Optimization of triazo Acid Black 210 dye degradation by Providencia sp. SRS82 and elucidation of degradation pathway

Process Biochemistry 49 (2014) 110–119

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Optimization of triazo Acid Black 210 dye degradation by Providencia sp. SRS82 and elucidation of degradation pathway Shweta Agrawal a , Devayani Tipre b , Bhavesh Patel c , Shailesh Dave b,∗ a b c

Department of Biotechnology, Sanghvi Institute of Management and Science, Indore 453331, India Department of Microbiology and Biotechnology, School of Sciences, Gujarat University, Ahmedabad 380009, India V.P. and R.P.T.P. Science College, Vallabh Vidyanagar 388120, India

a r t i c l e

i n f o

Article history: Received 9 April 2013 Received in revised form 3 October 2013 Accepted 13 October 2013 Available online 21 October 2013 Keywords: Providencia sp. Azo dyes Acid Black 210 Cytotoxicity FTIR GC–MS

a b s t r a c t The current work is aimed to evaluate the degradation of triazo textile dye Acid Black 210 (AB210) by Providencia sp. SRS82 that degrade 100 mg/L dye within 90 min under optimum conditions and was also found tolerant to as high as 2000 ppm of dye AB210. Optimum conditions for decolourization and degradation of AB210 with the isolate were viz. temperature 30 ◦ C, pH 8, NaCl concentration 2.5% (w/v) and initial cell load of 8 × 108 cells/mL under static condition. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase and tyrosinase, azoreductase, and DCIP reductase indicated their contribution in the biodegradation of AB210. The products obtained from Providencia sp. SRS82 degradation was monitored through UV–Vis spectrophotometer and were characterized by FTIR, HPTLC, HPLC, GC/MS and LCMS. The proposed metabolic pathway for the biodegradation of AB210 is elucidated for the first time, which showed production of 4 molecules of benzene, one of naphthalene and 4-aminophenyl-N-(4-amino phenyl) benzene sulphonamide. Microbial toxicity and cytotoxicity studies revealed the comparatively less toxic nature of metabolites generated after degradation of AB210. Providencia sp. SRS82 was found competent to degrade actual effluent and diverse dyes that could be present in textile industry effluent showing usefulness of the organism for possible commercial application. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Synthetic dyes are extensively used in textile dyeing, paper printing, colour photography, pharmaceutical, food, cosmetic and leather industries as well as additives in petroleum products [1,2]. Colours are perceptible at a dye concentration as low as 1 mg/L, whereas an average concentration of 300 mg dye/L has been reported in effluents from textile manufacturing processes [3]. Azo dyes, one of the major constituents of this pollution, are resistant to biodegradation due to their complex structures and xenobiotic nature. Various azo dyes and their degradation intermediates contribute to the mutagenic activity of ground and surface waters that are polluted by textile effluents. The existence of azo dyes and their by-products in aqueous ecosystems leads to aesthetically unacceptable colouration of waters, along with obstruction of light penetration and diminution of dissolved oxygen, consequently leading to death and putrefaction of aquatic animals. The azo dyes and/or their degradation products may be toxic and mutagenic to the aquatic animals and human. Therefore, treatment of textile dye effluents prior to their release in nearby water streams is

∗ Corresponding author. Tel.: +91 79 2630 3225; fax: +91 79 26303225. E-mail address: [email protected] (S. Dave). 1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2013.10.006

necessary [4,5]. Treatment of textile and dyeing industry effluent by the usually employed physico-chemical methods is a herculean task, primarily because of its high BOD (biochemical oxygen demand), COD (chemical oxygen demand), heat, colour, pH and the presence of metals. Additionally, physico-chemical methods used are associated with the shortcomings like heavy cost, formation of large amounts of sludge and the emission of toxic substances [6]. On the other hand microbial decolourization is an environment-friendly and cost-competitive alternative [7]. Over the past decades, various bacteria, fungi, yeasts, actinomycetes and algae have been reported competent to degrade azo dyes [8]. The degree of decolourization by microorganisms greatly depends on the type, molecular weight, and substitution groups of the dye. Azo compounds with an amino or hydroxyl group are more likely to be degraded than those with methyl, methoxy, sulpho or nitro groups [9]. Moreover, microorganisms can decolourize the dyes with different enzyme systems. The bacterial biodegradation of dye is associated with its intracellular and extracellular oxidoreductive enzyme system such as azo reductase, DCIP-reductase and laccase [10,11]. A large quantity of AB210 is used in dyeing cotton, leather and woolen fabric [12]. AB210 is an azo (metal complex) dye containing sulphonyl and amino groups. It is toxic, recalcitrant and has mutagenic and carcinogenic effects on both aquatic biota and humans. Some of the physical methods reported earlier for

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removal of AB210 from effluents include electrochemical oxidation [13] and sonochemical decolourization [14]. Very few reports viz use of; consortium SV5 [15], Cladosporium cladosporioides [16], Vibrio harveyi TEMS1 [17], Bacillus thuriengiensis [18] were reported to degrade AB210 dye. Moreover, the biotransformation of AB210 and the identification of degradation intermediates and/or products have not been reported. Thus the aim of this work was to explore the potential of isolated Providencia sp. SRS82 for the decolourization of the structurally complex triazo dye AB210. This study also includes characterization and identification of intermediates of AB210 degradation and involvement of intra- and extracellular enzymes responsible for degradation. Cytotoxicity and microbial toxicity effects of degraded metabolites were studied on selected crops and bacteria.

