Journal of Basic Microbiology 2012, 52, 1–11 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Differential catalytic action of Brevibacillus laterosporus on two dissimilar azo dyes Remazol red and Rubine GFL

Mayur B. Kurade1, Tatoba R. Waghmode2, Dhawal P. Tamboli1 and Sanjay P. Govindwar2

1 Department of Biotechnology, Shivaji University, Kolhapur, India 2 Department of Biochemistry, Shivaji University, Kolhapur, India

This comparative study disclosed the diverse catalytic activities of Brevibacillus laterosporus on two different azo dyes. It decolorized 100% of Remazol red and 95% of Rubine GFL within 30 and 48 h respectively, under static condition at 50 mg l–1 dye concentration. Significant increase was observed in azo reductase, NADH-DCIP reductase, veratryl alcohol oxidase and tyrosinase in cells obtained after decolorization of Remazol red; whereas these values were much different with complete inhibition of azo reductase during decolorization of Rubine GFL. The plausible pathway of dye degradation obtained from Gas chromatography-Mass spectroscopy (GC-MS) data confirmed the different metabolic fate of these structurally unidentical dyes. FTIR and HPTLC analysis of extracted metabolites confirmed the biodegradation, while phytotoxicity study assured the detoxification of both the dyes studied. The results obtained in this study suggests, i) sulpho and hydroxyl group present at ortho position to azo group stimulated reduction of azo bond by azo reductase in Remazol red, ii) the same reduction was totally hampered due to presence of ethyl-amino propanenitrile group at para position to azo group in Rubine GFL.

Keywords: Decolorization / Biodegradation / HPTLC / Azo dyes / GCMS

Received: August 11, 2011; accepted: October 29, 2011

DOI 10.1002/jobm.201100402

Introduction*

Colors are considered most undesirable contaminants present in wastewater from textile industry, mainly caused by dyes as compared to other contaminants [1]. Major classes of synthetic dyes include: azo, an-thraquinone and triarylmethane dyes; which constitute more than 50% of those used in the industries. Azo dyes are the largest group of dyes with great deal of structural and color variety used in industry represent-ing up to 70% of the annual production [2]. They are characterized by the presence of one or more azo bonds (–N=N–) in association with one or more aromatic structures making them more persistent in the envi-ronment that cannot be degraded easily under natural condition.

Correspondence: Prof. Sanjay Govindwar, Department of Biochemis-try, Shivaji University, Kolhapur-416-004, India E-mail: spg_biochem@unishivaji.ac.in Phone: +91-231-2609152 Fax: +91-231-2691533

Azo dyes are potential mutagens, carcinogens and cause other harmful disorders in mammalian cells [3], which necessitate proper degradation and safer dis-posal. Biological decolorization treatment is an envi-ronmentally friendly and cost competitive alternative to physical and chemical decomposition. Previous stud-ies have shown the biodegradation of numerous azo dyes using bacteria, fungi, yeasts and algae [2, 4–10], which have caused decolorization of azo dyes via the reduction of azo bond. Based on the previous report; decolorization effi-ciency of azo dyes is limited to several azo dye struc-tures and it is influenced by the chemical structures of the dyes [11]. Hence, the effect of different dye struc-tures or their various functional groups on decoloriza-tion rate is an important and interesting aspect in azo dye degradation. For similar structures of azo dyes, monoazo dyes were easily decolorized than polyazo dyes [12]. Evidently, the specificity of Orange II azore-ductase from Pseudomonas strain KF46 towards azo dye is strongly dependent upon the electron-withdrawing

2 M. B. Kurade et al. Journal of Basic Microbiology 2012, 52, 1–11

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

ability of functional groups in the proximity of azo linkage(s) [13]. It has been reported that the position of electron-withdrawing groups to azo bond significantly influenced the reductive decolorization and their rank-ing of the decolorization rate was para > ortho > meta [14]. Although these reports on effects of chemical struc-ture and their functional group on decolorization effi-ciency are well documented, detailed evidence of the microbial enzyme system involved in azo dye decolori-zation and the metabolic fate of azo dye still remains open for discussion. Therefore, the present study was carried out to investigate the versatile action of a bacte-rium-Brevibacillus laterosporus MTCC 2298 on two struc-turally different azo dyes (Remazol red and Rubine GFL). Remazol red, a sulphonated monoazo dye and Rubine GFL, containing nitro group have tremendous consumption rates in the textile dying processes. Dif-ference in structure and functional groups present upon them make them suitable model dyes for this study. The present study suggests that, the catalytic action of the microbial enzymatic machinery is de-pendent on functional groups on the dye molecule. The main objective of this work is to find the interesting difference in the fate of metabolism of two dyes. So we report the diverse pattern in metabolic fate of two dis-similar azo dyes using Gas chromatography-Mass spec-troscopy (GC-MS) with possible role of responsible en-zymes involved in these biodegradation processes.

