enzyme from Bacillus subtilis

Fig 2 . Dye structure

Benzidine

1. Salicylic acid 2. (Alkaline)Gamma acid Ci constitution no 22311 direct brown2 (Reddish brown ) colourist Medium: The Bacillus subtilis Pseudomonas aerogenosa, Pseudomonas putida culture was routinely grown at 37C in the basal culture medium, Bushnell and Hass medium (BHM) containing the following in g/l, MgSo 4 0.2, 4 CaCl2 0.02, KH PO 1.0, K HPO4 1.0, 2 2 4 2 4 NH No 1.0, FeCl3 0.05, Glucose 0.9, Yeast 4 3 3 extract 0.9 Direct brown 2 -100mg. Measurement of Dye Concentrations: The dye concentrations were measured with a UV/VIS spectrophotometer (Elico-159 Spectrophotometer) at regular intervals during the decolourization 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. (Super spin R-VIFm plasto crafts). The dye concentrations were determined from the attenuance (O.D) of the culture at 438nm. Decolorization activity was calculated as follows: Decolorization(%) = Initial absorbance Observed absorbance X 100 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 (100-200 mg/l), , pH (5-9), Temperature (37-45 0C), 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 Directbrown2 dye was autoclaved at 1210C for 15 minutes. Bacillus subtilis culture was plated on nutrient agar plates containing Directbrown2(200mg/l). The plates were wrapped with parafilm and were incubated at 37 0C for 7days. The plates were observed for clearance of the surrounding the colonies. photo plate 1 photo plate 2

Analysis of UV/Visible Spectrophotometer: Under static conditions, the culture with an initial dye (Directbrown2) concentration of 10% (v/v) was 90% decolorized in 72 hours. UV/Visible spectra of culture supernatants of 0 hour and 72 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 1480C 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 CODmg/l = (A-B) x N x Equi.wt of O 2 x1000 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) 1 ml 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 , 4 FeCl2 and seeding/L of water. Before use 3 bring dilution water temperature to 20C. Dilution water was aerated with organic free filtered air. All the bottles are kept in BOD incubator at 2000C for 5 days. After incubation 1ml of MnSO 4 , Alkali iodide 4 solution and sulphuric acid was added to form brown color solution. After color formation they were titrated against their Na SO for 2 4 their BOD values. The readings were noted. BODmg/l = B-T(v) x 250 S(v)

A Control , B - Test

A - Test, B - control

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B-volume of NaSo 4 used for blank (v)-volume of NaSo 4 used for sample S(v)-volume of sample BOD values were compared between the initial medium containing dye solution and decolourized medium. Thin Layer Chromatography (TLC) Degradation of dye, Direct brown 2, was analyzed 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 plate supplied by the residue was spotted on TLC plates in which micro syringe were used. The solvent system used was methanol, chloroform the ratio of 1:9 respectively. Effect of culture conditions on dye decolorization: The culture under agitation conditions demonstrated a better growth than that underStatic conditions. But the bacterial species Bacillus subtilis exhibited dye decolorizing activity only when incubated under the stationary conditions, whereas, negligible decolorization (30%) was noticed under the agitating conditions. Stationary cultures exhibited apparently complete decolorization (90%) of Direct brown2(fig 3) with in 72 hrs of incubation (fig 3),(Table 1) and maximum degradation was observed in 25 ppm (90% Bacillus subtilis, 78% Pseudomonasaerogenosa, 60% by Pseudomonas putida).
Fig : 4 Effect of different PH on Dye Degradation

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].

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 were similar to those studies on E. coli NO 3 and Pseudomonas luteola [27].

Table 2Analysis of Effect of ph on Percentage of degradation (Concentration of dye, 100 ppm)

Azo Dye Direct Brown II Percentage of degradation S.No Concentration of Dye Bacillus Pseudomonas (100ppm) subtilis putida 1 2 3 pH 5 pH 7 pH 9 35.6 66.5 64.3 33.9 70.2 69.4 Pseudomonas aerogenasa 35.6 63.2 61.2

Based on the above result ph 7 is ideal for all three organisms

Effect of Temperature on dye Decolorization The dye decolorization activity of our culture was found to increase with increase in incubation temperature (Figure-5) 370c to 45 0C with maximum activity attained at 45 0C (60%). Further decrease in temperature resulted in marginal reduction in decolorization activity of the bacterial culture Bacillus subtitles (Table-3), so the bacterial culture B.subtilis was more resistant to temperature, increase in decolorization activity at higher temperature can be attributed to the cell viability or to the resistant of the azo reductase enzymes (14). Maximum dye decolorization activity of the bacterial consortium NBNJ6 was noticed at 37o C [31].