From the preserved slant a loop full cell growth was inoculated in 100 mL nutrient broth and incubated on an orbital shaker agitating at 150 rpm and 32 ± 2 ◦ C temperature for 18 h. For AB210 dye decolourization study, 10% (v/v) of the actively growing culture having ∼108 cells/mL was used as inoculum in all the further experiments if otherwise mentioned.

2. Materials and methods

2.4. Optimization of decolourization parameters

2.1. Dyes, chemicals and media

For optimization of process parameters for AB210 dye decolourization one parameter at a time approach was taken. The process parameters optimization for AB210 decolourization by the Providencia sp. SRS82 were viz. initial dye concentration (50 to 1500 mg/L), temperature (10 to 70 ◦ C), initial system pH (6 to 12), inoculum size (2 × 108 to 1 × 109 cells/mL), NaCl concentration (0.5 to 10% w/v) in the system as well as the influence of agitation (150 rpm) and static conditions. If otherwise mentioned, all the experiments were performed in triplicates in 250 mL Erlenmeyer flask containing 100 mL nutrient broth (pH 7.0) as growth medium supplemented with 100 mg/L AB210 dye and 10% inoculum having 108 cells/mL. All the flasks were incubated at 32 ± 2 ◦ C under static condition. The capability of the bacterial isolates to decolourize different commercial textile dyes viz: Acid Yellow 11, Acid Red 131, Acid Black 15, Acid Black 298, Reactive Violet 5, Reactive Red 152, Disperse Yellow 126, Disperse Red 60 and Direct Black 19 were also examined in a similar manner. To study fed batch decolourization of the experiment, 100 mg/L AB210 dye was added to nutrient broth with 10% activated culture of Providencia sp. SRS82. After >90% decolourization of the added dye, again 100 mg/L of dye was added without supplement of any external nutrients. Dye aliquots were added continuously until the culture started to lose its decolourization efficiency. For liquid medium, the samples of the culture broth were analysed at the dye max 605 nm using the supernatant from the culture medium after centrifugation at 10,000g for l0 min at 4 ◦ C.

Commercial grade triazo acid dye AB210; CAS No. 99576-154-amino-6-((4-(((4-((2,4-diaminophenyl)azo)phenyl)amino) 5; sulphonyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)-2,7naphthalenedisulphonic); was generously provided by Sanchi Chemicals, Indore, India. Nutrient broth, nutrient agar, methyl red and l-tyrosine were obtained from Hi Media, India; ABTS (2,2azinobis (3-ethylbenzothiazolin-6-sulphonic acid)) and NADH was purchased from Sigma Chemical Company, USA while, tartaric acid, n-propane and other chemicals were obtained from SD Fine Chemicals, India. All chemicals used were of analytical grade. The Allium cepa (onion) bulbs were obtained from local markets, Indore, India. Microbial cultures of Escherichia coli, Bacillus subtilis, Azotobacter, and phosphate solubilizers were isolated from soil. 2.2. Strain isolation, characterization and preservation Soil and wastewater samples from the vicinity of textile dyeing industries located in Indore, India, were inoculated at a concentration of 1% (w/v) into a nutrient broth supplemented with 100 mg/L AB210 and were kept at 32 ± 2 ◦ C temperature for 48 h under static condition. Samples showing the dye decolourization were streaked on nutrient agar plates containing 50 mg/L AB210 dye and were incubated at 32 ± 2 ◦ C temperature for 48 h. The morphologically distinct bacterial isolates showing high ratio of zone of decolourization to colony diameter, were selected and purified by the streak plate method. Forty three isolates were selected for further comparison and were first cultured in nutrient broth (without dye) for 24 h at 30 ◦ C with shaking at 150 rpm. The cells were harvested, and a uniform cell density (1.0 OD at 560 nm) was maintained. The mean cell counts of these cultures were between 108 and109 cells/mL. The cell density standardized decolourization experiment was performed in 100 mg/L AB210 supplemented nutrient broth. The culture showing the highest rate of decolourization was selected for further studies. Suspension prepared from a distinctly isolated colony of the selected strain was used for inoculating various biochemical media. The selected isolate was transferred to BUG medium (Biolog®, USA) and prepared suspension was inoculated in GN Biolog plates as per the standard protocol provided [19]. All the media and Biolog® plates were incubated for 6 to 24 h at 32 ± 2 ◦ C. Results were recorded and interpreted for identification using Biolog® software (Biolog Inc., USA). The 16S rRNA sequencing of isolated bacteria was carried out at Ocimum Biosolutions, Hyderabad, India. Analysis of the nucleotide sequence was done using BlastN (NCBI server) and the sequence

was refined manually to remove ambiguities and submitted to the GenBank. Selected strain was preserved and maintained on nutrient agar slants containing 100 mg/L AB210 dye at 4–8 ◦ C.