Materials and methods

Microorganisms and culture conditions B. laterosporus MTCC 2298 was obtained from Microbial Type Culture Collection, Chandigarh, India. The pure culture was maintained on nutrient agar slant contain-ing (g l–1): NaCl, 5.0; bacteriological peptone, 5.0; yeast extract, 2.0; beef extract, 1.0 and agar powder 15.0 at 4 °C. All decolorization experiment was carried out in 100 ml nutrient broth [Composition (g l–1): NaCl, 5.0; bacteriological peptone, 10.0; yeast extract, 1.5; beef extract, 1.5].

Dyes and chemicals Textile dyes Remazol red and Rubine GFL were a mu-nificent gift from Manpasand textile processing indus-try, Ichalkaranji, India. All required chemicals were obtained from Sigma Aldrich, USA, Hi-media Laborato-ries Pvt. Ltd., Mumbai, India and Sisco Research Labora-tory (SRLs), India. All chemicals used were of the high-est purity available and of the analytical grade.

Decolorization experiment Decolorization experiments were performed in the 250 ml Erlenmeyer flasks containing 100 ml nutrient broth. A pregrown culture was prepared by cultivating a loopful of microbial culture in static condition for 24 h at 30 °C. Dye was added into this pregrown culture in log phase (24 h) at a concentration of 50 mg l–1. De-colorization of Remazol red and Rubine GFL was carried out under its pre-optimized condition i.e. 40 °C tem-perature at static condition, pH 9 and 7, respectively. The optimum pH and temperature of the culture broth was adjusted prior to dye addition. Aliquots (4 ml) withdrawn after regular time intervals, centrifuged (4000 × g for 20 min) and decolorization was deter-mined by measuring absorbance maxima (λmax) of the respective dyes (530 nm for both dyes) using UV visible spectrophotometer (Hitachi U-2800) by using following formula,

% Decolorization

(Initial absorbance Final absorbance)100

Initial absorbance−

= ×

All the decolorization experiments were carried out in triplicate. Abiotic controls (without microorganisms) were always included.

Preparation of cell free extract and enzyme assays The loopful cells of B. laterosporus were grown in nutri-ent broth (pH –7.0) at 30 °C under static condition for 24 h. The grown cells were harvested by centrifugation (10000 × g, 20 min at 4 °C) and suspended in 50 mM potassium phosphate buffer (pH 7.4), gently homoge-nized and sonicated (30 s, 40 amplitude, 7 strokes) at 4 °C. The sonicated cells were centrifuged and the su-pernatant was used as source of intracellular enzyme. The culture supernatant obtained after centrifugation during harvesting of cell biomass was directly used as source of extracellular enzymes. Same procedure was followed for samples obtained after degradation of the dye. Activities of dye degrading enzymes, such as veratryl alcohol oxidase and tyrosinase were assayed spectro-photometrically (Hitachi U-2800) in cell free extract and culture supernatant. Veratryl alcohol oxidase activity was determined by monitoring absorbance increase at 310 nm due to oxidation of the veratryl alcohol to vera-traldehyde in citrate phosphate buffer (pH –3.0) [15]. Tyrosinase activity was determined by the method re-ported by Waghmode et al. [16] in which formation of o-benzoquinone and dehydro-ascorbic acid in 3 ml reac-tion mixture containing 50 mM of catechol and 2.1 mM

Journal of Basic Microbiology 2012, 52, 1–11 Differential action of B. laterosporus on azo dyes 3

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

of ascorbic acid in 50 mM potassium phosphate buffer (pH 6.5) was measured by monitoring the decrease in optical density at 265 nm. Azo reductase activity was assayed by modifying earlier reported method Telke et al. [17]; the assay mix-ture (2 ml) contained 25 μM of methyl red (MR), 100 μM NADH, 1.7 ml of potassium phosphate buffer (20 mM, pH 7.5). The reaction mixture was pre-incubated for 4 min followed by the addition of NADH and monitored for the decrease in color absorbance (430 nm) at room temperature. The reaction was initiated by addition of 0.1 ml of the enzyme solution. Methyl red reduction was calculated by using its molar extinction coefficient of 0.023 μM–1 cm–1. One unit of enzyme activity was defined as amount of enzyme required to reduce 1 μM of substrate min−1 mg of protein–1. NADH-DCIP reduc-tase was monitored at 590 nm and calculated using an extinction coefficient 0.019 μM–1 cm–1. The reaction mixture (5.0 ml) contained 25 μM substrate (DCIP) in the 50 mM potassium phosphate buffer (pH 7.4) and 0.1 ml enzyme. From this, 2.0 ml reaction mixture was assayed at 590 nm by addition of 250 μM NADH. Ribo-flavin reductase NAD(P)H:flavin oxidoreductase was measured by monitoring the decrease in absorbance at 340 nm [18]. All the enzyme assays were run in tripli-cates and reference blanks contained all components except the enzyme. Enzyme induction was calculated using following formula,

Enzyme induction (%)

