Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e10, 2012 www.elsevier.com/locate/jbiosc

Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS Rijuta G. Saratale,1 Soniya S. Gandhi,2 Madhavi V. Purankar,2 Mayur B. Kurade,2 Sanjay P. Govindwar,2, 3 Sang Eun Oh,4 and Ganesh D. Saratale2, 3, * Department of Biotechnology, Shivaji University, Kolhapur 416004, Maharashtra, India,1 Department of Environmental Biotechnology, Shivaji University, Kolhapur 416004, Maharashtra, India,2 Department of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India,3 and Department of Biological Environment, Kangwon National University, Chuncheon 200-701, South Korea4 Received 9 August 2012; accepted 8 December 2012 Available online xxx

A novel bacterium was isolated from the soil of Ichalkaranji textile industrial area. Through 16S rRNA sequence matching and morphological observation it was identified as Lysinibacillus sp. RGS. This strain has ability to decolorize various industrial dyes among which, it showed complete decolorization and degradation of toxic sulfonated azo dye C.I. Remazol Red (at 30
C, pH 7.0, under static condition) with higher chemical oxygen demand (COD) reduction (92%) within 6 h of incubation. Various parameters like agitation, pH, temperature and initial dye concentrations were optimized to develop faster decolorization process. The supplementation of cheap co-substrates (e.g., extracts of agricultural wastes) could enhance the decolorization performance of Lysinibacillus sp. RGS. Induction in oxidoreductive enzymes presumably indicates involvement of these enzymes in the decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Remazol Red into various metabolites. The phytotoxicity assay (with respect to plants Phaseolus mungo and Sorghum vulgare) revealed that the degradation of Remazol Red produced nontoxic metabolites. Finally Lysinibacillus sp. RGS was applied to decolorize mixture of dyes and actual industrial effluent showing 87% and 72% decolorization (in terms of decrease in ADMI value) with 69% and 62% COD reduction within 48 h and 96 h, respectively. The foregoing result increases the applicability of the strain for the treatment of industrial wastewaters containing dye pollutants. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Decolorization; Remazol Red; Lysinibacillus sp. RGS; ADMI; Chemical oxygen demand; Phytotoxicity]

Azo dyes are the largest chemical class of dyes (3000 different varieties) because of their stability and the variety of colors available compared to natural dyes (1). It was estimated that about 60e70% of all textile dyestuffs contains azo dyes having one or more azo bond (eN]Ne) and thus making them the largest group of synthetic colorants released into the environment (2). Generally they are widely used in the textile, paper, food and leather industries (3). It was observed that the higher electronic density functional group of azo dye leads to low or no capability for microbial decolorization (4). Due to this reason sulfonated group of azo dyes are normally considered to be more recalcitrant than carboxylated group of azo dyes (5). The industrial effluent contains a significant amount of residual azo dyes because of the inefficiency during dyeing processes and improper discharge of these textile dye effluent in aqueous ecosystems leads to the reduction in sunlight penetration, dissolved oxygen concentration, biochemical oxygen demand (BOD), chemical oxygen demand (COD), water quality and are lethal to resident organisms (6). They also become threat to public health * Corresponding author at: Department of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India. Tel.: þ91 231 2609152; fax: þ91 231 2691533. E-mail address: [email protected] (G.D. Saratale).

and natural ecology because synthetic azo dyes and their metabolites are toxic, carcinogenic and mutagenic in nature (7). In India, an average textile mill discharges about 1.5 million liters of contaminated effluent per day was recorded (8). Moreover it was observed that the traditional textile finishing industry consumes about 100 L of water to process about 1 kg of textile material (9). Thus treatment of industrial effluents containing azo dyes and their metabolites becomes necessary prior to their final discharge to the environment to reduce their levels of toxicity and to minimize their pollution impact. The conventional physical/chemical treatment methods cannot be completely remove azo dyes and their metabolites, generate significant amount of sludge that may cause secondary pollution problems and require complicated procedures (10). Bioremediation is the microbial clean up approach is on the front line and priority research area in the environmental sciences. In this process microbes can acclimatize themselves to the toxic wastes and can transform various toxic chemicals to less harmful forms using the biotransformation enzymes system (11). In the current scenario, microbial or enzymatic treatment offers an indispensable, ecofriendly and cost-effective solution towards restoring azo dye polluted ecosystems. Also, the biological treatment system could help to reduce the enormous water

1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

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SARATALE ET AL.

consumption compared to conventional physicochemical methods (5,12). Many biological agents including bacteria, fungi, yeasts, actinomycetes and algae are capable of degrading azo dyes, among which bacterial cells represent an inexpensive and promising tool for the removal of various azo dyes from textile dye effluents (2). Transfer of two electrons produces the stable dianion radical during reduction of azo dyes (2). It was observed that higher electronic density functional group of azo dye becomes unfavorable to second electron transfer to form the dianion and leads to low or no capability for decolorization (4). Sulfonated reactive group of azo dyes are normally considered to be more recalcitrant than carboxylated azo dyes because of this reason. The rate limiting step during bacterial decolorization of sulfonated azo dyes is the permeation through the bacterial cell membrane (2,6). Moreover the isolation of potent species and thereby degradation is one of the interest in biological aspect of industrial effluent treatment. Recently a substantial amount of research on the subject of color removal has been carried out using single bacterial cultures like; Pseudomonas luteola, Bacillus fusiformis KMK5, Micrococcus glutamicus NCIM2168, Bacillus sp. VUS, Proteus mirabilis, Lysinibacillus sp. strain AK2 and Aeromonas hydrophila has shown very promising results for the azo dye (1,5,6,12e14). Lysinibacillus sp. was found to be catabolically versatile with the ability to utilize a wide range of unusual substrates such as; ethanediol, organophosphorus pesticide malathion; omeprazole, fomesafen and dibenzothiophene (15,16). It is also involved in the reduction of heavy metal pollution (Cr) (16). Recently Kim et al., (17) reported the involvement of Lysinibacillus fusiformis in the production of hydroxy fatty acids from oleic acid increases the biotechnological value of this strain. Decolorization and degradation of sulfonated azo dye Metanil Yellow by Lysinibacillus sp. strain AK2 and Reactive Violet 5R by mixed cultures of six organisms including Lysinibacillus sp. V3DMK was reported earlier (10,18). In this study, we focused on the isolation and identification of dye-degrading microorganism from textile effluent contaminated soil having decolorizing ability for several different dyes commonly used in various textiles and allied industries in India. We have investigated the potential of isolated Lysinibacillus sp. RGS in the decolorization and degradation of C.I. Remazol Red. Effects of various physicochemical parameters have been studied to achieve maximum dye degradation. The possible enzymatic mechanism in the decolorization as well as the nature and toxicity of the degradation products were systematically investigated. The various intermediates formed have been analyzed during the degradation of Remazol Red by using various analytical techniques. We have also determined the ability of this strain to decolorize mixture of dyes as well as actual dye wastewater. MATERIALS AND METHODS Dyestuff and chemicals The textile dyes used, i.e., C.I. Remazol Red, C.I. Rubin, C.I. Scarlet RR, C.I. Brilliant Blue, C.I. Brown 3 REL, C.I. Golden Yellow HER, C.I. Methyl Red and textile industry effluent were generous gift from Mahalaxmi textile industry, Ichalkaranji, India. Veratryl alcohol, o-tolidine, dichloroindophenol (DCIP), catechol, n-propanol, and tartaric acid, were obtained from SRL Chemicals (India). 2, 2-Azinobis (3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) and NADH were purchased from Sigma Chemical Company (USA). All other chemicals were of highest purity and of an analytical grade. Isolation, screening, and identification of microorganism Soil samples were collected from the textile effluent disposal site of Mahalaxmi textile processing plant, Ichalkaranji. Two hundred milligrams of soil sample was added into 250 ml Erlenmeyer flask containing 100 ml of nutrient broth and mixture of textile dyes that contains C.I. Remazol Red, C.I. Rubin, C.I. Scarlet RR, C.I. Brilliant Blue, C.I. Brown 3 REL, C.I. Golden Yellow HER, C.I. Methyl Red at a final concentration of 30 mg/l. After 48 h of incubation, the 1 ml of cell suspension was transferred into fresh dye containing nutrient broth to screen the color removal ability of culture. After complete decolorization, the 102 fold diluted sample from decolorized culture broth was transferred into nutrient agar plates that contain C.I. Remazol Red, C.I. Rubin, C.I.