2.3. Inoculum preparation

2.5. Textile dye effluent and other dyes decolourization Industrial effluent collected from textile dyeing industry, Ahmedabad, India, and acid reactive and direct dyes were screened for decolourization by Providencia sp. SRS82 with above mentioned optimized conditions. True colour values of the textile effluent were determined by the American Dye Manufacturers’ Institute (ADMI 3WL; New York, USA) tristimulus filter method. To observe reduction in ADMI in the test flask, an aliquot of 3 mL was withdrawn at the interval of 24 h and subjected for determination of ADMI value by observing % transmittance at 590, 540, and 438 nm [20]. The ADMI removal ratio was calculated using the following equation: ADMI removal(%) = [(Initial ADMI − Final ADMI)/Initial ADMI] × 100

The textile effluent was characterized by BOD and COD [21] before and after its degradation.

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2.6. Preparation of cell free extract The cells of Providencia sp. SRS82 were activated in 250 mL Erlenmeyer flasks containing 100 mL sterile nutrient broth of pH 7.0, incubated at 32 ± 2 ◦ C for 24 h under static condition and cells were harvested by centrifugation at 10,000g for 15 min. The culture supernatant obtained after centrifugation was directly used as a source of extracellular enzymes. The harvested cells suspended in potassium phosphate buffer (50 mM, pH 7.4) were homogenized and sonicated (Labsonic®M, Sartrious, Germany) at an output of 70 W with seven strokes each of 30 s with a 1 min interval at 4 ◦ C. These extracts were used as a source of the intracellular enzyme after centrifugation at 15,000g for 15 min at 4 ◦ C. All the enzyme assays were run in triplicates and average rates were calculated. Protein concentration was determined using the Lowry method [22] using bovine serum albumin as the standard protein. 2.7. Enzyme activity The oxidoreductive enzymes are responsible for generating highly reactive free radicals that undergo a complex series of spontaneous cleavage reactions. The oxidoreductive enzymes, such as lignin peroxidase, laccases, tyrosinase, azoreductase, NADH–DCIP reductase have been mainly playing major role in the bacterial decolourization and degradation of azo dyes [23]. The azoreductase activity was assayed by monitoring NADH disappearance at 440 nm based on the modified procedure described by Zimmermann et al. [24]. Reaction mixtures for the standard assay contained in a total volume of 2.2 mL: 50 mM sodium phosphate buffer pH 5.5, 20 ␮M NADH, 152 ␮M methyl red and 200 ␮l of enzyme solution. The reaction was started by the addition of NADH followed by monitoring the decrease in colour intensity at 440 nm. The blank reaction (no protein added) was insignificant. The activity of NADH–DCIP reductase was determined by modifying the procedure reported earlier by Lade et al. [11]. Briefly, 5.0 mL reaction mixture contained 25 mM DCIP (2,6dichloro-phenol indophenol) and 200 ␮l enzyme solution in potassium phosphate buffer (50 mM, pH 7.4). From this, 2.0 mL reaction mixture was assayed at 590 nm by adding 250 mM NADH. One unit of reductase enzyme activity was defined as the amount of enzyme required to reduce 1 ␮g of substrate per minute per milligram of protein. Laccase assay was carried out by modified procedure of Senan and Abraham, [6] following oxidation of 2,2 -azinobis-(3-ethyl benzthiazoline-6sulphonate) (ABTS) with a reaction mixture containing 500 M ABTS buffered with 50 mM sodium phosphate buffer of pH 4.5 and 200 ␮l of enzyme extract. Oxidation of ABTS was followed by an increase in absorbance at 420 nm. The activities of lignin peroxidase and tyrosinase were determined as reported by Kadam et al. [10]. All enzyme assays were carried out at 30 ◦ C where reference blanks contained all components except the enzyme. One unit of enzyme activity was defined as the amount yielding a reduction of 1 ␮mol of substrate per min per ml of enzyme. 2.8. Extraction and analysis of metabolites obtained after decolourization 2.8.1. UV–Vis spectrophotometer analysis For liquid medium, the samples of the culture broth were analysed at the dye max 605 nm using the supernatant from the culture medium after centrifugation at 10,000g for l0 min at 4 ◦ C. The colour removal was calculated as percent decolourization and was expressed as [18] decolourization(%) =

initial absorbance − final absorbance × 100 initial absorbance

2.8.2. FTIR, HPTLC, HPLC and GC–MS analysis The decolourized broth was centrifuged at 7000 g for 15 min at 4 ◦ C. The metabolites formed after decolourization of dyes were extracted thrice using equal volumes of ethyl acetate, concentrated in a rotary vacuum evaporator and then dissolved in a small volume of HPLC grade methanol. Same sample was then subjected for FTIR, HPLC and HPTLC analysis to confirm degradation. Identification of the metabolites was carried out by GC–MS. FTIR analysis (Perkin Elmer, Spectrum GX) was done in the mid IR region of 400–4000 cm−1 with 16 scan speed. HPTLC was carried out by modified procedure described by Lade et al. [11], using silica gel plates (HPTLC Lichrospher silica gel 60 F254S). The solvent system as mobile phase used was toluene:ethyl acetate:methanol (11:2:5). The twin trough chamber was saturated with mobile phase for 20 min prior to plate development. After development scanning was done using a TLC scanner (Camag, Switzerland) at 254 and 530 nm using deuterium and tungsten lamp, respectively, with slit dimension of 5 × 0.45 mm. The chromatogram was analysed using HPTLC Win-CATS 1.4.4.6337 software. HPLC (Shimadzu, Japan) analysis was carried out on a C18 column (250 mm × 4.6 mm, 5 mm) equipped with dual wavelength detector by the isocratic method. The mobile phase was methanol with a flow rate of 0.75 mL/min and having 10 min run time. GC–MS analysis of metabolites was carried out using a QP 5000 mass spectrophotometer (Autosyztem XL GC+ , Perkin Elmer, and USA). The ionization voltage was 70 eV. Gas chromatography was conducted in temperature programming mode with a Resteck column (0.25 mm × 30 mm; XTI-5). The initial column temperature was 40 ◦ C for 4 min, then increased linearly at 10 ◦ C/min to 270 ◦ C and held at 4 min. The temperature of injection port was 275 ◦ C and GC–MS interface was maintained at 300 ◦ C. The helium was used as carrier gas with a flow rate of 1 mL/min and 30 min run time [25] carried out at SICART Vallabh Vidyanagar, India.