Enzyme activity after dye decolorization 100= 100

Enzyme activity before dye addition×

−

Extraction and analysis of products obtained after dye decolorization Biomass was removed after decolorization by centrifu-gation (10000 × g at 4 °C for 20 min) and degradation metabolites were extracted from supernatant with equal volume of ethyl acetate. The extracted residues were dried over anhydrous Na2SO4 and evaporated in a rotary evaporator. The crystals obtained were dissolved in small volume of HPLC grade methanol and used for further analyses. FTIR analysis was done in the mid IR region of 400–4000 cm−1 with 16 scan speed [8]. To confirm the biodegradation of dyes, the same metabo-lites were analyzed by HPTLC using silica gel plates (HPTLC Lichrospher Silica gel 60 F254S, Merck). 15 μl of sample was applied on the plate by micro syringe using sample applicator (Linomat V, Camag, Switzerland). The solvent system used for Remazol red was metha-

nol :ethyl acetate (7 :3 v/v) whereas for Rubine GFL, toluene:ethyl acetate:methanol (7 :2:1 v/v/v). The chro-matogram was analyzed using scanner (Camag, Swit-zerland). 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, nonpolar). Helium was used as car-rier gas at a flow rate of 1 ml min−1. The injector tem-perature was maintained at 280 °C with oven condi-tions as: 80 °C kept constant for 2 min and increased up to 200 °C with 10 °C min–1-raised up to 280 °C with 20 °C min−1 rate. The compounds were identified on the basis of mass spectra and using the NIST library.

Phytotoxicity study To verify toxicity of dyes and its metabolites phytotox-icity test was performed according to the method re-ported by Waghmode et al. [16] at a final concentration of 2000 ppm of metabolites and dye. Ten seeds of each plant were sowed into a plastic sand pot. The sand pot was prepared by adding 15 g washed sand into the plas-tic pot. Metabolites were extracted using procedure described previously. Toxicity study was done by water-ing the seeds of each plant with original dye and ex-tracted metabolites of dye mixture with a distilled water control (5 ml per d). Germination (%), lengths of the shoots and roots were recorded after 7 d.

Statistical analysis Data were analyzed by one-way analysis of variance using Tukey–Kramer multiple comparison test. Values are mean of three experiments. Values were considered significant when P was <0.05.

Results

Decolorization experiment The present study demonstrates the different metabolic capability of the B. laterosporus with respect to two azo dyes having dissimilar structural characteristics. Ta-ble 1 illustrates the decolorization of selected model dyes for this study having diverse structures and col-ored properties using B. laterosporus. Both of the azo

Table 1. Decolorization of textile azo dyes by Brevibacillus laterosporus MTCC 2298.

Name of the dye λmax (nm) Time (h) Decolorization (%)

Remazol red 530 30 100 Rubine GFL 530 48 95

4 M. B. Kurade et al. Journal of Basic Microbiology 2012, 52, 1–11

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Figure 1. Effect of pH and Temperature on decolorization of Remazol red and Rubine GFL by B. laterosporus.

dyes were efficiently degraded by B. laterosporus in static condition. The optimum pH and temperature for Re-mazol red was found to be pH 9 and 40 °C, respectively, whereas B. laterosporus needed neutral pH and 40 °C temperature for faster decolorization of Rubine GFL (Fig. 1). After providing the respective optimum condi-tions for decolorization of individual azo dyes, B. latero-sporus exhibited 100 and 95% decolorization of Remazol red and Rubine GFL, respectively, within 30 and 48 h, at 50 mg l–1 dye concentration. However, the abiotic controls of both the dyes did not show any decoloriza- tion.

Enzyme analysis In order to get additional insights into mechanism of decolorization using B. laterosporus; various oxidative and reductive enzymes were monitored (Table 2). En-

zyme activities studied before dye addition and after its complete decolorization showed significant increase in the activities of veratryl alcohol oxidase (680%) and tyrosinase (211%) during the decolorization of Remazol red. There was very little induction of veratryl alcohol oxidase (18%) and no activity of tyrosinase in the cells obtained after decolorization of Rubine GFL. Azo reduc-tase activity was increased during the biodegradation of Remazol red (36%) but there was no activity of azo reductase detected during the decolorization of Rubine GFL (Table 2). There was 273% and 562% induction of NADH-DCIP reductase during decolorization of Rema-zol red and Rubine GFL. Induction of riboflavin reduc-tase was observed in the decolorization of Rubine GFL; but there was neither activity of the same in control cells nor in the cells obtained after decolorization of Remazol red.

Table 2. Enzyme activities of control cells and cells obtained after decolorization of dyes.

Control cells Cells obtained after decolorization of Remazol red

Cells obtained after decolorization of Rubine GFL

Veratryl alcohol oxidasea 0.237 ± 0.014 1.85 ± 0.154** 0.279 ± 0.030 (Intracellular) 0.358 ± 0.025 1.12 ± 0.018* NA Tyrosinasea

(Extracellular) 0.579 ± 0.062 0.253 ± 0.014 NA NADH DCIP reductaseb 62.67 ± 4.64 230.94 ± 34.36*** 411.89 ± 5.39*** Azo reductasec 2.87 ± 0.398 3.89 ± 0.157* NA Riboflavin reductased NA NA 7.39 ± 0.30***

a Enzyme units/min/mg protein; b μg of DCIP reduced/min/mg protein; c μM of Methyl red reduced/min/mg protein; d μg of riboflavin reduced/min/mg protein; Values are mean of three experiments ± SEM. Significantly different from control cells at * P < 0.05, ** P < 0.01 and *** P < 0.001 by One-way ANOVA with Tukey–Kramer comparison test.