J. BIOSCI. BIOENG., Scarlet RR, C.I. Brilliant Blue, C.I. Brown 3 REL, C.I. Golden Yellow HER, C.I. Methyl Red up to 50 mg/l. Colonies surrounded by decolorized zones were selected and isolated by streak plate method. Decolorization efficiency of selected bacteria (RGS, Iso-3, Iso-4, Iso-5, and Iso-6) for individual dye was checked in the nutrient broth. The best isolated microorganism (RGS) was further identified on the basis of morphological and biochemical characteristics. 16S rRNA sequencing and phylogenetic analysis The isolate was identified using 16S rRNA sequence analysis. 16S rRNA gene sequencing of isolated bacteria was carried out at Bangalore Genei, Bangalore, India. The nucleotide sequence alignment of the sequence was done at Blast-n site at NCBI server (http://www.ncbi. nlm.nih.gov/BLAST). The alignment of the sequences was done by using CLUSTALW program V1.82 at European bioinformatics site (http://www.ebi.ac.uk/clustalw). The bootstrap consensus tree inferred from 1000 replicates and was considered to represent the evolutionary history of the analyzed taxa. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates. The Phylogenetic tree was constructed using the aligned sequences by the neighbor-joining method using Kimura-2-parameter distances in MEGA 4 software (19). The sequence was refined manually after crosschecking with the raw data to remove ambiguities and submitted in Genbank databases. Microorganism and culture condition The isolated Lysinibacillus sp. RGS was maintained on nutrient agar slants and stored in test tubes at 4
C and subcultured monthly. The pure culture of Lysinibacillus sp. RGS was grown in 250 ml Erlenmeyer flask, containing 100 ml of nutrient broth (beef extract, 3 g/l; peptone, 10 g/l; NaCl, 5 g/l; pH 6.6) for 24 h at 30
C, under static anoxic condition (no aeration and no agitation). To study the effect of carbon and nitrogen sources on the decolorization of Remazol Red, semi-synthetic medium having following composition was used (g/l): C.I. Remazol Red, 0.050; (NH4)2SO4, 0.28; NH4Cl, 0.23; KH2PO4, 0.067; MgSO4$7H2O, 0.04; CaCl2$2H2O, 0.022; FeCl3$6H2O, 0.005; NaCl, 0.15; NaHCO3, 1.0 and 1 ml per liter of a trace element solution containing (g l1) ZnSO4$7H2O, 0.01; MnCl2$4H2O, 0.1; CuSO4$5H2O, 0.392; CoCl2$6H2O, 0.248; NaB4O7$10H2O, 0.177; and NiCl2$6H2O, 0.02 with different carbon and nitrogen sources (1% each) such as starch, sucrose, glucose, lactose, peptone, beef extract and yeast extract (6). Ten percent inoculum with an optical density of 1.0 (at 620 nm) grown in the nutrient broth for 24 h was used for inoculation of the synthetic medium. In addition, 1 g of wheat straw, paddy straw, sugarcane bagasse powder and wood shavings were mixed with 100 ml distilled water individually and autoclaved at 121
C for 20 min then, 5 ml extract of each agricultural waste was added in semi-synthetic medium and check the decolorization performance of C.I. Remazol Red by Lysinibacillus sp. RGS to make the process economically feasible. Nucleotide sequence accession number The 16S rRNA gene sequence of the isolated ‘Lysinibacillus sp. RGS’ was determined in this study and submitted to the GenBank database under accession number JX912157.1. Decolorization experiments In order to examine the decolorization performance of Lysinibacillus sp. RGS screening of various individual dyes (each 50 mg/l; Remazol Red, Rubin, Scarlet RR, Brilliant Blue, Brown 3REL, Reactive Golden Yellow HER, Methyl Red) was studied. Lysinibacillus sp. RGS culture was inoculated (1 ml inoculum; 6.0  107 CFU/ml) in 250 ml Erlenmeyer flask containing 100 ml of sterile nutrient broth and incubated under static and shaking (120 rpm) condition. Remazol Red (50 mg/l) was added in each flask after 24 h of incubation and reincubated under static and shaking condition. Aliquots (3 ml) of the culture broth were withdrawn after regular time intervals for analysis of pH and percent decolorization. The aliquot was centrifuged at 3000 g for 15 min to separate cell mass. Supernatant was used to determine the decolorization by measuring the change in the absorbance of culture supernatants at the maximum absorption wavelength (lmax) of the respective dyes. Growth of microorganism in dye containing medium was measured by the gravimetric method after drying at 80
C until constant weight. The decolorization performance of mixture of azo dyes (Remazol Red, Rubin, Scarlet RR, Brilliant Blue, Brown 3 REL, Golden Yellow HER and Methyl Red) with a concentration of 50 mg/l each was studied in 250 ml Erlenmeyer flask containing 100 ml nutrient broth at 30
C under static condition. The original textile effluent was filtered by Whatmann grade one filter paper. The 70 ml of filtered effluent was distributed into 250-ml Erlenmeyer flasks and then sterilized at 121
C for 20 min. The 30 ml of inoculum was added in each flask and incubated under static condition. Samples were withdrawn after regular time intervals and analyzed for color intensity. Controls without effluent and inoculum were run under similar conditions. All decolorization experiments were performed in triplicates. Abiotic controls (without microorganism) were always included. Establishment of optimum operation conditions To evaluate the effects of operational and environmental conditions on the decolorization performance of C.I. Remazol Red by Lysinibacillus sp. RGS, the batch decolorization experiments were carried out (24 h grown cells; dye concentration 50 mg/l); at different agitation speeds (0 and 100 rpm), temperatures (25e45
C), pH values (3e10), and initial dye concentrations (50e250 mg/l) at 30
C under static condition. Further, the decolorization of repeated addition of dye aliquots (50 mg/l) to culture media was also studied by Lysinibacillus sp. RGS in the nutrient broth under static condition without supplement of additional nutrients. All decolorization experiments were

