Ecotoxicology and Environmental Safety 114 (2015) 23–30

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Improved biodegradation of textile dye effluent by coculture S.R. Vijayalakshmidevi, Karuppan Muthukumar n Department of Chemical Engineering, Alagappa College of Technology Campus, Anna University, Chennai 600025, India

art ic l e i nf o

a b s t r a c t

Article history: Received 29 May 2014 Received in revised form 23 September 2014 Accepted 26 September 2014

The present study demonstrates the de-colorization and degradation of textile effluent by coculture consisting of three bacterial species isolated from textile effluent contaminated environment with an aim to reduce the treatment time. The isolates were identified as Ochrobactrum sp., Pseudomonas aeruginosa and Providencia vermicola by 16S rRNA analysis. Their secondary structure was predicted and GC content of the sequence was found to be 54.39, 52.10, and 52.53%. The co-culture showed a prominent increase in the degradation activity due to the action of oxidoreductase enzymatic mechanism of laccase, NADH– DCIP reductase and azoreductase activity. The biodegradability index of 0.75 was achieved with 95% chemical oxygen demand (COD) reduction in 16 h and 78 and 85% reduction in total organic carbon (TOC) and total solids was observed. Bioaccumulation of metals was identified by X-ray diffraction (XRD) analysis. The effective decolorization was confirmed from the results of UV–vis spectroscopy, high performance liquid chromatography and Fourier transformed infrared spectrometer analyzes. The possible degradation pathway was obtained from the analysis of liquid chromatography–mass spectroscopy analysis and the metabolites such as 2-amino naphthalene and N-phenyl-1.3,5 triazine were observed. The toxic nature of the effluent was analyzed using phyto-toxicity, cell-death assay and geno-toxicity tests. & 2014 Elsevier Inc. All rights reserved.

Keywords: Textile effluent Coculture Biodegradation Toxicity

1. Introduction The textile industry is one of the largest water consuming industries and releases large amount of effluent (Hai et al., 2006). The textile effluent contains carcinogenic dyes, toxic heavy metals, phenolic compounds, softeners and other chemicals used in the dyeing process (Correia et al., 1994). During the dyeing process, about 50% of the dye remains with the spent dye bath effluent, in its hydrolyzed form, which loses its affinity towards the fabric that cannot be re-used in the dyeing process (Watanapokasin et al., 2008). This effluent has to be disposed safely since dyes are toxic, mutagenic and cause major health hazards. The treatment methods include chemical oxidation, coagulation–flocculation, membrane filtration, adsorption, photo-catalysis, biodegradation etc., Some of these processes are complex and expensive (Neill et al., 1999, Eichlerova et al., 2006). Biodegradation is a cost-effective process (Swamy and Ram, 1999) and a large variety of microorganisms such as bacteria, fungi, yeasts, actinomycetes and algae are capable of degrading dyes (Dafale et al., 2007). Microorganism can be used, either as a pure culture or as a consortium to degrade dye containing wastewater (Moosvi et al., 2005; Moosvi et al., 2007; Chen and Chang, 2007; Telke et al., 2008; Kalyani et al., 2009). Bacteria such as Bacillus sp., Pseudomonas sp., Stenotrophomonas sp., Serratia sp., n

Corresponding author. Fax: þ 91 44 22352642. E-mail address: [email protected] (K. Muthukumar).

http://dx.doi.org/10.1016/j.ecoenv.2014.09.039 0147-6513/& 2014 Elsevier Inc. All rights reserved.