Journal of Basic Microbiology 2012, 52, 1–11 Differential action of B. laterosporus on azo dyes 5

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Figure 2. FTIR spectra of Remazol red, its metabolites obtained after decolorization (A) and Rubine GFL and its metabolites obtained after decolorization (B). Remazol red in medium (C), Remazol red with dead microbial cells in medium (D), Rubin GFL in medium (E) and Rubin GFL with dead microbial cells in medium (F).

Product analysis Fig. 2A elucidates the FTIR spectrum of Remazol red and its metabolites obtained after decolorization. The FTIR spectrum of the Remazol red showed the major peaks at 1590.32 cm–1 for N=N stretching in azo com-pounds, 1400.85 cm–1 for O–H deformation in phenols and 1137.07 cm–1 for S=O asymmetric stretching in sulphones. On the other hand, FTIR spectrum of Rubine GFL (Fig. 2B) showed major peaks at 1595 cm–1 for N=N stretching in azo compounds and N=O stretching in nitrites, 2242 cm–1 for C≡N stretching in α, β unsatu-rated alkyl compounds and 2933 cm–1 or CH stretching in alkanes. Controls of dye in medium and with dead

microbial cells also showed presence of these groups. On the other hand, significant disappearance of major peaks and formation of new peaks in the FTIR spec-trum of metabolites obtained after dye decolorization suggests the biotransformation of both the dyes studied (Fig. 2C–F). The metabolites obtained after biodegradation of dyes were analyzed by HPTLC for supplementary sub-stantiation of the biotransformation of dyes. In case of biodegradation of Remazol red, the HPTLC chroma-togram (Fig. 3A) in the visible light showed absence of colored band in the metabolites lane whereas, single colored band was observed in the control dye lane. In

6 M. B. Kurade et al. Journal of Basic Microbiology 2012, 52, 1–11

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Figure 3. HPTLC profiles of Remazol red, its metabolites (A) and of Rubine GFL and its metabolites (B) obtained after decolorization. Control dye track (a) and metabolites track (b). HPTLC profiles of Remazol red in medium and with dead microbial cells (C), Rubin GFL in medium and with dead microbial cells (D). Dye in medium (a) and dye with dead microbial cells (b). HPTLC profiles at 254 nm (I), HPTLC plate exposed under UV light (II).

Journal of Basic Microbiology 2012, 52, 1–11 Differential action of B. laterosporus on azo dyes 7

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

the UV range, fluorescent bands in the metabolites lane were detected (Fig. 3A-II). The dye control in medium and in dead microbial cells showed two peaks each (excluding parent dye peak) having similar Rf values, but different than the Rf values of products obtained after decolorization of Remazol red (Fig. 3C). In case of Rubine GFL (Fig. 3B), the HPTLC chromatogram in visi-ble light showed a decrease in intensity of pink colored band in the lane corresponding to the track of metabo-lites as compared to control dye band. When HPTLC plate of Rubine GFL and its degraded metabolites was seen in the UV range, same fluorescence was seen as observed in case of Remazol red (Fig. 3B-II). Rubin GFL dye control in medium showed four distinct peaks (ex-cluding parent dye peak), whereas five peaks were de-tected in dye control with dead cells in which one peak was identical to peak obtained in dye control in me-dium lane with respect to Rf value (Fig. 3D). In addition, Rf values of two peaks were similar with two peaks out of fourteen peaks detected in the lane of metabolites of Rubine GFL. We proposed the biodegradation pathway of Remazol red on the basis of GC-MS data (Table 3A) of metabolites obtained after decolorization of the dye and the action of induced enzymes (Fig 4A). The formation of the in-termediate [A], 2-(phenylsulfonyl)ethanesulfonate [Rt-28.108, MW-249, m/z-249] indicates initial asymmetric cleavage of azo group present in the parent structure of the dye by the action of azo reductase. Further, desul-fonation from this intermediate by oxidase activity gave [B] (ethylsulfonyl)benzene [Rt-25.700, MW-170, m/z-170]. Dechlorination, dehydroxylation and deami-nation of intermediate [1] yields the intermediate [C] 4-({4-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino) naphthalene-2,7-disulfonate [Rt-20.783, MW-550, m/z-549], which on further cleavage by oxidase activity gave final product [D] 3-(1,3,5-triazin-2-ylamino)benzene-sulfonate [Rt-28.650, MW-251, m/z-251] and product [2]. The mass spectra of Remazol red control in medium and dead cells showed two and three peaks respec-tively. These mass spectra were totally unrelated to the mass spectra of dye metabolites (Table 3C). The proposed pathway of Rubine GFL (Fig. 4B) from GC-MS data (Table 3B) suggested the formation of the intermediate [A], 2-{[4-(methylamino) phenyl] diazenyl}-5-nitrobenzonitrile [Rt-27.700, MW-281, m/z-281] in the preliminary step which was further transformed into intermediate [B], 5-nitro-2-(phenyldiazenyl)benzonitrile [Rt-26.442, MW-251, m/z-249] by the action of oxidase enzymes. The elimination of molecular nitrogen from the later intermediate gave the final product [C], 1-(2-methylphenyl)-2-phenyldiazene [Rt-21.525, MW-196,

Table 3. (A) The GC-MS analysis of metabolites obtained after decolorization of Remazol red. (B) The GC-MS analysis of metabolites obtained after decolorization of Rubine GFL. (C) The GC-MS analysis of Remazol red control samples. (D) The GC-MS analysis of Rubin GFL control samples.

