Int J Pharm Bio Sci 2013 July; 4(3): (B) 524 - 534

Research Article

Microbiology

International Journal of Pharma and Bio Sciences

ISSN 0975-6299

BIOTRANSFORMATION OF SULPHONATED AZO DYE DIRECT RED 5B BY MARINOBACTER SP. DR-7- A BIOREMEDIAL ASPECT IN MARINE ENVIRONMENT *SHERTATE R.S. AND THORAT P.R. P.G. Department of Microbiology and Research Center, Shri Shivaji Mahavidyalaya, Barshi – 413411, Dist. - Solapur, MS, India.

ABSTRACT A marine bacterium capable of degrading the textile azo dye Direct Red 5B was isolated from natural environments on nutrient medium containing the high salinity and the dye. It was identified as Marinobacter sp. DR-7 (Accession No. HF558993) on the basis of biochemicals and phylogenetic analysis based on 16s rRNA gene sequence. The decolorization of the azo dye Direct Red 5B in nutrient broth and half strength nutrient broth having 8.0% salt concentration was up to 94.00% and 92.00% respectively in 24 hours. The degradation products formed were analyzed by GC-MS technique and it was found that culture degraded Direct Red 5B to the products having molecular weights 98, 99, 100, 149, 150, 149, 150, 149, 150, 223, 149, 150, 57, 113, 149, 167 and 279.The isolate reduced the COD of the dye up to 70%. KEYWORDS : Marine Bacteria, Sulphonated azo dye, Bioremediation, GC-MS, COD Reduction.

SHERTATE R.S. P.G. Department of Microbiology and Research Center, Shri Shivaji Mahavidyalaya, Barshi – 413411, Dist. - Solapur, MS, India.

*Corresponding author

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1. INTRODUCTION The textile industry accounts for two-thirds of the total dyestuff market. Over 10,000 dyes with an annual production of over 7 X 105 metric tons are commercially available1 for use by this industry, of these azo dyes which are resistant to chemicals and environmental conditions, are most commonly used by this industry. Azo dyes are characterized by the presence of one or more azo groups substituted with aromatic amines. A substituent often found in azo dyes is the sulfonic acid group (–SO3H). The azo dyes containing this substituent are called as sulfonated azo dyes. Sulfonated azo dyes are widely used in the different industries. The fixation rate of these reactive dyes (including azo dyes) in dying is as low as 50%2 which results the release of 10–15% water soluble azo dyes into the environment through wastewater discharge3. Sulfonated and unsulfonated azo dyes have a negative aesthetic effect on the wastewater and some of these compounds and biodegraded products are toxic, carcinogenic and mutagenic4. Azo dyes represent a major group of dyes causing environmental concern because of their color, biorecalcitrance and potential toxicity to animal and human5. The treatment of such dye containing effluent was initially carried by using physical and chemical treatment processes including adsorption, concentration, chemical transformation, but with time, potential hazards and disadvantages of these methods were noted as, formation of toxic sludge and formation of even more toxic metabolites6,7. Bioremediation is becoming important, because it is costeffective and environmentally friendly, and produces less sludge8,9. Bioremediation in salty environments inevitably requires the application of these halotolerant and halophilic microorganisms which are able to grow under such harsh conditions. Halotolerant and halophilic

bacteria have been utilized in the bioremediation of oil10,11 and oxyanion pollution12,13 but there is a dearth of information on their effectiveness in decolourization of solutions containing azo dyes and high concentrations of sodium chloride. The use of microorganisms able to degrade azo dyes in presence of salt could help to prevent costly dilution to lower the salinity, or the removal of salt by physicochemical methods before biological treatment. A degradative pathway has been elucidated for sulfonated azo dyes using Pseudomonas strains14.Dye containing waste if disposed in marine water adversely affect the marine life. erefore, industrial effluents containing dyes must be treated before their safe discharge into the environment. In the present study, a bacterium was isolated from marine environment capable of decolorizing and degrading a sulphonated textile azo dye Direct Red 5B. This strain was studied for decolorization and degradation of the dye Direct Red 5B in various different conditions like in complete nutrient medium, in half strength nutrient medium, cell-free extract and in presence of different co-substrates. The decolorization of the dye was monitored spectrophotometrically (Systronics-106) at its specific absorbance maxima (λmax) 510nm. Percent COD reduction of the dye was calculated.