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

VOL. xx, 2012

DECOLORIZATION OF REMAZOL RED BY LYSINIBACILLUS SP. RGS

performed in triplicates. The percentage decolorization was calculated (20) as follows: % Decolorization ¼

Initial absorbance  observed absorbance  100% Initial absorbance

(1)

The average decolorization rate (mg/h) was calculated (6) as follows: Average decolorization rate ¼

C% D 100  t

(2)

where C is the initial concentration of dye (mg/l) and % D is the dye decolorization (%) after time t (h). Preparation of cell free extract Lysinibacillus sp. RGS cells were grown in the nutrient broth, pH 6.6 incubated at 30
C for 24 h and harvested by centrifuged at 3000 g for 15 min. These cells (ca. 9.8 g/l) were suspended in potassium phosphate buffer (50 mM, pH 7.4) for sonication (Sonics-vibracell ultrasonic processor), keeping sonifier output at 40 amp and giving 8 strokes each of 40 s with a 2 min interval at 4
C. After centrifugation of this crude extract at 3000 g for 15 min the supernatant was used as a source of an enzyme. Similar procedures were followed to the cells obtained after complete decolorization (6 h). Protein content was estimated by the Biuret method. Protein concentration of cell free extract was kept constant (2.0 mg/ml) for the enzymatic studies. Oxidative enzymes during decolorization The oxidative enzymes were assayed spectrophotometrically in the cell free extract. Laccase activity was determined in a reaction mixture of 2 ml containing 10% ABTS in 20 mM potassium phosphate buffer (pH 4.0) and increase in the optical density was measured at 420 nm. Molar extinction coefficient of ABTS was 0.036 mM/cm at 420 nm (21). Lignin peroxidase activity was determined by monitoring the formation of propanaldehyde at 300 nm in a 2.5-ml reaction mixture containing 100 mM n-propanol, 250 mM tartaric acid, 10 mM H2O2 (6). Veratryl alcohol oxidase activity was determined by using veratryl alcohol as a substrate (22). The reaction mixture contained 1 mM veratryl alcohol in 0.1 M citrate phosphate buffer (pH 3.0) and 0.2 ml enzyme. Total volume of 2 ml was used for the determination of oxidase activity. Oxidation of the substrate at room temperature was monitored by an absorbance increase at 310 nm due to the formation of veratraldehyde. All enzyme assays were conducted in triplicate and the average rates were calculated to represent the enzyme activity. One unit of enzyme activity was defined as a change in absorbance Unit min/mg protein of the enzyme. Reductase enzymes during decolorization The azo reductase activity was assayed by modifying earlier method (23); monitoring the decrease in the Methyl Red concentration at 440 nm in a reaction mixture of 2.2 ml containing 152 mM Methyl Red, 50 mM sodium phosphate buffer (pH 5.5) and 20 mM NADH. Enzyme activity was calculated by using molar extinction coefficient of Methyl Red. NADHeDCIP reductase activity was assayed by following the procedures reported earlier (11). Riboflavin reductase NAD(P)H:flavin oxidoreductase was measured by the method of Fontecave et al. (24) with some modification. In this aerobic assay, the flavin reductase catalyzes the reduction of riboflavin, and the reduced riboflavin is immediately reoxidized by oxygen. Cell extract was added to a solution (final volume, 1 ml) containing 100 mM of TriseHCl (pH 7.5), 25 mmol of NADPH and 0.003 unit riboflavin. The decrease in absorbance at 340 nm (A340) was measured spectrophotometrically. Reaction rates were calculated by using a molar extinction coefficient of 6.3 mM cm1. One unit of enzyme activity was defined as mg of riboflavin reduced min/mg protein. All enzyme assays were run in triplicate. COD measurement (% of mineralization) To understand the degree of biodegradation (mineralization) of Remazol Red reduction in chemical oxygen demand of the culture before and after decolorization after 6 h of incubation with Lysinibacillus sp. RGS were measured (25). The nutrient medium was used as blank and similar condition was used for test. Similar procedure was employed for the mixture of dyes and dye wastewater study. The COD was calculated as follows: CODðmg=lÞ ¼

ðA  BÞ  N  1000  8 Volume of sampleðmlÞ

(3)

where A is the ml of FAS was used for blank, B is the ml of FAS was used for test sample, N is the normality of FAS and 8 is the milliequivalent weight of oxygen. Analysis of color removal in the medium dye wastewater For the mixture of dyes and dye wastewater study, the true color level independent of hue was measured using the American Dye Manufacturers’ Institute (ADMI 3WL) tristimulus filter method. This method is applicable to colored waters and wastewaters having color characteristic. The decolorization of mixture of dyes and actual dye wastewater by Lysinibacillus sp. RGS was determined by measuring ADMI from the aqueous solutions. ADMI removal percent (%) is the ratio between the removal ADMI value at any contact time and the ADMI value at initial concentration was calculated (25) as follows: ADMI removal ratioð%Þ ¼

Initial ADMIð0

hÞ

 Observed ADMIðtÞ

Initial ADMIð0

hÞ

 100%

(4)

where ADMI(0 h) and ADMI(t) are the initial ADMI value (at 0 h) and the ADMI value after a particular reaction time (t), respectively.