Metabolite Retention time (min)

m/z

(A) [A] 2-(phenylsulfonyl)ethanesulfonate 28.108 249 [B] (ethylsulfonyl)benzene 25.700 170 [C] 4-({4-[(3-sulfonatophenyl)amino]-

1,3,5-triazin-2-yl}amino)naphthalene-2,7-disulfonate

20.783 549

[D] 3-(1,3,5-triazin-2-ylamino)benzenesulfonate

28.650 251

(B) [A] 2-{[4-(methylamino) phenyl]diazenyl}-

5-nitrobenzonitrile 27.700 281

[B] 5-nitro-2-(phenyldiazenyl)benzonitrile 26.442 249 [C] 1-(2-methylphenyl)-2-phenyldiazene 21.525 196

Dye in medium Dye in medium with dead microbial cells

Retention time (min) m/z Retention time (min) m/z

(C) [1] 22.413 154 23.483 93 [2] 29.812 879 22.455 154[3] – – 29.789 875

(D) [1] 22.394 154 23.509 93 [2] 26.316 348 22.422 154[3] 23.408 85 22.975 202[4] – – 26.316 346

m/z-196]. In case of Rubin GFL control in medium and dead cells, the mass spectra showed three and four peaks respectively which were not identical to the mass spectra of dye metabolites (Table 3D).

Phytotoxicity Use of untreated effluents has direct impact on fertility of soil. Thus, it was of concern to assess the phytotoxic-ity of the dye before and after degradation. Hence, two plants (P. mungo and S. vulgare) were chosen which are having commercial values, for the toxicity assessment of azo dyes and their metabolites. The relative sensitiv-ity towards the dye Remazol red and Rubine GFL and its degradation products in relation to Sorghum vulgare and Phaseolus mongo seeds are presented in Table 4. Phyto-toxicity test showed good germination rate for both the plants in the metabolites extracted after decolorization of both the dyes; whereas germination was hampered in the untreated dyes samples. In addition to this, the metabolites had a nutritive role in seed germination as

8 M. B. Kurade et al. Journal of Basic Microbiology 2012, 52, 1–11

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Figure 4. Proposed pathway of biodegradation of Remazol red (A) and Rubine GFL (B).

shoot length in both the plants was grown better as compared to the seeds grown in plain water.

Discussion

B. laterosporus is an ecofriendly strain and has a tremen-dous capability to convert the environmental pollutants

such as textile dyes having versatile range of structures into simple forms [19]. Both the dyes studied were de-colorized in the static condition. The inhibition of dye reduction at shaking condition might be due to the presence of oxygen in shaking condition which nor-mally inhibits the azo bond reduction capability of azo reductase since; aerobic respiration may dominate utilization of NADH, thus hindering the electron trans-

Journal of Basic Microbiology 2012, 52, 1–11 Differential action of B. laterosporus on azo dyes 9

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Table 4. Phytotoxicity study of Remazol red and Rubine GFL and the metabolites obtained after decolorization.

Remazol red Rubine GFL

Germination (%)

Shoot length (cm)

Root length (cm)

Germination (%)

Shoot length (cm)

Root length (cm)

I 90 3.47 ± 0.180 5.11 ± 0.133 90 3.47 ± 0.180 5.11 ± 0.133 II 50 2.32 ± 0.182* 1.38 ± 0.22*** 40 2.64 ± 0.182** 2.38 ± 0.127***

Sorghum vulgare

III 90 4.48 ± 0.217$$ 4.69 ± 0.271$$$ 90 3.67 ± 0.206$$ 4.21 ± 0.122$$$

I 100 12.52 ± 0.271 5.72 ± 0.116 90 12.52 ± 0.271 5.72 ± 0.116 II 60 10.31 ± 0.469* 2.35 ± 0.120** 60 11.47 ± 0.217* 3.40 ± 0.190**