2. MATERIALS AND METHODS Sample source and collection Soil samples from salterns (Saltpan), areas nearby waste disposal sites of the textile industry, sewage, sludge from effluent treatment plants (ETP), marine water and compost were collected as the source of microorganisms; these soil samples were kept in a container and refrigerated till use.

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2.1.

Structure and properties of the dye Direct Red 5B HO3 S

N N

N N OH

HO3 S

NH CO

Figure 1 Structure of the dye Direct Red 5B. Dye Molecular Weight Molecular formula Composition λmax

– Direct Red 5B – 631.63574 – C29H21N5O8S2 – C (55.14%), H (3.35%), N (11.09%), O (20.26%), S(10.15%) – 510nm

2.2. Acclimatization and isolation of microorganisms Soil samples from salterns (Saltpan), area nearby waste disposal site of textile industries, marine water, sewage, and sludge from ETP along with compost were collected and homogenized. The micro-flora from the homogenized samples, were acclimatized by adding the dye Direct Red 5B in increasing concentration for the period of one month. One gram of acclimatized soil was inoculated in the nutrient broth containing different NaCl concentrations and after incubation isolation was carried out on nutrient agar having the same NaCl which was amended with Direct Red 5B (Figure 1) at a final concentration of 1000µg/ml. Colony showing decolorization was designated as DR-7 and used for further studies. 2.3. Decolorization of Dye in Nutrient Broth, Half strength nutrient broth and in presence of different co-substrates Isolate DR-7 was used to inoculate in 20ml nutrient broth containing 8.0% NaCl and 1000µg/ml concentration of dye. The tube was then incubated at ambient temperature for 24 hrs and observed for decolorization of the dye. In addition, half strength nutrient broth was also used to test the ability of

isolate to decolorize the dye Direct Red 5B at lower concentration of nutrients. Also the dye decolorization was also studied in presence of different 1% co-substrates viz. glucose, yeast extract and starch with the same NaCl and dye concentration. 2.4. Decolorization studies by Cell-free extract The cells grown in nutrient broth were separated by centrifugation using cooling centrifuge (BIO-LABS 165-R) at 7000 rpm for 20 min at 30C. These cells were then suspended in 50mM phosphate buffer pH – 7.4. The cell suspension in the buffer was properly cooled and lysed by using Ultrasonicator (Sonic-Vibra Cell System – 130) the output was kept 50amp with 6 strokes of 25 s each, time interval kept was 2min at 40C. This homogenate was centrifuged at 10000 rpm for 10 min so as to separate the cell debris and the intracellular enzymes. The supernatant was used as a crude enzyme source. The supernatant containing the crude enzyme was then added with 1000µg/ml concentration of dye solution and 8.0% NaCl and observed for dye decolorization. The percent decolorization studies were monitored by using spectrophotometer (Systronics – 106 model).

The percent decolorization of the dye was determined by using following formula.