Fig : 3 Effect of culture conditions on dye decolorization Table 1 Effect of culture conditions on dye decolorization
Azo Dye Direct Brown II Percentage of degradation S.No Concentration Bacillus Pseudomonas of Dye(ppm) subtilis Putida 1 2 3 4 25ppm 50ppm 100ppm 200ppm 90% 60% 40% 30% 78% 60% 55% 30% Pseudomonas aerogenasa 60% 48% 34% 30%

Fig : 5 Effect of temperature in dye degradation Table 3 Effect of temperature in dye degradation
Solano 1 2 3 Organism Pseudomonas putida Pseudomonas aerogenasa Bacillus subtilis Temperature C 37 68% 63% 50% 45 15% 8% 60%

Effect of pH on Dye decolorization Bacterial culture generally exhibited maximum decolorization rate at pH values near 7. Decolorization of CI Direct brown 2 at various pH value by the Bacillus subtilis is in Fig. 4. It shows that an increase in pH from 5.0 (35.6%) to 7 (66.5%) (Table 2) while the decolorization rate value decreased as pH was increased further from 7.0 (66.5%) to 9.0 (64.3%). The rate of decolorization for B.subtilis was optimum in the narrow pH range from 7.0 (66.5%) to 8.0 (64.3%) with marked reduction in decolorization activity at pH 5.0. Both Pseudomonas putida and Pseudomonas aeruginosa exhibited best decolorization rate at pH 7 and with constant decolorization rates up to pH 9.5 [26]. Klebsiellapneumoniae 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

Effect of Different concentration of Dye decolorization Decolorization activity of the bacterial culture Bacillus subtilis was studied using Directbrown2 at different initial concentrations varying from 25 ppm to 200 ppm (Fig.6). Rate of decolorization increased with increase in initial dye concentration up to 25 ppm ( (90%), Bacillus subtilis, 78%

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Pseudomonasaerogenosa, 60% pseudomonas putida (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 within narrow range of 0.1-0.2 g/l. [31] They reported that the Direct red 81 decolorization rate was increased with increase in initial dye concentration up to 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 exposed for treatment of dye bearing industrial waste waters. 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 24% of Directbrown2 within 72 hour of incubation. [36] They reported lactose (5g/l) and yeast extract (5g/l) in this study 2% sucrose was best carbon source shows 96% of degradation. 200 (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 most effective carbon-nitrogen source in decolorization of Direct red 81 by bacterial consortium NBNJ6. In the present study Beef extract organic nitrogen(26%) and sucrose is the best carbon source.(96%). Ammonium nitrate is the best inorganic nitrogen source( 85% ).
Table 5. Effect of Different Sources of Carbohydrate
Sl.No 1 2 3 4 Concentration of Dye(ppm) 100 100 100 100 Source of carbohydrate Sucrose Glucose Arabinose Citrate Percentage of degradation 96% 80% 39% 53%

Based on the above result sucrose was found to be the best carbohydrate source and was used for further studies.

Fig : 6 Table 4

Effect of Different concentration of Dye decolorization Effect of Different concentration of Dye decolorization
Pseudomonas aerugenasa 60% 48% 34% 30%

Azo Dye Direct Brown II Percentage of degradation S.No Concentration of Bacillus Pseudomonas Dye(ppm) subtilis Putida 1 2 3 4 25ppm 50ppm 100ppm 200ppm 90% 60% 40% 30% 78% 60% 55% 30%

Fig 8. Effect of different concentration of Sucrose at 100 ppm Table 6. Effect of different concentration of Sucrose at 100 ppm
Sl.No 1 2 3 4 5 Concentration of dye (ppm) 100 100 100 100 100 Concentration sucrose in % 0.00 0.20 0.50 1.00 2.00 Percentage of degradation 3.00% 11.80% 21% 69% 88%

In order to find out the optimum Bacillus subtilis inoculum needed for faster and higher percentage decolorization by decolorizing ability was tested at different inoculums concentrations starting from 5 to 30% (v/v) (Fig 6). The decolorization rate increased with increase in the inoculum size, reaching maximum (1.984 mg/l/h) (Table 4) at 20% (v/v) inoculum size. However, beyond 20% (v/v) inoculum size rate 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.