2.9. Toxicity assay 2.9.1. Microbial toxicity The microbial toxicity of control dyes and products obtained after its degradation (1500 ppm) was carried out using B. subtilis, E. coli, Azotobacter and phosphate solubilising strains and the mean of inhibition zone (diameter in cm) was recorded after 24 h of incubation at 30 ◦ C [26].

2.9.2. Comet assay (single cell gel electrophoresis) Small bulbs of A. cepa with uniform size and shape were exposed to water initially for the development of roots. The bulbs thus prepared were grouped into three sets, followed by 48 h exposure to dye sample as (1500 mg/L of AB210, Set I), biodegradation metabolites of dye (1500 mg/L, Set II) and water (control, Set III). After exposure, the bulbs were removed and thoroughly washed in running tap water and used for further studies. For each slide, 25 randomly chosen nuclei were analyzed using an inverted microscope (Labovision, India). A computerized image analysis system (Comet version 1.5) was employed to measure % DNA damage (% T) and tail length (TL) [27,28].

2.10. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons test. Readings were considered significant when P was ≤0.05 using SPSS 16.0.

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3. Results and discussion 3.1. Screening, isolation and identification of decolourizing bacteria Soil and wastewater samples heavily contaminated with dyes from dye manufacturing industry facilitated isolation of nine morphologically distinct isolates showing decolourization zone of 3–11 mm on nutrient agar plates containing 100 ppm AB210. The isolate 1, which gave the largest ratio of decolourization zone:colony diameter of 1.79 for a colony size of 3–4 mm was selected for further study. Remaining five isolates showed decolourization zone:colony diameter ratio between 1.15 and 1.45. All the nine isolates were resistant to 100 ppm AB210 and grew on nutrient agar plates containing 100 ppm AB210 concentration. This could be a consequence of natural adaptation of the organisms as the samples, from which the isolates obtained were profoundly contaminated with dyes. Among the 3 isolates which showed high ratio of decolourization zone:colony diameter on agar plate the one which showed the highest (93.9%) decolourization in 48 h in liquid medium was selected for subsequent study (detail data not shown). The selected isolate was identified as Providencia sp. on the basis of conventional biochemical tests and Biolog® GP plate. The identity of the purified isolate was confirmed by 16S rRNA gene sequence analysis and it was designated as Providencia sp. SRS82. The 1403 bp sequence is deposited in GenBank with accession no. JN381552.1. 3.2. Optimization of physico-chemical parameters for AB210 decolourization by Providencia sp. SRS82 Various parameters mentioned below were optimized for decolourization of AB210 by Providencia sp. SRS82 before the microbial system was employed to treat textile industrial wastewater. 3.2.1. Effect of static and 150 rpm shaking condition on AB210 decolourization by Providencia sp. SRS82 Monitoring decolourization of AB210 under static condition showed >99% decolourization within 24 h, whereas when it was kept in shaking condition only 9.36% decolourization was observed even after 24 h of incubation (Fig. 1a). Azo dyes are generally resistant to attack by bacteria under aerobic conditions. Azo dye decolourization by bacterial species is often initiated by an enzymatic reduction of azo bonds, the presence of oxygen normally favours bacterial growth but inhibits the azo bond reduction activity since aerobic respiration may dominate utilization of NADH; thus impeding the electron transfer from NADH [29]. Wang et al. [30] reported >96% decolourization of Reactive Red 180 under anaerobic conditions and only 13% in aerobic conditions (150 rpm), similarly oxygen inhibitory effects were observed for decolourization with Pseudomonas luteola [31], V. harveyi TEMS1[17] and with Basidiomycete strain PV002 [32]. Therefore, all further experiments for dye degradation study were carried out under static microaerophilic condition. However, Kodam et al. [33], Pourbabaee et al. [34], Tony et al. [3] have reported efficient decolourization of dyes under aerobic condition. 3.2.2. Effect of temperature Temperature can affect the growth and degradability of microorganisms. As shown in Fig. 1b, Providencia sp. SRS82 showed decolourization ability of AB210 in a temperature range of 10 to 60 ◦ C and a temperature of 30 ◦ C was found to be optimum showing 96.75% decolourization of AB210 in 24 h. Temperatures above 37 ◦ C and below 30 ◦ C resulted in a decline in the extent of decolourization. Providencia sp. SRS82 showed 5% decolourization activity even at 60 ◦ C indicating the ability of the organism to tolerate high temperature. These results further showed that there is no