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2.5. Percent COD reduction Studies Percent COD reduction value of the decolorized dye in nutrient broth by isolate DR-7 was calculated. 2.6. GCMS Analysis The samples for GCMS analysis were prepared by extracting the products in DiChloro Methane (DCM) solvent. The decolorized broth was centrifuged at 10,000 RPM for 20 min. The supernatant was decanted and was taken in a separating funnel. Equal amount of DCM was added to the separating funnel. The separating funnel was shaken vigorously for 20 min to extract the products in DCM. Then contents were kept undisturbed for the separation of solvent phase and liquid phase. The separated solvent was then taken out from the funnel. The products extracted in the solvent were concentrated in the vial by evaporating of the solvent at room temperature and then analyzed by Gas chromatography and Mass spectroscopy (GCMS). The GC-MS analysis of metabolites was carried out using a Shimadzu 2010 MS Engine, equipped with integrated gas chromatograph with HP1 column (60 m long, 0.25 mm id, non-polar). Helium was used as carrier gas at a flow rate of 1 ml min−1. The injector temperature was maintained at 2800C with oven conditions as: 800C kept constant for 2 min and increased up to 2000C with 100C min-1 raised up to 2800C with 200C min−1 rate. 2.7. Identification of the isolate sequencing of 16S rRNA gene The 16S rRNA sequencing was done at National Center for Cell Sciences, University of Pune Campus, Pune. The sequence was submitted to EBI (European Bioinformatics Institute). Sequence was analyzed at the National Center for Biotechnology Information (BethesdaMD) (http//: www.nbi.nlm.nih.gov/BLAST) for closed homology using BLASTn algorithm. The sequences downloaded from the NCBI

database were aligned by using CLUSTAL X2 multiple sequence alignment tool, the phylogenetic evolutionary history was inferred using the Maximum Composite Likelihood analyses were analysis15Phylogenetic conducted in MEGA 4.0. Phylogenetic tree building was performed using MEGA 4.016. 2.8 Microbial Toxicity of Dyes and their Biodegradation Products Testing It is very important to know whether biodegradation of a dye leads to detoxification of the dye or not. Agar well bioassay is the most common technique used to evaluate the microbial toxicity. This can be achieved by microbial toxicity tests with the original dyes and their biodegradation products. The microbial toxicity was tested on three test organisms viz. Pseudomonas sp., Rhizobium sp. and Azotobacter sp. After the confirmation of dye degradation, the degraded solution (decolorized broth) was poured in the wells prepared in nutrient agar previously spreaded with the test organisms. These plates were incubated at ambient temperatures for 24 hours. The zone of inhibition around the wells proved the toxicity of the dye and their degraded products.

3. RESULTS 3.1. Isolation and Identification The organism was isolated from the soil on nutrient agar having 8.0% NaCl and was identified by using biochemical observations and 16s rRNA analysis technique. The sequence was submitted to EBI (European Bioinformatics Institute) and received the accession number HF563062. The biochemical results showed Catalase, oxidase, Urease, Nitrate Reduction tests positive, able to ferment Glucose, sucrose and lactose with production of Acid and Gas whereas unable to ferment mannose and maltose. The phylogenetic tree was developed by using MEGA 4.0. (Figure 2)

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Int J Pharm Bio Sci 2013 July; 4(3): (B) 524 - 534 AF479689 Marinobacter litorali

66 70

AY180101 Marinobacter excellen DQ235263 Marinobacter vinifirm

19

EU293412 Marinobacter mobilis

18

EU293413 Marinobacter zhejiang

77 30

AY147906 Marinobacter lipolyti AM503093 Marinobacter guineae EF660754 Marinobacter goseonge

48

12

DQ414419 Marinobacter gudaonen

35

EF157832 Marinobacter segnicre 9

DQ458821 Marinobacter pelagius

22

EU496088 Marinobacter santorin

55

99 DQ325514 Marinobacter koreensi

DR-7 80 AJ609271 Marinobacter bryozoor

EF486354 Marinobacter salicamp AY517632 Marinobacter flavimar EF028328 Marinobacter salsugin