3

Biodecolorization and biodegradation analysis Decolorization was monitored by UVevisible spectroscopic analysis (Hitachi U-2800), whereas biodegradation was monitored by high performance liquid chromatography (HPLC). After complete decolorization (pH 6.6) at 30
C by Lysinibacillus sp. RGS, the culture broth was centrifuged at 3000 g for 15 min and equal volume of ethyl acetate was used to extract the metabolites from clear supernatant using rotary evaporator. The extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained were dissolved in small volume of HPLC grade methanol and used for analysis. During UVevisible spectral analysis, changes in absorption spectrum in the decolorized medium (400e800 nm) were recorded in comparison with the results from the control runs. HPLC analysis was performed in an isocratic Waters’ 2690 system equipped with dual absorbance detector, using C18 column (symmetry, 4.6  250 mm) with HPLC grade methanol as a mobile phase at flow rate of 1.0 ml/min for 10 min at 585 nm. Fourier transform infrared spectroscopy (FTIR) (8400S Shimadzu, Japan) was used for investigating the changes in surface functional groups of the samples, before and after microbial decolorization. FTIR analysis was done in the mid-IR region of 400e4000 cm1 with 16 scan speed. The pellets were prepared using spectroscopic pure KBr (5:95, w/w) and fixed in the sample holder for analyses. The identification of metabolites formed after degradation was carried using a QP2010 gas chromatography (GC) coupled with mass spectroscopy (MS) (Shimadzu). The ionization voltage was 70 eV. GC was conducted in the temperature programming mode with a Restek column (0.25 mm, 60 m; XTI-5). The initial column temperature was 80
C for 2 min, then increased linearly at 10
C/min to 280
C, and held for 7 min. The temperature of the injection port was 280
C, and the GCeMS interface was maintained at 290
C. The helium carrier gas flow rate was 1.0 ml/min. Degradation products were identified by comparison of retention time and fragmentation pattern, as well as with mass spectra in the NIST spectral library stored in the computer software (version 1.10 beta, Shimadzu) of the GCeMS. Phytotoxicity and microbial toxicity studies Phytotoxicity of Remazol Red was performed in order to assess the toxicity of the untreated and treated dye to common agricultural crops. The obtained product was dissolved in water to prepare a final concentration of 300 ppm. Ten seeds of Sorghum vulgare and Phaseolus mungo plants were sowed into a plastic sand pot with daily watering of (5 ml) Remazol Red (300 ppm) and its degradation metabolites (300 ppm) obtained after degradation by L. fusiformis. Control set was carried out using distilled water (daily 5-ml watering) at the same time. Germination (percent) and length of shoot and root were recorded after 7 days. The study was carried out at room temperature. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with TukeyeKramer multiple comparisons test. Readings were considered significant when P was 0.05.

RESULTS AND DISCUSSION Isolation, screening, and identification of microorganism The bacterial strains were selected based on their ability to form a clear zone on nutrient agar plates containing industrial dyes. Among the five isolated bacterial strains, one strain (RGS) was selected based on its ability to decolorize various industrial dyes with faster decolorization rate. The nearly full length sequence of 16S rRNA gene for RGS was determined. Based on the sequence identity of 16S rRNA gene against the GenBank database indicates that the isolate was closely related to the members of the genus Lysinibacillus. The highest similarity towards the type strain of L. fusiformis was observed. A phylogenetic tree illustrating the relationship of strain RGS to other Lysinibacillus species is depicted. Selected strain was identified as Lysinibacillus sp. RGS on the basis of 16S rRNA sequence, morphological and biochemical characteristics (data not shown). The effectiveness of microbial decolorization depends on the survival, adaptability and the activity of enzymes produced by selected microorganisms (3). The decolorization capacity of Lysinibacillus sp. RGS tested by examining its potential to degrade various dyes. Lysinibacillus sp. RGS decolorized six textile dyes out of these, Remazol Red and Golden Yellow HER were decolorized by 100% and as Remazol Red took minimum time (6 h) with average decolorization rate of about 8.33 mg/h so we studied further parameters by using this dye (data not shown). Growth and decolorization experiment Better growth of Lysinibacillus sp. RGS and decolorization of Remazol Red was observed under static condition (no aeration and agitation) (9.8 g/l;

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

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SARATALE ET AL.

J. BIOSCI. BIOENG.,

100% decolorization) compared with under shaking condition (100 rpm) (4.0 g/l; 20% decolorization) (data not shown). There was no change in pH under static condition, indicating decolorization was because of microbial action and not because of change in pH. Similar results were observed in the studies on pure bacterial strains such as, P. luteola, M. glutamicus NCIM-2168, and P. mirabilis (1,6,26). It was claimed that under aerobic condition there may be competition between azo dyes and oxygen for reduced electron carriers and also inhibits the azo bond reduction activity since aerobic respiration may dominate utilization of NADH and impeding the electron transfer from NADH to azo bonds (2). Thus static conditions were necessary for the decolorization; therefore, static conditions were adopted to investigate the bacterial decolorization in the following experiments. The textile effluent is not stable and consisting crude mixture of dyes along with a number of chemicals and thus affects on treatment process. Adaptation of microorganisms to broad range of pH and temperature can make them more suitable for degradation of dye pollutants. The Lysinibacillus sp. RGS showed the decolorization up to 90% in the broad pH range (7.0e9.0) and temperature (30e35
C), but maximum decolorization was achieved at pH 7.0 and at 30
C (Fig. 1). The broad pH stability in decolorization increases the applicability of this strain since dye wastewater is generally higher pH. For the runs with a dye concentration of 50, 100, and 150 mg/l, complete decolorization by Lysinibacillus sp. RGS was observed within 6, 7, and 8 h, respectively. The time required for decolorization of Remazol Red was found to be raised with subsequent increase in dye concentration. At higher dye concentrations (e.g., 200 and 250 mg/l), Lysinibacillus sp. RGS showed less

pH 3.0 pH 5.0 pH 7.0 pH 8.0 pH 9.0 pH 10.0

Decolorization (%)

100 80 60 40 20 0 120

25 30 35 40 45

Decolorization (%)

100

C C C C C

80 60 40 20 0 0

1

2

3

4

5

6

Time (h) FIG. 1. Effect of pH and temperature on decolorization performance of C.I. Remazol Red (50 mg/l) by using Lysinibacillus sp. RGS.