Phaseolus mungo

III 100 13.81 ± 0.196$$ 4.47 ± 0.211$$ 100 14.35 ± 0.229$$$ 4.94 ± 0.152$

I: Seeds germinated in distilled water. II: Seeds germinated in dye. III: Seeds germinated in metabolites obtained after decolori-zation of dye. Data were analyzed by one way analysis of variance (ANOVA) with Turkey–Kramer multiple comparison test using mean values of germinated seeds of three experiments. Seeds germinated in Remazol red are significantly different from the seeds germinated in plain water at * P < 0.05, ** P < 0.01, *** P < 0.001 and the seeds germinated in metabolites are significantly different from the seeds germinated in Remazol red at $$ P < 0.01 and $$$ P < 0.001. Seeds germinated in Rubin GFL are significantly different from the seeds germinated in plain water at * P < 0.05, ** P < 0.01, *** P < 0.001 and the seeds germinated in metabolites are significantly different from the seeds germinated in Rubin GFL at $ P < 0.05, $$ P < 0.01, $$$ P < 0.001. fer from NADH to azo bonds [20]. For maximum decol-orization of dyes, bacterial cultures generally prefer neutral to slightly alkaline pH range and the tempera-ture range of 25 to 50 °C. Mali et al. [21] found that a pH value between 6 and 9 was optimum for decolorization of triphenylmethanes and azo dyes by Pseudomonas sp., whereas Bacillus sp. VUS also had similar temperature optima for the decolorization of Brown 3 REL [22]. It can be presumed that the major mechanism of decolorization by microorganisms is mostly because of the biotransformation enzymes to mineralize the syn-thetic dyes. The difference in enzyme activity profile in decolorization of these two dyes might be due to the difference in dye structure. These oxidative enzymes have been found to play in an active role in the biodeg-radation of various textile dyes such as Reactive red HE7B, Direct blue GLL [15] and Direct red 5B [18]. Major functional groups may play a key role in providing the proper specificity and orientation to the enzyme for substrate catalysis. In the case of Remazol red, sul-phonyl and hydroxyl group may have accelerated the azo bond reduction by azo reductase [14]; whereas, the same enzyme could not initiate this azo bond catalysis due to absence of these groups in Rubine GFL. Telke et al. [17] reported the similar induction of NADH-DCIP reductase and azo reductase during the detoxification of Congo red using Pseudomonas sp. SU-EBT. The metabolites obtained after decolorization of azo dyes were characterized by FTIR. The biotransforma-tion of dyes was confirmed when the FTIR spectrum of metabolites obtained after dye decolorization showed significant disappearance of major peaks and formation of new peaks. For additional confirmation of these re-sults, HPTLC analysis was carried out. The results ob-

tained in that experiment justify that the chromo-phoric groups were totally removed from the intact dye structure. The multiple bands obtained in the metabo-lite lane confirmed the biodegradation of both the azo dyes as compared to single band of control dye. The difference in Rf values (data not shown) of control dye and metabolites supports the FTIR data which suggests biodegradation of Remazol red and Rubine GFL. The biocatalytic action of B. laterosporus depends upon the substrate properties which includes structure, func-tional group associated with it. This interesting task was cleared when metabolic pathways of two azo dyes oper-ated by B. laterosporus were proposed using the data ob-tained by GC-MS studies of metabolites of dyes obtained after their decolorization and enzymes involved in this catalysis. Azo reductase played the key role in reduction of most resistant azo group present in Remazol red. Azo reductase can cleave the substrate molecule in both ways, symmetrically as well as asymmetrically [23, 24]. Asymmetric action of azo reductase in the degradation of dye Direct red 5B have been reported by Tamboli et al. [18], while veratryl alcohol oxidase is also important oxidative enzyme that carries out biotransformation of various hazardous dyes [15]. From the proposed pathway of both the dyes, it is clear that B. laterosporus could min-eralize Remazol red effectively than Rubin GFL as simple products were formed after biodegradation of Remazol red. Recently Khandare et al. [25] have obtained different intermediate products formed after biodegradation of Remazol red using a plant system – Aster amellus Linn. except one final product 3-(1,3,5-triazin-2-ylamino)ben-zenesulfonate [MW-251, m/z-251]. The reduction of azo bond which is the primary step in the bacterial degradation of azo dyes, forces the for-

10 M. B. Kurade et al. Journal of Basic Microbiology 2012, 52, 1–11

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

mation of aromatic amines [26]. These amino groups are linked to dye, which are further removed by oxida-tive enzyme system. This is true in case of biodegrada-tion of Remazol red, where azo bond was cleaved at the beginning by azo reductase which forced to form aro-matic amine (intermediate [1]). Unlike Remazol red, in case of Rubine GFL, the azo bond was not cleaved by the same microbial enzyme system, as FTIR spectrum of metabolites of Rubine GFL showed a peak (1593 cm–1) corresponding to the azo group (Fig. 4B). The possible reason for this was the difference in structure of the dyes. The chemical structures of the dyes greatly influ-ence their decolorization rates and the decolorization efficiency is limited to several azo dye structures [11]. The basic structure of Remazol red contains naphthol ring attached to azo group and all azo dyes having naphthol ring holds a hydroxyl group at 2-position (ortho) of azo bond, because they are synthesized from H acids. Due to the hydroxyl group on the 2-position of naphthol ring in azo dyes, azo-hydrazone tautomerism (i.e., –N=N– ↔ =N–NH) could take place [27], which leads to conversion of hydroxyl group (electron-releas-ing group) to carbonyl group (electron-withdrawing group). Thus, this crucial conversion helped to reduce azo bond and it was only possible in the presence of electron-withdrawing group (carbonyl group instead of hydroxyl group). The results obtained in this study are in agreement with the findings of Zimmermann et al. [13] who reported that, a hydroxyl group in the 2-position of the naphthol ring is required for azo bond reduction. In addition, they found that sulpho groups in the proximity of azo bond hindered the azo reduc-tase; however, according to recent literature, steric hindrance created by nearby sulpho groups was elimi-nated by inductive and resonance effect [14]. Although these sulpho groups present at ortho position to azo bond in naphthol ring causes steric hindrance, reduc-tion of azo bond was still achievable, because inductive and resonance effect due to high electronegetivity of sulpho group could withdraw electrons from azo group. Due to this resonance effect, azo bond became more electrophilic leading to reduction of azo bond in Remazol red. In contrast, the same microbial enzyme system failed to reduce azo bond within Rubine GFL. In case of Rubine GFL; even though it has nitro group (an electron withdrawing group) at 4-position (para) in first phenyl ring to azo group, the positive inductive effect of elec-tron releasing group (ethyl amino propanenitrile) at 4-position (para) to azo group in adjacent phenyl ring totally hindered the reduction of azo bond. Rubine GFL holds a nitrile group at 2-position (ortho), which could