Based on above result 2% was found to be the best concentration and was used for further studies

Fig 9. Effect of different Concentration of Inorganic Nitrogen\Source at 100ppm Fig 7 . Effect of Different Sources of Carbohydrate Table 7. Effect of different Concentration of Inorganic NitrogenSource at 100ppm
Sl.No 1 2 3 4 Concentration of Dye(ppm) 100 100 100 100 Sources of Inorganic Nitrogen Ammonium Sulphate Ammonium chloride Ammonium nitrite Sodium nitrite Percentage of degradation 41% 75% 85% 41%

Effect of dye & inoculum size concentrations on dye decolorization Bacterial culture Bacillus subtilis exhibited maximum decolorization of Directbrown2 dye when starch & peptone were supplemented in the medium (Table 5). In absence of co-substrate the bacterial culture was unable to decolorize the dye, with indicates the availability of supplementary carbon source seems

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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 Directbrown2= 0.85) 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 10. Effect of different Concentration of Organic Nitrogen Source at 100 ppm Table 8. Effect of different Concentration of Organic Nitrogen Source at 100 ppm
Sl.No 1 2 3 4 Concentration of Dye(ppm) 100 100 100 100 Sources of Organic Nitrogen Tryptone Peptone Beef Extract Yeast Extract Percentage of degradation 20% 6% 26% 24%

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 Directbrown2. The decolorization ability of Bacillus subtilis was shown in plate1 and plate 2. Analysis of UV/VIS-spectra The UV-VIS spectra corresponding to initial (Fig 10) & final samples of decolorization experiments for Direct Brown 2 are shown in Fig 11. The absorbance analysed from 400 to 700nm. The initial dye solution showed high peak at the wavelength of 438nm. 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 i.e., K Cr O consumed during oxidation of 27 organic matter (biodegradable and non biodegradable) under acidic conditions. Chemical oxygen demand of degraded dye solution gets considerably reduced after degradation by Bacillus subtitles. COD of the solutions after degradation shows significant decrease from 12600 mg/l to 2200 mg/l. Similarly [37] They reported that the COD of the synthetic effluent (5200 mg/l) and React fix 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 from21050 mg/l to 1075 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). Characterization of Effluent from Dyeing Industry: 1.pH- 10 2.COD 1220 mg/L 3.Filter COD -1000 mg/L 4.Chloride -6248mg/l 5.Sulphate (SO4)- 3451 mg/l 6.TDS 5742 mg/L 7.Metal TDS 2.90 g/l 8.VFA - 840mg/l 9.TOC 452 mg/l 10.VDS 0.0237 g/l 11.BOD 410mg/lit 12.ORP 579 Rmv

Fig 11. TLC analysis after 72 hours Incubation

Fig 12. TLC analysis after 72 hours Incubation

(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 , Pseudomonasaerogenosa, pseudomonas putida to decolorize the Textile dye Directbrown2 with decolorization efficiency of 90%,78% 60% thus suggesting its application for decolorization of dye bearing of industrial wastewaters. Presence of a Co-Substrate (sucrose & ammonium nitrate) is the essential conditions for attaining maximum decolorization efficiency. The anaerobic decolorization of Directbrown2 dye occurs as a result of reduction of N=Nbond accompanied by the formation of aromatic amines The amine intermediates formed in static conditions treatment can be removed by agitating conditions & approximately 90% decolorization under agitating conditions after a reaction period of 72hrs. ACKNOWLEDGEMENT The Author is grateful for the guidance and facilities provided by Dr.Kantha D. Arunachalam, Professor and Co-ordinator , Centre for interdisciplinary Research, SRM University, Kattankulathur 603 203. REFERENCES
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16. 17. 18. 19. 20. 21.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 9. September 2011

2969-2974