decolorization performance, giving 82 and 45% decolorization after 24 h of incubation, respectively (data not shown). Decrease in the decolorization rates in Lysinibacillus sp. RGS probably due to the toxic effect of dyes and accumulation of large quantity of metabolites after decolorization of high concentration of dye followed by their adverse effect on the enzymes involved in decolorization (2,12). Decolorization with repeated addition of dye aliquots Up to sixth cycles were studied in order to assess the capability of Lysinibacillus sp. RGS to decolorize repeated additions of Remazol Red dye aliquots (50 mg/l) under static condition. In this experiment it was found that the dye could be decolorized when added repeatedly for consecutive sixth cycles with that the time required for decolorization went on increasing (6 h, 8 h, 12 h, 17 h, 24 h and 36 h). After sixth cycle there is no decolorization was observed even after 48 h (data not shown). The decrease in the decolorization is likely due to number of viable cells might have decreased due to unavailability of nutrients in the medium or due to entry of cells in stationary phase and then subsequently to death phase resulting in gradual decrease in the decolorization activity (5). However the foregoing results demonstrate an increase in the commercial applicability of Lysinibacillus sp. RGS in practical wastewater decolorization. Effect of supplementation of carbon and nitrogen sources on the decolorization of Remazol Red by Lysinibacillus sp. RGS Microbial decolorization/degradation of dye generally depends on the availability and type of a co-substrate used, because they acts as an electron donor for the azo dye reduction as well as azo dyes are deficient in carbon source (8,27). The performance of Lysinibacillus sp. RGS in decolorizing Remazol Red in the presence of an additional carbon and nitrogen sources (1%) and 5 ml extract of agricultural by-products (1%) were examined to obtain efficient and faster decolorization. Although there was similar growth of this strain in all tested sources, large difference was observed in the decolorization pattern. In control set synthetic media with each carbon and nitrogen supplements, there is no abiotic loss of Remazol Red within 24 h of incubation was observed. There was reduced decolorization in the presence of carbon source in contrast, amplified decolorization was obtained in the medium supplemented with nitrogen source like peptone and beef extract. Addition of carbon sources seemed to be less effective to promote the decolorization, probably due to the preference of the cells in assimilating the added carbon sources over using the dye compound as a carbon source. Percent decolorization was maximum with peptone (100%), and beef extract (100%) within 6 h of incubation, followed by the tests in the presence of 5 ml extract of wheat straw (82%) and paddy straw (76%) after 24 h of incubation. With other supplements of carbon and nitrogen source, less decolorization efficiency was observed within 24 h (Table 1). The faster decolorization in the medium containing pure (beef extract and peptone) and agricultural extract (wheat straw and paddy straw) might be due to the metabolism of these nitrogen sources, which is considered essential for the regeneration of NADH required for azo dye decolorization (2). Supplementation of agricultural by-products extract could enhances the decolorization of Direct Blue GLL by Comamonas sp. UVS along with production of lignolytic enzymes reported earlier (22). The use of agricultural by-products (wheat straw and paddy straw) instead of pure substrates (peptone and beef extract) for the enhancement of the decolorization of Remazol Red becomes an inexpensive and ecofriendly process since utilizing of lignocellulosic waste for the degradation of industrial effluents could resolve the problem of the disposal of agro-residues which is present in larger quantities (28).

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

VOL. xx, 2012

DECOLORIZATION OF REMAZOL RED BY LYSINIBACILLUS SP. RGS

TABLE 1. Effect of supplementation of different carbon and nitrogen sources on the decolorization of Remazol Red by Lysinibacillus sp. RGS. Media SM SMþ SMþ SMþ SMþ SMþ SMþ SMþ SMþ SMþ SMþ SMþ

Decolorization (%)

Time (h)

ND 14 37 18 100 ND 100 84 35 82 76 40

24 24 24 24 06 24 06 24 24 24 24 24

Glucose Starch Sucrose Peptone Lactose Beef extract Yeast extract Wood strawa Wheat strawa Paddy strawa Sugarcane bagassea

SM, synthetic media; ND, no decolorization. a 5 ml extract of each agricultural residue (1%).

Analysis of enzymes related to dye decolorization and degradation The literature survey suggests that different enzymes have been found to be involved in the degradation of dyes in microbial sources. Besides uptake, the presence and activity of a network of detoxification enzymes is crucial for the metabolism and eventually the degradation of chemicals under consideration. To understand the decolorization mechanism, enzyme activities of laccase, veratryl alcohol oxidase, lignin peroxidase, NADHeDCIP reductase, azo reductase and riboflavin reductase were monitored over time. In the present study, significant induction in the enzyme activity of azo reductase (1030%), riboflavin reductase (702%), was observed over the period of Remazol Red decolorization by Lysinibacillus sp. RGS as compared to laccase (293%), NADHeDCIP reductase (108%), and veratryl alcohol oxidase (143%) after complete decolorization (6 h) (Table 2). The enzymatic profile presumably indicates communal action of oxidoreductive enzymes for the degradation of Remazol Red into simple metabolites by Lysinibacillus sp. RGS. The role of oxidoreductive enzymes in the decolorization of azo dyes have been characterized in various bacteria are well documented (2). Determining degree of mineralization of Remazol Red by Lysinibacillus sp. RGS via COD measurement Variety of synthetic dyestuff released by the textile industries pose a threat to the environmental safety. It was observed that the transformed intermediates of azo dyes (aromatic amines) using microbial system are long-lived and highly toxic and mutagenic in nature (29). To evaluate the decolorization and biodegradation level of the Remazol Red by Lysinibacillus sp. RGS, the percentage of mineralization (represented by COD removal ratio) by measuring the initial and final COD content. The Lysinibacillus sp. RGS

TABLE 2. Oxidoreductive enzyme profile in control cells of Lysinibacillus sp. RGS at (0 h) and the induced cells obtained after complete decolorization (6 h). Enzymes Lignin peroxidasea Veratryl alcohol oxidasea Laccasea NADHeDCIP reductaseb Riboflavin reductasec Azo reductased

Control cells (0 h) 0.38 1.81 0.31 24.61 0.67 0.39

     

0.09 0.30 0.01 0.40 0.07 0.05

Cells obtained after complete decolorization (6 h) 0.15 2.60 0.91 26.52 4.71 4.02

     

0.07 0.31* 0.52* 0.42** 1.02** 0.24**

Values are mean of three experiments  standard error of mean (SEM), significantly different from control cells at *P < 0.05 and **P < 0.001 by one-way analysis of variance (ANOVA) with TukeyeKramer multiple comparisons test. a U min/mg protein. b mg of DCIP reduced min/mg protein. c mg of riboflavin reduced min/mg protein. d mg of Methyl Red reduced min/mg protein.