withdraw electrons from azo bond; however due to its low electronegetivity it was unable to withdraw elec-trons from azo bond via resonance and further forma-tion of resonance structure R=C=N– may have oc-curred, which was nothing but a second polar group in the dye molecule. According to Zimmermann et al. [13], a second polar substituent on the dye molecule im-pedes the azo bond reduction reaction. Hsueh et al. [28] also found that second substituent containing nitro group in the proximity of azo bond impeded the reduc-tion. In addition, due to the absence of hydroxyl group in the close proximity of azo group, azo reduction process could not be initiated. The efficiency of biodeg-radation by B. laterosporus was more towards Remazol red than Rubine GFL which was much more complex than later one. Even though Chen et al. [29] and Pearce et al. [12] reported that the faster decolorization of dye is dependent upon simplicity of dye structure; it is not the only determining factor, as our findings suggests efficiency of biodegradation of azo dyes depends upon the functional groups present in the nearby vicinity of azo bond. This reveals the structural influences of these two dyes on the catalytic efficiency of B. laterosporus. The different metabolic pathways operated by this bac-terial strain were attributed to structural differences between two dyes. Despite the fact that untreated dying effluents may cause serious environmental and health hazards, they are being disposed off in water bodies and used for the agriculture purposes. For this concern, phytotoxicity studies were conducted. The inhibition in germination of both the plants in control dye solution suggests the toxic nature of dyes. The induction in root and shoot lengths of both the plants in the dye metabolites con-firmed the non/less toxicity of metabolites. In addition, the metabolites provided a nutritive support to plants as the better growth of root and shoot in metabolites solution was obtained. This signifies the detoxification of both the dyes studied in this work by B. laterosporus.

Conclusion

The present study corroborates the biodegradation of two structurally different azo dyes by B. laterosporus. B. laterosporus can degrade the sulphonated azo dyes having naphthol ring much faster; supporting that the sulpho and hydroxyl group present at ortho position to azo group stimulated the reduction of azo bond by azo reductase; however the same reduction was totally hampered due to presence of ethyl amino propaneni-trile group at para position to azo group as in Rubine

Journal of Basic Microbiology 2012, 52, 1–11 Differential action of B. laterosporus on azo dyes 11

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

GFL. The functional groups present in the nearby vicinity of azo bond play a central role in azo bond reduction.

Acknowledgement

M. B. Kurade and T. R. Waghmode wishes to thank Department of Biotechnology, New Delhi, India. M. B. Kurade also wishes to thank Rakesh M. Patil for his technical assistance.

References

[1] Gupta, V., Mittal, A., Gajbe, V., 2005. Adsorption and desorption studies of a water soluble dye, Quilnoline yel-low, using waste materials. J. Colloid. Interf. Sci., 284, 89–98.

[2] Tony, B., Goyal, D., Khanna, S., 2009. Decolorization of textile azo dyes by aerobic bacterial consortium. Int. Bio-deter. Biodegr., 63, 462–469.

[3] Mansour, H., Corroler, D., Barillier, D., Ghedira, K. et al., 2007. Evaluation of genotoxicity and pro-oxidant effect of the azo dyes: Acids yellow 17, Violet 7 and Orange 52, and of their degradation products by Pseudomonas putida mt-2. Food Chem. Toxicol., 45, 1670–1677.

[4] Chang, J., Lin, Y., 2000. Fed-batch bioreactor strategies for microbial decolorization of azo dye using a Pseudomonas luteola strain. Biotechnol. Prog., 16, 979–985.

[5] Saratale, R., Saratale, G., Chang, J., Govindwar, S., 2011. Bacterial decolorization and degradation of azo dyes: A review. J. Taiwan Inst. Chem. Eng., 42, 138–157.

[6] Vijaykumar, M., Vaishampayan, P., Shouche, S., Karegou-dar, T., 2007. Decolourization of naphthalene-containing sulfonated azo dyes by Kerstersia sp. strain VKY1, Enzyme Microb. Technol., 40, 204–211.

[7] Lodato, A., Alfieri, F., Olivieri, G., Donato, A. et al., 2007. Azo-dye conversion by means of Pseudomonas sp. OX1. En-zyme Microb. Technol., 41, 646–652.