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thermal deactivation of decolourization activity under operational temperatures. This strain could acclimatize to a broad range of temperature. Lade et al. [11] observed 37 ◦ C as the optimum temperature for decolourization of Rubine GFL by using consortium-AP and increase and decrease in temperature from 37 ◦ C decreased the extent of decolourization. Ozdemir et al. [17] have reported maximum 94% AB210 decolourization efficiency of V. harveyi TEMS1 at 20 ◦ C. Likewise, Keharia et al. [35] noticed that the decolourization rate for Reactive Violet 5 (RV5) increased with increase in incubation temperature from 8 to 37 ◦ C, at which decolourization rate was maximum (7.07 mg/L/h). Rate of RV 5 decreased with further increase in temperature up to 45 ◦ C, which then remained constant till 55 ◦ C. Wang et al. [30] reported maximum azo dye decolourization efficiency of Citrobacter sp. at temperature 32 ◦ C. 3.2.3. Effect of pH Optimization studies with regard to initial medium pH versus dye decolourization showed maximum (97.34%) decolourization of AB210 within 24 h by Providencia sp. SRS82 at pH 8.0. The isolate showed 63.78%, 71.23%, 80.23% and 54.17% decolourization at pH 6, 7, 8 and 10, respectively, at 6 h incubation (Fig. 1c). When data of 24 h are considered the differences were not significant at pH 6, 7 and 8. The optimum pH for colour removal is often at a neutral or slightly alkaline pH, and the decolourization rate tends to decrease at strongly acidic/alkaline pH conditions [5]. This could be due to enhanced solubilization and transport across the cell membrane of dye at slightly alkaline pH. The rate of decolourization was comparatively much lower at strongly acidic (6.0) and alkaline pH (10.0 and 12.0). These results corroborate earlier reports of Wang et al. [30] who reported maximum azo dye decolourization efficiency of Citrobacter sp. at pH 7.0. Lade et al. [11] observed an optimum pH 8.5 for decolourization of Rubine GFL by using consortiumAP, Anjaneya et al. [36] also reported optimum decolourization of Metanil Yellow at pH 7.2 for both Bacillus sp. strain AK1 and Lysinibacillus sp. strain AK2. Contrary to our findings, Kalpana et al. [37] observed higher decolourization rate at pH values of 5, 6, and 9 as compared to pH 7 and 8 for Levafix Blue E-RA granulate dye by the white rot fungus Irpex lacteus, possibly due to the optimum enzyme activity of Lucas at pH 5 and 6. 3.2.4. Effect of initial bacterial biomass on AB210 decolourization by the isolate The growth response of Providencia sp. SRS82 concomitantly enhanced in synchrony with extent and dye decolourization at each time of incubation, when inoculum size was increased (Fig. 1d). At every inoculum size studied, there were 2.3–3 fold enhancements in the dye decolourization during 2–6 h. Maximum dye decolourization (99.2%) was evident with 8 × 108 cells/mL within 2 h. Similarly, Mathew and Madamwar [15] studied the effect of 5 to 20% (v/v) of the bacterial consortium SV5 and noticed an increase in the decolourization with an increase in inoculum concentration but the rate of increase in the decolourization was not very significant above 10% (v/v) inoculum. However, a reduction in rate was generally noticed with an increase in incubation period at all cell concentrations, which might be due to the gradually decreased in dye concentration. Garg et al. [5] noticed maximum growth response and dye decolourization (86.9%) with 4.0% (v/v) dose of Pseudomonas putida SKG-1 inoculum. Our results also agree with reports of Radha et al. [38], Dafale et al. [39] and Keharia et al. [35]. 3.2.5. Effect of salt concentration on AB210 decolourization In both the dye manufacturing and dye-consuming industrial processes the salt concentration is up to 15–20%. Generally, sodium concentration above 10 g/L, cause moderate inhibition of most bacterial activities. Therefore, it is important to assess the decolourization by biomass at high salt concentration. As depicted in Fig. 1e,

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Fig. 1. Effect of physico-chemical factors on decolourization of AB210 by Providencia sp. SRS82. (a) agitation/aeration, (b) incubation temperature, (c) initial pH, (d) initial bacterial biomass, (e) salt concentration, (f) fed batch dye addition.

the effect of NaCl concentration on the decolourization of AB210 by Providencia sp. SRS82 was examined from 0.5–10% NaCl concentration. The strain showed maximum, 99.6% decolourization at 2.5% salt concentration within 22 h of incubation. The degradation efficiency decreased successively on either side of optimal salt concentration. These results corroborate with the results obtained by

Dafale et al. [39] and Anjaneya et al. [36] who observed retention of dye decolourization efficiency by microorganisms up to 5% salt concentration followed by the inhibitory effect of salinity at higher salt concentration. However, Meng et al. [40] reported a twofold decrease in the initial decolourization rate at 5% NaCl concentration.