5

displayed complete decolorization of Remazol Red sequentially with a significant COD reduction (92%) within 6 h. The reduction in COD of Remazol Red is higher than, Navy Blue HE2R by Exiguobacterium sp. RD3 (55.55); Reactive Red 2 by Pseudomonas sp. SUK1 (52%) and Reactive Green 19A by M. glutamicus NCIM2168 (68%), respectively (6,23,30). The results suggest that the Lysinibacillus sp. RGS can utilize this dye and their reaction intermediates as a carbon source, achieving mineralization of the dye compound. Analysis of metabolites resulting from decolorization and biodegradation of Remazol Red by Lysinibacillus sp. RGS To disclose the possible mechanism of the dye decolorization, we have also analyzed the products of biotransformation of Remazol Red by UVevisible spectroscopy, HPLC, FTIR and GCeMS. Microbial decolorization of dyes is mainly due to adsorption and biodegradation mechanism. In adsorption mechanism the absorption spectrum of dye decrease approximately in proportion of dye adsorbed as well as cell becomes deeply colored, whereas in biodegradation, either the major visible light absorbance peak will completely disappear or a new peak will appear (8). UVevisible scan (400e800 nm) of the culture supernatants withdrawn at different time intervals indicated the decolorization and decrease in the dye concentration from batch culture. Peak observed at 540 nm (0 h) decreased without any shift in lmax up to complete decolorization of the dye (6 h). Evidence of the removal of dye can be observed with absorbance at lmax being virtually zero after 6 h of incubation (data not shown). HPLC elution profile showed prominent peak at retention time at 1.892, 2.482 and small peak of 2.297 min when products were separated from the sample obtained after decolorization, compared to control peaks with retention times at 2.593 and 2.650 min (data not shown). The analysis showed the presence of new peak with marked decrease in intensity of the peak, confirming the degradation of Remazol Red by Lysinibacillus sp. RGS. The FTIR spectrum of control dye Remazol Red compared with extracted metabolites obtained from 6 h culture is shown in Fig. 2. The spectrum of control dye displayed a peak at 3419 cm1 for NeH stretching, a peak at 2926 cm1 for asymmetric eCH3 stretch and overtone band for CeH stretching at 1754 cm1 was observed. Whereas a peak at 1592 cm1 represented eN]Ne stretching of azo group. Peak at 1486 cm1 showed eOH stretching vibration. In addition a peak at 1286 cm1 represented primary aromatic amines with CeN vibration. A peak at 1045 cm1 for eS]O stretch and a peak at 844 cm1 for eCeS stretch support the presence of sulfur containing group in control dye (Fig. 2A). The FTIR spectrum of the decolorized sample (extracted metabolites from 6 h culture of Lysinibacillus sp. RGS) displayed a peak at 1668 cm1 represented of formation charged aromatic amines derivatives and a peak at 1443 cm1 stretching eCH2 deformation and a peak at 1298 cm1 of CeN vibration of secondary aromatic amines. In addition a peak at 3231 cm1 showed eNH stretching and the disappearance peak of azo bond proves the cleavage of azo linkage a peak at 748 cm1 represents CeH deformations indicating loss of aromaticity of benzene (Fig. 2B). Finally disappearance of peak of 844 cm1 also proves the removal of sulfur containing groups of Remazol Red. To verify the degradation products formed during dye decolorization by Lysinibacillus sp. RGS, GCeMS analysis was carried out, revealing the presence of several peaks in TIC (data not shown). The low molecular weight aromatic compounds were produced from the degradation of Remazol Red by Lysinibacillus sp. RGS. Accordingly, the pathway for the degradation of Remazol Red is proposed as depicted in Fig. 3, showing various steps involved in the degradation mechanism. However, very little is known about the nature of the degradation products formed in these reactions and the reaction mechanism about oxidoreductive enzymes. We propose

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

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J. BIOSCI. BIOENG.,

FIG. 2. FTIR analysis of products (extracted with ethyl acetate) formed by degradation of C.I. Remazol Red: (A) at 0 h (control), (B) metabolites formed by Lysinibacillus sp. RGS after complete decolorization (6 h).

that initially primary reductive cleavage in azo bond of Remazol red results in the intermediate-I and intermediate-II as the intermediate products. The intermediate-II after demethylation resulted in the formation of [A] methanesulfinic acid with a retention time of 1.57 min and a mass peak of 79 (Fig. 3). Whereas the asymmetric cleavage of product intermediate-I by oxidative enzymes (veratryl alcohol oxidase/laccase) resulted in the formation of [B] 4-[(6amino-4-chloro-1,2,3,4-tetrahydro-1,3,5-triazin-2-yl) amino] decahydronaphthalene-2,7-disulfonate] with a retention time of 26.42 min and a mass peak of 429, followed by deamination produces [C] 4-[(4-chloro-1,3,5-triazin-2-yl)amino] naphthalene2,7-disulfonate] with a retention time of 22.49 min and a mass peak of 415 (Fig. 3). Further sequential deamination and dechlorination reaction resulting in the formation of low molecular weight compound such as [D] 4-chloro-1,3,5-triazin-2-amine followed by [E] 1,3,5-triazine as a final product (Fig. 3). Therefore, analytical studies confirmed the biodegradation of Remazol Red dye, in which the smaller molecular weight intermediates are formed by the consecutive action of oxidoreductive enzymes present in Lysinibacillus sp. RGS, which may be present in minute quantity in the decolorized solution. Phytotoxicity studies Disposal of untreated dyeing effluents in water bodies might cause serious environmental and health

hazards and this water is being used for an agriculture purpose shows toxic effect on the germination rates and biomass of several plant species, which play an important role in ecological function such as providing the habitat for wildlife, protecting soil from erosion and providing bulk of organic matter that is so significant to soil fertility (23,29,31,32). This study is of particular relevance since the Panchganga river and Ichalkaranji area near Kolhapur, India are heavily industrialized, with significant wastewater discharge from textile industries to the environment which causes the harmful impacts on the nearby flora and fauna. Thus, it was of concern to assess the phytotoxicity of the azo dye before and after degradation. Table 3 represents the phytotoxicity analysis of the Remazol Red and its metabolites obtained after decolorization. Phytotoxicity test in relation to P. mungo and S. vulgare demonstrated that the biodegradation products did not interfere with the germination of plant seeds. The germination rate of P. mungo and S. vulgare was inhibited as well as plumule and radical length were drastically affected when treated with a Remazol Red (300 ppm). In contrast, plumule length and radical length in P. mungo and S. vulgare were found equivalent to control set (distilled water) with 90% germination when treated with 300 ppm concentration of the degradation products (Table 3). The phytotoxicity studies reveal that the metabolites generated after the biodegradation of Remazol Red are less toxic

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

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FIG. 3. Proposed pathway for biodegradation of C.I. Remazol Red by Lysinibacillus sp. RGS.