[8] Jadhav, S., Kalme, S., Govindwar, S. 2008. Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360. Int. Biodeter. Biodegr., 62, 135–142.

[9] El-Sheekh, M., Gharieb, M., Abou-El-Souod, G., 2009. Bio-degradation of dyes by some green algae and cyanobacte-ria. Int. Biodeter. Biodegr., 63, 699–704.

[10] Elisangela, F., Andrea, Z., Fabiana, F., Isis, S. et al., 2009. Microaerophilic–aerobic sequential decolourization/bio-degradation of textile azo dyes by a facultative Klebsiella sp. strain VN-31. Process Biochem., 44, 446–452.

[11] Chivukula, M., Renganathan, V., 1995. Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl. Environ. Microbol., 61, 4374-4377.

[12] Pearce, C. Lloyd, J. Guthrie, J. 2003. The removal of colour from textile wastewater using whole bacterial cells: a re-view. Dyes Pigments, 58, 179–196.

[13] Zimmermann, T., Kulla, H., Leisinger, T., 1982. Properties of purified Orange I1 azoreductase, the enzyme initiating azo dye degradation by Pseudumunas KF46. Eur. J. Bio-chem., 129, 197–203.

[14] Hsueh, C., Chen, B., Chia-Yi, Y., 2009. Understanding effects of chemical structure on azo dye decolorization characteristics by Aeromonas hydrophila. J. Hazard. Mater., 167, 995–1001.

[15] Jadhav, U., Dawkar, V., Tamboli, D., Govindwar, S., 2009. Purification and characterization of veratryl alcohol oxi-dase from Commamonas sp. UVS and its role in decoloriza-tion of textile dyes. Biotechnol. Biopro. Eng., 14, 369–376.

[16] Waghmode, T., Kurade, M., Govindwar, S., 2011. Time dependent degradation of mixture of structurally differ-ent azo and non azo dyes by using Galactomyces geotrichum MTCC 1360. Int. Biodeter. Biodegr., 65, 479–486.

[17] Telke, A., Joshi, S., Jadhav, S., Tamboli, D., Govindwar, S., 2009. Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium Pseudo-monas sp. SU-EBT. Biodegradation, 21, 283–296.

[18] Tamboli, D., Kagalkar, A., Jadhav, M., Jadhav, J., Govind-war, S., 2010. Production of polyhydroxyhexadecanoic acid by using waste biomass of Sphingobacterium sp. ATM generated after degradation of textile dye Direct red 5B. Bioresour. Technol., 101, 2421–2427.

[19] Gomare, S., Tamboli, D., Kagalkar, A., Govindwar, S., 2009. Eco-friendly biodegradation of a reactive textile dye Golden yellow HER by Brevibacillus laterosporus MTCC 2298. Int. Biodeter. Biodegr., 63, 582–586.

[20] Chang, J., Lin, Y., 2001. Decolorization kinetics of recom-binant E. coli strain harboring azo dye decolorization de-terminants for Rhodococcus sp. Biotechnol. Lett., 23, 631–636.

[21] Mali, P., Mahajan, M., Patil, D., Kulkarni, M., 2000. Biode-colorization of members of triphenylmethanes and azo groups of dyes. J. Sci. Ind. Res., 59, 221–224.

[22] Dawkar, V., Jadhav, U., Jadhav, S., Govindwar, S., 2007. Biodegradation of disperse textile dye Brown 3 REL by newly isolated Bacillus sp. VUS. J. Appl. Microbiol., 105, 14–24.

[23] Kalme, S., Ghodake, G., Govindwar, S., 2007. Red HE7B degradation using desulfonation by Pseudomonas desmolyti-cum NCIM 2112. Int. Biodeter. Biodegr., 60, 327–333.

[24] Kalme, S., Parshetti, S., Jadhav, S., Govindwar, S., 2007. Biodegradation of benzidine based dye Direct blue 6 by Pseudomonas desmolyticum NCIM 2112, Bioresour. Technol., 98, 1405–1410.

[25] Khandare, R., Kabra, A., Tamboli, D., Govindwar, S., 2011. The role of Aster amellus Linn. in the degradation of a sul-fonated azo dye Remazol red: A phytoremediation strat-egy, Chemosphere, 82, 1147–1154.

[26] Junnarkar, N., Murty, D., Bhatt, N., Madamwar, D., 2006. Decolorization of diazo dye Direct red 81 by a novel bac-terial consortium. W. J. Microb. Biotechnol., 22, 163–168.

[27] Libra, J., Borchert, M., Vigelahn, L., Storm, T., 2004. Two stage biological treatment of a diazo Reactive textile dye and the fate of the dye metabolites. Chemosphere, 56, 167–180.

[28] Hsueh, C., Chen, B., 2008. Exploring effects of chemical structure on azo dye decolorization characteristics by Pseudomonas luteola. J. Hazard. Mater., 154, 703–710.

[29] Chen, K., Wu, J., Liou, D., Hwang, S., 2003. Decolorization of the textile dyes by newly isolated bacterial strains. J. Biotechnol., 101, 57–68.