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Table 1 Extracellular and intracellular enzyme activity during decolourization of AB210 by Providencia sp. SRS82. Enzymes

Enzyme activity Control

a

Laccase Tyrosinasea Intracellular Extracellular Lignin peroxidisea Intracellular Extracellular NADH–DCIP reductaseb Azoreductasec

0.64 ± 0.20 58.9 ± 4.4 11.9 ± 4.37 7.82 ± 1.4 0.42 ± 0.26 31.26 ± 2.18 2.4 ± 0.8

Test 1.53 ± 0.58 114.86 ± 2.6 19.53 ± 8.5 18.48 ± 4.2 0.26 ± 0.09 44.66 ± 4.72 3.2 ± 0.001

Control = enzyme extracted from culture medium without dye after 24 h; Test = enzyme extracted from the dye decolourized culture medium after 24 h. Values are mean of three experiments ± SD. Percent induction calculated from mean values. a Enzyme activity: ␮M/min/mg protein. b Enzyme activity: ␮g of DCIP reduced/min/mg protein. c Enzyme activity: ␮g of methyl red reduced/min/mg protein. Fig. 2. Effect of initial AB210 dye concentration on dye decolourization rate by Providencia sp. SRS82.

3.2.6. Fed-batch decolourization As illustrated in Fig. 1f, in this experiment it was found that the dye could be decolourized by Providencia sp. SRS82 when added repeatedly for eight consecutive cycles with a decrease in decolourization rate and an increase in the time required for decolourization. The possibility is that the number of viable cells might have decreased due to reduction of nutrients in the medium or due to entry of cells in stationary phase and then subsequently to death phase resulting in a gradual decrease in decolourization activity. Similar observations have been recorded previously by Vijaya and Sandhya [41] while using mixed bacterial culture with 90% decolourization of methyl red up to 4 cycles. Likewise, Anjaneya et al. [36] reported 100% dye decolourization (200 mg/L) up to initial four cycles with Bacillus sp. AK1 and Lysinibacillus sp. AK2 at 12 and 24 h, respectively, but for the next remaining five cycles time required for dye decolourization increased with reduction in decolourization percentage. Contrastingly, Dave and Dave [18] reported that B. thuringiensis took 6 h to decolourize first dose of 300 mg/L of AR 119 but then after up to 1600 ppm (5th cycle) in gradual addition, the time required for ≥90% decolourization were less than 2.5 h. Thereafter, gradually the time for decolourization increased, requiring 7 h for 50% decolourization during the 7th cycle, indicating either scarcity of the available nutrient in the medium or the attainment of stationary or decline phase of the growth of the culture. 3.2.7. Effect of different initial dye concentrations on AB210 decolourization The effect of different initial dye concentrations of AB210 on decolourization was observed using 50 to 2000 mg/L (Fig. 2). With the increase in initial dye concentration an increase in the initial decolourization rate was noticed up to 750 ppm with a maximum decolourization rate of 12.1 mg/L/h during initial 24 h of incubation, following which the decolourization rate decreased. Additionally, the rate of decolourization was also observed to decrease with time of incubation, probably due to depletion of nutrients and redox mediators and also because of the increasing poisonous effect of the dye and its metabolites on the degrading microorganisms. Our strain showed high colour removal activity, and was able to decolourize the dye at a much higher initial dyestuff concentration than other bacterial strains. Our observations are of significance for decolourization of textile dye effluents, as it indicates the potential of Providencia sp. SRS82 to withstand and

remove high concentrations of azo dyes. Garg et al. [5] reported P. putida SKG-1 (MTCC 10510) could tolerate 1000 mg/L of monoazo dye Orange II. Ozdemir et al. [17] reported 94% of 100 mg/L and 56% decolourization of 400 mg/L AB210 by V. harveyi TEMS1under static conditions at the end of 24 h of incubation period at four different dye concentrations. Dave and Dave [18] have reported 60% and 36% decolourization at 500 and 1000 ppm concentration of AR 119 by B. thuringiensis. Keharia et al. [35] showed that the anaerobic sludge bacteria could effectively decolourize RV 5 with decolourization efficiency varying from 95% for 100 mg/L to 75% for 1000 mg/L initial dye concentration during 48 h of incubation. Wang et al. [30] reported that the time required to achieve complete decolourization of Reactive Red 180 using Citrobacter sp. CK3, increased with increase in initial dye concentration from 25 to 500 mg/L while the decolourization extent was only 41.69% at 1000 mg/L dye after 120 h incubation. 3.3. Enzyme analysis Induction of various enzymes during decolourization gives additional insight into the decolourization mechanism and also supports the active role of Providencia sp. SRS82 in the degradation process (Table 1). In the present study, significant induction in the activity of laccase by 190% was observed in Providencia sp. SRS82 when decolourization was compared with control. Intracellular and extracellular tyrosinase activity was induced by 95% and 64.12%, respectively, when culture was grown in the presence of the dye. Existing literature on biodegradation of dyes suggest that reductive cleavage of azo bond is the primary step in bacterial metabolism of azo dyes under microaerophilic conditions. An increase of 136% intracellular peroxidase activity along with a 61% increase in extracellular lignin peroxidase activity was noticed. In our study, significant induction of azo reductase (325%) and NADH–DCIP reductase (24.27%) activities suggests their involvement in the decolourization of dye molecule. In the same contest, the inductive pattern of reductase was reported during the decolourization of azo dye Navy Blue HE2R by developed consortium-PA of A. ochraceus NCIM-1146 fungi and Pseudomonas sp. SUK1 bacterium [10,11]. 3.4. Biodegradation analysis 3.4.1. Decolourization analysis using UV–visible spectroscopy Decolourization of AB210 dye was monitored by observing changes in UV–Vis absorption spectrum (400–700 nm) and the

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3227.02 cm−1 with supporting peak at 3441.12 cm−1 and near 1650 cm−1 indicated N–H stretching of amines. A C=C stretch of aromatic compound forms peaks at 1450.52, 1305.85 and 1151.54 cm−1 shows S=O stretching of sulphonamides and sulphonic acids, respectively. Peak at 1097.53 cm−1 indicates C–OH stretching of primary alcohol while the one at 829.42 cm−1 showed C–H deformation of trisubstituted benzene. The appearance of new peak in degraded metabolites and the disappearance of peak from control spectrum confirmed the degradation of dye.