TABLE 3. Phytotoxicity studies of C.I. Remazol Red and its metabolites formed after biodegradation on Phaseolus mungo and Sorghum vulgare. For Remazol Red Phaseolus mungo Parameters studied Germination (%) Shoot length (cm) Root length (cm)

Water 100 3.52  0.05 2.36  0.05

Remazol Red 40 2.15  0.03 1.17  0.04

a

Sorghum vulgare Extracted Metabolites 90 3.68  0.07 1.86  0.05

a

Water 100 4.31  0.11 1.67  0.06

Remazol Reda 40 2.25  0.03 No roots

Extracted Metabolitesa 90 3.66  0.10 1.18  0.03

Values are mean of three experiments, SEM (), significantly different from the control (seeds germinated in distilled water) at *P < 0.05, **P < 0.001, by one-way analysis of variance (ANOVA) with TukeyeKramer multiple comparisons test. a 300 ppm concentration.

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

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TABLE 4. Comparison of the decolorization performance and toxicity studies of different bacterial strains with Lysinibacillus sp. RGS. Strain Bacillus sp. AK1 Lysinibacillus sp. AK2 Bacillus sp. VUS

Brevibacillus laterosporus MTCC 2298 Bacillus fusiformis KMK5 Geobacillus stearothermophilus (UCP 986) Micrococcus glutamicus NCIM-2168 Mixed bacterial cultures SB4a Bacillus sp. YZU1 Lysinibacillus sp. RGS

Time (h)

ADR (mg/h)

7.2, 37, static

27

7.40

Reductive

7.2, 37, static

12

16.66

Reductive

7.0, 40, static 7.0, 40, static

18 18

2.77 2.61

Oxidative and reductive

7.0, 30, static,

48

0.90

Oxidative and reductive

9.0, 37, static

48

31.25

NA

5e6, 50, aeration (150 rpm)

24

0.70

NA

Toxicity using the brine shrimp Artemia salina

34

Reactive Green 19 A; (50 mg/l)

6.8, 37, static

42

1.19

Oxidative and reductive

6

Reactive Violet 5R (200 mg/l) Reactive Black 5 (100 mg/l) Remazol Red (50 mg/l)

7.0, 37, static

18

11.11

NA

Phytotoxicity (Sorghum vulgare and Phaseolus mungo) NA

7.0, 40, static

120

0.79

Reductive

NA

35

7.0, 30, static

06

8.33

Oxidative and reductive

Phytotoxicity assay (Phaseolus mungo and Sorghum vulgare)

Dye and concentration Metanil Yellow (200 mg/l) Metanil Yellow (200 mg/l) Red HE7B (50 mg/l) Navy blue 2GL; (50 mg/l) Golden Yellow HER; (50 mg/l) Acid Orange 10 (1500 mg/l) Orange II (17.5 mg/l)

Condition [pH, temp. (
C), agitation]

Type of enzymes involved

Toxicity test performed

References

Phytotoxicity (Chick pea and Pigeon pea) Phytotoxicity (Chick pea and Pigeon pea) Phytotoxicity (Triticum aestivum and Sorghum bicolor) Phytotoxicity (Sorghum vulgare and Phaseolus mungo) NA

18 18 12

33

5

10

This study

a Mixed cultures SB4 consists of Bacillus sp. V1DMK, Lysinibacillus sp. V3DMK, Bacillus sp. V5DMK, Bacillus sp. V7DMK, Ochrobacterium sp. V10DMK, Bacillus sp. V12DMK. SB4; NA e Not available.

100

80 COD reduction (%) ADMI removal (%)

80

60 60 40 40 20

ADMI removal (%)

COD reduction (%)

20

0

0 0

12

24

36

48

Time (h)

B

70 60

80 COD reduction (%) ADMI removal (%)

70 60

50

50 40 40 30 30 20

20

10

ADMI removal (%)

Decolorization of mixture of dyes and dye wastewater Synthetic dyes and pigments released to the environment in the form of effluents by textile, leather and printing industries cause severe ecological damages. The removal of the polluting dyes is an important problem, however in India particularly for small scale textile industries, where working conditions and economic status do not allow them to treat their wastewater before disposal and they have no choice rather than dumping all effluent into the main stream of water resources. The composition of textile effluent consists of a mixture of many synthetic dyes and the effluent characteristics (such as pH, dissolved oxygen, organic, and inorganic chemical content etc.) which depend greatly upon the textile processing (36). Thus, the microbial population used in the treatment process for removing color from the effluent must have the capability of decolorizing a mixture of different dyes. We have evaluated the decolorization ability of mixture of various seven industrial dyes (C.I. Remazol Red, C.I. Rubin, C.I. Scarlet RR, C.I. Brilliant Blue, C.I. Brown 3 REL, C.I. Golden Yellow HER, C.I. Methyl Red) each at concentration (50 mg/l) and actual textile wastewater by using Lysinibacillus sp. RGS in batch culture at 30
C under static condition. The mixture of dyes and textile wastewater did not have a well-defined peak at the visible absorption spectra. The ADMI color value provides a true measure of water color, independent of hue and thus opens the way to the more accurate definition of water and wastewater color. The true color of the mixture of dyes and textile wastewater measured by using ADMI 3WL suggesting that Lysinibacillus sp. RGS could achieve higher color removal value (87% and 72%) and higher COD reduction (69 and 62%) within 48 h and 96 h, respectively (Fig. 4A and B). Literature survey showed that Phanerochaete sordida exhibited 90% decolorization of the mixture of four reactive dyes within 48 h (32); Ischnoderma resinosum showed 100% decolorization of the mixture of two dyes after 20 days (37); isolated A. hydrophila

A

COD reduction (%)

than the original dye which also supported by other studies (Table 4). The results suggest that using Lysinibacillus sp. RGS to treat wastewater containing the reactive dye is safe, thereby enhancing its feasibility in practical applications.

10

0

0 24

48

72

96

Time (h) FIG. 4. Color removal of (A) mixture of dyes; (B) textile industrial effluent in terms of ADMI removal ratio values and COD reduction at different incubation time by Lysinibacillus sp. RGS.

Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009

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DECOLORIZATION OF REMAZOL RED BY LYSINIBACILLUS SP. RGS

exhibited significant decolorization performance for mixture of dyes within 2 days (13); M. glutamicus NCIM-2168 showed 63% decolorization of mixture of 10 dyes (6) and Galactomyces geotrichum MTCC 1360 showed 88% decolorization of seven dyes with 69% COD reduction (36). Decolorization performance of textile wastewater by Lysinibacillus sp. RGS is quiet comparable with Pseudomonas sp. SU-EBT decolorized 90% effluent within 60 h with 50% COD reduction (27); Citrobacter sp. strain KCTC 18061P strain removed 70% of effluent color within 5 days with 35% COD reduction (38). The foregoing results suggest the potential of utilizing Lysinibacillus sp. RGS to decolorize textile effluent via appropriate bioreactor operations and will be useful to small industries present in Ichalkaranji nearby area of Kolhapur in an ecoefficient and cost-effective manner. This study demonstrates that isolated Lysinibacillus sp. RGS was able to degrade and detoxify the toxic sulfonated azo dye Remazol Red under static condition. Supplementation of extracts of agricultural waste (namely, wheat straw and paddy straw), instead of purified peptone and beef extract, was found to be more economically feasible supplements to enhance the decolorization of Remazol Red by Lysinibacillus sp. RGS. Enzyme analysis indicated prime involvement of oxidoreductive enzymes in the decolorization process. The COD measurement showed mineralization of Remazol Red and phytotoxicity tests shows nontoxic residual metabolites. Analytical studies of extracted products confirmed the biodegradation of Remazol Red by Lysinibacillus sp. RGS. A possible pathway for biodegradation of this dye was proposed with the help of GCeMS analysis. This strain also showed better color removal of mixture of dyes and textile wastewater with significant reduction in COD could be potential strain for the treatment of textile dyestuffs and textile industry effluent by using appropriate bioreactor. ACKNOWLEDGMENTS The authors would like to thank, Common Facility Center (CFC), Shivaji University, Kolhapur for availing GCeMS facility. The author RGS thankfully acknowledges the funding support received from University Grants Commission (UGC), Government of India for providing financial assistance (41-1268/2012 SR). One of the author GDS would like to acknowledge UGC and DST, New Delhi, India for their financial support. References 1. Chang, J. S., Chou, C., Lin, Y., Ho, J., and Hu, T. L.: Kinetic characteristics of bacterial azo-dye decolorization by Pseudomonas luteola, Water Res., 35, 2841e2850 (2001). 2. Saratale, R. G., Saratale, G. D., Chang, J. S., and Govindwar, S. P.: Outlook of bacterial decolorization and degradation of azo dyes: a review, J. Chin. Inst. Chem. Eng., 42, 138e157 (2011). 3. Khehra, M. S., Saini, H. S., Sharma, D. K., Chadha, B. S., and Chimni, S. S.: Comparative studies on potential of consortium and constituent pure bacterial isolates to decolorize azo dyes, Water Res., 39, 5135e5141 (2005). 4. Pearce, C. I., Lloyd, J. R., and Guthriea, J. T.: The removal of colour from textile wastewater using whole bacterial cells: a review, Dyes Pigm., 58, 179e196 (2003). 5. Kolekar, Y. M., Pawar, S. P., Gawai, K. R., Lokhande, P. D., Shouche, Y. S., and Kodam, K. M.: Decolorization and degradation of Disperse Blue 79 and Acid Orange 10, by Bacillus fusiformis KMK5 isolated from the textile dye contaminated soil, Bioresour. Technol., 99, 8999e9003 (2008). 6. Saratale, R. G., Saratale, G. D., Chang, J. S., and Govindwar, S. P.: Ecofriendly decolorization and degradation of Reactive Green 19A using Micrococcus glutamicus NCIM-2168, Bioresour. Technol., 100, 3897e3905 (2009). 7. Vandevivere, P. C., Bianchi, R., and Verstraete, W.: Treatment and reuse of wastewater from the textile wet-processing industry: review of emerging technologies, J. Chem. Technol. Biotechnol., 72, 289e302 (1998). 8. Sani, R. K. and Banerjee, U. C.: Decolorization of triphenylmethane dyes and textile and dyestuff effluent by Kurthia sp., Enzyme Microb. Technol., 24, 433e437 (1999).

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9. Abadulla, E., Tzanov, T., Costa, S., Robra, K. H., Cavaco-Paulo, A., and Gubitz, G. M.: Decolorization and detoxification of textile dyes with a laccase from Trametes hirsute, Appl. Environ. Microbiol., 66, 3357e3362 (2000). 10. Jain, K., Shah, V., Chapla, D., and Madamwar, D.: Decolorization and degradation of azo dye e reactive Violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil, J. Hazard. Mat., 214, 378e386 (2012). 11. Saratale, G. D., Bhosale, S. K., Kalme, S. D., and Govindwar, S. P.: Biodegradation of kerosene in Aspergillus ochraceus (NCIM-1146), J. Basic Microbiol., 47, 400e405 (2007). 12. Dawkar, V. V., Jadhav, U. U., Tamboli, D. P., and Govindwar, S. P.: Efficient industrial dye decolorization by Bacillus sp. VUS with its enzyme system, Ecotoxicol. Environ. Saf., 73, 1696e1703 (2010). 13. Chen, B. Y., Chen, W. M., Wu, F. L., Chen, P. K., and Yen, C. 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33. Gomare, S. S., Tamboli, D. P., Kagalkar, A. N., and Govindwar, S. P.: Ecofriendly biodegradation of a reactive textile dye Golden Yellow HER by Brevibacillus laterosporus MTCC 2298, Int. Biodeteriorat. Biodegrad., 63, 582e586 (2009). 34. Evangelista-Barreto, N. S., Albuquerque, C. D., Vieira, R. H. S. F., and CamposTakaki, G. M.: Cometabolic decolorization of the reactive azo dye Orange II by Geobacillus stearothermophilus UCP 986, Textile Res. J., 79, 1266e1273 (2009). 35. Wang, Z. W., Liang, J. S., and Liang, Y.: Decolorization of Reactive Black 5 by a newly isolated bacterium Bacillus sp. YZU1, Int. Biodeteriorat. Biodegrad., 76, 41e48 (2013).

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Please cite this article in press as: Saratale, R. G., et al., Decolorization and detoxification of sulfonated azo dye C.I. Remazol Red and textile effluent by isolated Lysinibacillus sp. RGS, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.12.009