12

AJ609270 Marinobacter sediminu

48

0.005

Figure 2 Phylogenetic analysis of the 16S rRNA sequence of Marinobacter sp. DR-7. The percent numbers at the nodes indicate the level of bootstrap support based on Maximum Composite Likelihood analysis of 1000 replicates. The scale bar indicates the base pair substitutions per site. 3.2. Percent Decolorization in Nutrient Broth, Half (½) Strength Nutrient Broth, Cell Free Extract and in presence of different co-substrates Marinobacter sp. DR-7 was examined for its percent decolorization capacity in Nutrient broth, in half (½) strength nutrient broth and in cell free extract containing 8.0% NaCl and dye concentration of 1000µg/ml. Also the decolorization of the dye were examined in presence of 1% co-substrates viz. glucose, yeast extract and starch as a carbon source and nitrogen source. The results of percent decolorization of dye Direct Red 5B is given in Table I. Table 1 Percent Decolorization in Nutrient Broth, Half (½) Strength Nutrient Broth, Cell Free Extract and 1% Co-substrates by Marinobacter sp. DR-7 in 24 hrs at λ max 510nm. Culture code

DR-7

Dye

Direct Red 5B (λmax 510nm)

% Decolorization (after 24 hrs) Nutrient Broth

½ Strength Nutrient Broth

Co-Substrates (1%) Yeast Glucose Extract

Cell-Free Extract (%) Starch

92.01

94.00

90.00 94.00

92.00

95.00

3.3. Percent COD Reduction After decolorization of the dye in nutrient broth having 8.0% NaCl, the percent COD reduction of the dye Direct Red 5B by Marinobacter sp. DR-7 was observed to be 95.65% after 24 hours.

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3.4. Molecular weights of the degraded products of dye The GCMS analysis report showed that the dye Direct Red 5B was degraded by Marinobacter sp. DR-7and not decolorized (Figure 3). The molecular weights of the degraded products 98, 99, 100, 149, 150, 149, 150, 149, 150, 223, 149, 150, 57, 113, 149, 167, and 279 while the probable degradation pathway of the dye Direct Red 5B which depicts the possibility of the products formed (Figure 4). Figure 3 GC-MS Analysis Report of the Direct Red 5B.

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Figure 4 Probable Degradation Pathway of Direct Red 5B by Marinobacter sp. DR-7.

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Int J Pharm Bio Sci 2013 July; 4(3): (B) 524 - 534 OH HO 3 S

N N

N

N NH CO

HO 3 S Direct Red 5B Molecular Formula = C29H21N5O8S2 Formula Weight = 631.63574+?

H 2N HO 3 S

N

N

NH2

HO 3 S

4-[(4-aminophenyl)diazenyl]benzenesulfonic acid Mol. wt.- 277

HO 3 S

H2N

NH2 H 2N

4-aminobenzenesulfonic acid Mol. wt.- 173

aniline Mol. wt.- 93

CO

NH2

benzene-1,4-diamine Mol. wt.- 108 NH3 ammonia Mol. wt.- 17

SO 3 H Mol. wt.- 81 NH2

NH CO Mol. wt.- 342.36

H 2N aniline Mol. wt.- 93

HO 3 S Mol. wt.- 105

H2C

NH2

3,7-diaminonaphthalene-2sulfonic acid Mol. wt.- 238

CH2

H3C CH3 ethane - ethene (1:1) Mol. wt.- 58

H2N HO 3 S

3-aminonaphthalene-2-sulfonic acid Mol. wt.- 223 H 3C

CH3

ethane Mol. wt.- 30

H 2N

naphthalen-2-amine Mol. wt.- 143

benzene Mol. wt.- 78

CH2 (1E,3Z,5E,7Z,9Z)-cyclodeca -1,3,5,7,9-pentaene Mol. wt.- 130

CH3 (3Z)-penta-1,3-diene Mol. wt.- 68

CH2 naphthalene Mol. wt.- 128

CH2 buta-1,3-diene Mol. wt.- 54

CH3 CH2 1-ethenyl-2-methylbenzene Mol. wt.- 118

3.5 MICROBIAL TOXICITY TESTING Microbial toxicity of the dye Direct Red 5B and its degradation products was studied on Pseudomonas sp., Rhizobium sp. and Azotobacter sp by the agar well assay. The results showed that the wells which were poured with decolorized broth had no zone of

inhibition and wells with original dye solution had zone of inhibition. This confirmed that the original dye solution 1000µg/ml was toxic to the bacteria but its degradation products were non toxic to the bacteria.