Fig. 3. FTIR spectrum of (a) dye and its (b) degraded metabolites extracted after 24 h.

results were compared with respective controls. The colour of reaction mixture changed from black to colourless as a function of time (Supplementary data; Fig. S1) shows that during the decolourization of AB210, the absorbance peak at 605 nm disappeared completely after 24 h of reaction time. The highly significant changes occurring both in ultra-violet and visible spectra indicated that the molecular structure of AB210 changed evidently after decolourization probably, due to degradation. However, the UV–Vis absorbance spectra of the blank control, which was not added with Providencia sp. SRS82 did not change throughout the test period. 3.4.2. HPLC, HPTLC, FTIR analysis HPTLC chromatogram of metabolites (Supplementary data; Fig. S2), extracted after 24 h degradation of dye showed fluorescent bands with Rf values different from that of dye indicating that removal of colour of the dye was not just decolourization but was due to dye degradation. The HPLC analysis of dye sample collected at the beginning showed a single peak at retention time 1.195 min (Supplementary data; Fig. S3a). As, the decolourization progressed the emergence of additional peaks were observed due to degradation of parent dye (after 24 h) at retention time 1.854 and 1.122 min (Supplementary data; Fig. S3b). Comparison of FTIR spectrum of control dye (Fig. 3a) with degraded metabolites (Fig. 3b) clearly indicated the degradation of dye by Providencia sp. SRS82. Peaks in control spectrum showed an O–H stretch at 3516.35 cm−1 , the peak at 1597.11 cm−1 showed a N=N stretch (indicates the azo group present in dye). The presence of N=O2 stretch at 1489.10 and 1556.61 cm−1 confirmed the aromatic nitro compound, N–H deformation of primary and secondary amines was observed at the peak of 1649.19 cm−1 , O–H deformation was observed at 1411.9 cm−1 , S=O stretch was observed at the peak of 1330.93, 1174.69 and 1139.97 cm−1 supports the dye contains the sulphonated dye compound, peak 1294.63 cm−1 showed C–N vibration 1045.45 cm−1 indicated C–OH stretching of primary alcohol and peak at 842.92 cm−1 indicated C–H deformation of benzene ring. C–H stretching is also indicated by peaks 2924.18 and 2854.74 cm−1 . The FTIR spectra of extracted metabolites showed disappearance of sharp peaks at 1597.11 cm−1 for azo compounds, which confirmed cleavage of azo bonds of the dye. The peak at

3.4.3. GC–MS analysis and predicted degradation pathway The gas chromatograph of degraded metabolites of AB 210 revealed the presence of several peaks and the structures of the detected compounds were assigned from the fragmentation pattern and m/z values (Supplementary data; Table S1). The pathway has been proposed for the biodegradation of Acid Black 210 (Fig. 4). Decolourization of AB210 by a bacterial consortium under microaerophilic condition suggested the higher probability of the reductive degradation during the preliminary stage of degradation. Symmetric cleavage of the AB210 by azoreductase yields four intermediates viz. benzene 1,2,4-triamine, 4-amino phenyl-N-4 (aminophenyl) benzene sulphonamide, 4-nitroaniline and an unidentified compound with M.W. 364. The metabolite 4-aminophenyl-N-(4-amino phenyl) benzene sulphonamide was also detected through LCMS analysis conducted at SICART, Vallabh Vidyanagar, Gujarat. Similar reductive cleavage by azoreductase and production aromatic amines has been reported earlier by Phugare et al. [28] during biodegradation of Red HE3B by consortium SDS composed of Providencia sp. SDS and Pseudomonas aeuroginosa strain BCH. In this study, the presence of an unidentified product could not be detected from GCMS analysis probably because of its very transient existence as it may have undergone rapid degradation through sequential demination and desulphonation to naphthol and subsequently to naphthalene as these intermediates were detected by GCMS. Travkin et al. [42] demonstrated reductive deamination as a novel initial step in anaerobic biodegradation of halogenated anilines by Rhodococcus sp. Desulphonation was previously reported as a step in the biodegradation of other sulphonated aromatic compounds, including substituted naphthalene sulphonates and benzene sulphonates by Haug et al. [43]. Muralikrishna and Renganathan [44] during desulphonation of a hydroxy-amino-substituted benzene-sulphonic acid by lignin peroxidase from P. chrysosporium concluded that the peroxidases might be involved in the desulphonation of aromatic rings, thus preparing them for further degradation. Benzene1,2,4-triamine undergoes deamination forming benzene, while 4-nitroaniline undergoes deamination forming 4-nitrobenzene. 4-Nitrobenzene forms benzene after removal of nitro group. Asymmetric cleavage of N-4(aminophenyl) benzene sulphonamide by laccase yields sulphanilamide and aniline, which further undergo deamination and desulphonation to produce benzene finally. The unidentified compound probably undergoes deamination and desulphonation to produce 1-napthol, which on dehydroxylation yields naphthalene at the end. Formation of naphthalenes has been reported earlier by Lade et al. [11] and Phugare et al. [28].

3.5. Toxicity study 3.5.1. Microbial toxicity Significant reduction in zone of inhibition was observed in the presence of biotransformed products of AB210, when compared with the untreated dye AB210 (Table 2).

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Fig. 4. Proposed pathway for Acid Black 210 degradation by Providencia sp. SRS82.

3.5.2. Comet assay Single cell electrophoresis or comet assay was employed to assess the genotoxic potential of the compound and was found to be useful for in vivo and in vitro biomonitoring of the environmental pollutants. The assay revealed that significant damage to nuclei occurs when exposed to dye, while the damage seems to be under control in case of the cells exposed to Providencia

sp. SRS82 degraded metabolites as compared to the control (Table 2). The untreated dyeing effluents may be hazardous to the environment, when directly used for irrigation. Thus, it was of concern to assess the toxicity of the effluent before and after decolourization. This study reveals that the metabolites generated after the degradation of dye are less toxic than the original dye [27,28,45]].

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Table 2 Toxicity study of AB210 dye and its metabolites. Parameter analyzed

Test organism

AB210a

Control

Metabolitesa

Microbial toxicity (zone of inhibition cm) Zone of inhibition

Comet tail length (␮m) DNA in comet tail (%)

B. subtilis E. coli Azotobacter sp. Phosphate-solubilizer Comet assay Allium cepa roots

0.80 0.80 0.80 0.80

± ± ± ±

0.005 0.01 0.007 0.005

17.39 ± 2.8 7.68 ± 0.99

1.2 1.36 1.1 0.93

± ± ± ±

0.05 0.02 0.057 0.015

37.66 ± 4.0 12.92 ± 1.66

0.86 0.81 0.88 0.81

± ± ± ±

0.05 0.03 0.03 0.008

20.45 ± 3.56 9.36 ± 1.2

a 1500 mg/L; values are mean of three experiments and SEM (±) is significantly different from the control at, * P < 0.05, by one-way analysis of variance (ANOVA) with Tukey–Kramer comparison test.

3.5.3. Biodegradation of different dyes and actual effluent by Providencia sp. SRS82 Biodecolourization ability of Providencia sp. SRS82 was also checked for 100 ppm of Acid Yellow 11 (AY11), Acid Red 131 (AR131), Acid Black 15 (AB15), Acid Black 298 (AB298), Reactive Violet 5 (RV5), Reactive Red 152 (RR152), and Direct Black 19 (DB19), dyes and the culture was found to be effective on all the four acid dyes resulting in 80–100% decolourization in 24 h (data not shown). Additionally, Providencia sp. SRS82 also showed more than 90% decolourization of the studied reactive and direct dyes. The difference in the decolourization of different dyes might be due to the structural diversity of the dyes [30]. The developed culture also showed 81.0% and 69.0% reduction in ADMI and COD, respectively, of the studied unsterilized actual textile effluent which had initial ADMI and COD value of 8442 and 6600 mg/L, respectively. This proves the utility of the culture for the treatment of industrial waste containing various acid azo dyes and could be useful for degradation of mixtures of such dyes in the effluent. According to the guidelines provided by Central Pollution Control Board (CPCB), India, the textile industry effluent should have pH 5.5–9.0 and phenols less than 1 mg/L, BOD (3 day) at 27 ◦ C should be 30 and COD 250 mg/L. The dye containing effluent treated with Providencia sp. SRS82 met the above guidelines and hence proved its potential for application [46].

4. Conclusions The ability of Providencia sp. SRS82 to decolourize the azo dye under a broad range of temperature, pH, dye and salt concentrations, suggested that the isolated strain could be useful in biological treatment of industrial wastewater. Presence of AB210 in the medium found to induce azoreductase, laccase, tyrosinase, and NADH–DCIP reductase in the strain studied. UV–visible spectroscopy analysis confirmed decolourization, HPLC analysis and FTIR analysis confirmed degradation of AB210 dye by the isolate. The degradation pathway reveals a pattern of dye decolourization and degradation by the organism. Further, the strain proved to be efficient for colour removal from actual industrial dye waste with cheap substrates like urea and ammonium sulphate as supplements with substantial reduction in ADMI and COD values. Moreover, Providencia sp. SRS82 has the capability to degrade AB210 into non-toxic metabolites, which are validated by microbial and cytotoxicity studies. Also, it showed the probable potential to decolourize other azo and acid dye. The decolourization and degradation of triazo metal complex dye AB210 have rarely been reported. The present work showed decolourization and degradation of AB210, by Providencia sp. SRS82 within 90 min at pH 7.0 and 30 ◦ C under static microaerophilic condition. The process could be further developed for periodic anoxic and aerobic cycles for better biodegradation of AB210. To our knowledge, this is the fastest degradation rate of dye AB210

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