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DISCUSSION The Marinobacter sp. DR-7showed, 94.00% and 92.00% decolorization of dye Direct Red 5B in complete nutrient broth medium having 8.0% NaCl and in half strength nutrient medium having the same salinity respectively. Murthi, et al., (2006) reported that the textile reactive dyes and their effluents decolorized very efficiently with the use of Tramets hirsute and Pleorotus florida sp. They also showed that there was significant increase in the percent decolorization by Pleorotus florida when this culture was supplemented with 2% glucose. Gondaliya and Parikh (2012) reported the highest percentage decolorization 97.04% of Reactive Orange–16 was obtained by Serratia marcescens when additional supplement of glucose (1 g/l) was added in Nutrient broth. These observations are similar with our results. There was significant increase in the percent decolorization by Marinobacter sp. DR-7, when this culture was supplemented with 1% glucose, 1% yeast extract and 1% Starch. Dyes are deficient of carbon source, so it is necessary to supplement additional carbon or nitrogen source to assist biodegradation (Senan and Abraham, 2004). Dhanve et al., (2008) reported that the Diazo Reactive Dye Navy Blue HE2R which is decolorized to 91.2 % by the Exiguobacterium spp. RD-3 in static aerobic condition showed toxicity to the agriculturally important plants in India like Triticum astivum and Ervum lens linn. It has been reported that halophilic microorganism Shewanella putrefaciens to be capable of the complete removal of Reactive Black-5, Direct Red-81, Acid Red-88 and Disperse Orange-3 (all 100 mg L-1) within 8 h in presence of 40 g L-1 NaCl (Ammozegar et al., 2010). These observations are very much similar with our results. Vigneeswaran et al., (2012) studied the biodegradation and bioremediation of azo dye entrenched soil by Pseudomonas sp. and reported that this species can decolourize the azo dye upto 75.00%. Mubarak Ali et al., (2011) showed the decolorization of the dye Amido Black and textile dye effluent by the

marine cyanobacterium, Oscillatoria formosa NTDM02, which decolorize the textile effluent efficiently in short period of time. These results were similar to our results. The results of microbial toxicity indicate that the azo dye degradation products formed after biodegradation by Marinobacter sp. DR-7 were less toxic compounds compared to the original azo dye. These results are in agreement with result of Kalyani et al., (2009) who found that the metabolite products after biodegradation of Reactive Red 2 and Reactive Blue 59 were less toxic compared to the original dye.

CONCLUSION Bioremediation is the microbial clean-up approach. Microbes can acclimatize themselves to stoxic wastes and new resistant species develop naturally, which can transform various toxic chemicals to less harmful forms. The isolated novel species DR-7 was a member of the genus Marinobacter which was identified by 16S rRNA sequence analysis and biochemical tests. The dye was confirmed for degradation by the Marinobacter sp. DR-7. In cell free extract the decolorization was observed, which proved the activity of enzymes on the dye. The COD of the dye was reduced after the treatment of Marinobacter sp. DR-7. The dye was degraded by Marinobacter sp. DR7.The highest decolourization was shown with additional supplements of carbon source such as glucose, nitrogen source such as yeast extract by Marinobacter sp. DR-7.

ACKNOWLEDGEMENT We would like to offer our sincere thanks to Staff of Indian Institute of Technology Powai, Mumbai for GCMS analysis and National Center for Cell Sciences, University of Pune for the 16s rRNA sequencing of the isolate. Our sincere thanks to Principal, Shri Shivaji Mahavidyalaya , Barshi for providing the Laboratory facilities.

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