Yersinia sp., Erwinia sp., Alcaligenes sp., Sphingomonas sp., Enterobacter cancerogenus, Paenibacillus polymyxa and Micrococcus sp. were successfully used for the degradation of different textile dyes (Moosvi et al., 2005, 2007; Khehra et al., 2005; Kumar et al., 2007; Dafale et al., 2007, Jiranuntipon et al., 2008; Tony et al., 2009; Gou et al., 2009; Jadhav et al., 2010). Several studies report that synergistic metabolic activities of mixed microbial consortium could completely mineralize dyes (Tony et al., 2009). For example, a bacterial consortium consisting of Proteus vulgaris and Micrococcus glutamicus was found to degrade dye containing effluent with 60% chemical oxygen demand (COD) reduction in 96 h (Saratale et al. 2010). The reviews on biodegradation stated to require more incubation time to mineralize the dyes. Therefore, the present study focuses on the incubation time reduction and this is achieved using novel coculture consisting of microorganisms isolated from textile effluent. The comparison of the axenic strain and the coculture degradation efficiency was done. The toxicity analysis of the treated effluent evaluated by cell death assay,genotoxicity test andphytotoxicity study.

2. Materials and methods 2.1. Microorganism The microorganisms isolated from the textile effluent contaminated environment were identified based on morphological

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S.R. Vijayalakshmidevi, K. Muthukumar / Ecotoxicology and Environmental Safety 114 (2015) 23–30

and physiological characteristics and by 16S rRNA gene sequencing. The sequence similarity searches of partial 16S rRNA nucleotide sequences initially done using public nucleotide database using nBLAST. The corresponding sequences were submitted to Bankit, and the accession id retrieved for the isolates. The bacterial nucleotide sequences were aligned using multiple sequence alignment tool CLUXTAL X2 program (www.clustal.org) and sequence similarity among three novel isolates was viewed for an evolutionary relationship through phylogenetic tree using Mega 4.1 software (www.megasoftware.net). The bacterial strains isolated were maintained routinely on nutrient agar medium in the form of slants at 4 °C. Energy minimization was performed using the RNA secondary structure by using Kinefold program (Xayaphoummine et al., 2005). The G þC content of the sequence for each isolate was determined using GeneMark.hmm (Version 2.8) (Lukashin and Borodovsky, 1998). 2.2. Acclimatization The acclimatization of microorganism to the textile effluent was done by incrementally increasing their exposure to the effluent. Initially, the acclimatization process was performed with nutrient broth containing 10% effluent at 37 °C in 250 ml conical flask. However, the nutrient broth concentration was decreased gradually from 90% to 0% until the organism provided with only the effluent as the sole source of nutrient. 2.3. Biodegradation studies Biodegradation was carried out with Ochrobactrum sp., P. vermicola and P. aeruginosa as axenic culture and mixed culture. A loopful of each microorganism was inoculated into 50 ml of nutrient broth and incubated for 6 h. Then coculture was developed by mixing each culture together in a nutrient broth and the same was incubated for 6 h. About 20 ml of inoculum was then added into 250 ml Erlenmeyer flask containing 100 ml textile effluent (undiluted) and the contents were incubated in an orbital shaker at 150 rpm, at 37 °C. The degradation expressed as % COD reduction was calculated using Eq. (1):

%COD reduction =

(COD(initial) − COD(t) ) COD(initial)

× 100

(1)

where COD(initial) and COD(t) represent the initial COD value and the COD value at time ‘t’ (h), respectively. Abiotic controls (without microorganism) were included during the experimental investigation. 2.4. Wastewater characteristics The textile dye effluent was collected from a textile dyeing unit located in the southern part of Tamil Nadu, India. The color of the effluent was purple red. The performance of the treatment was assessed based on biological oxygen demand (BOD) and chemical oxygen demand (COD) reduction. The characteristics of the effluent such as hardness, alkalinity, total solids (TS), total dissolved solids (TDS), total suspended solids (TSS), pH, total kjeldal nitrogen (TKN), total organic carbon (TOC), electrical conductivity (E.C), were analyzed by following the procedure prescribed by APHA. The metal ion content in the untreated and treated effluent was analyzed using atomic absorption spectrometer (Perkin-ElmerAnalyst400).

2.5. Growth kinetics The specific growth rate of the coculture was determined using the following equation:

ln

x = μt xo

(2)

where x is the concentration of biomass (g/L) at time (t) and xo is the initial concentration of biomass (g/L) at time t ¼0 and μ is the specific growth rate (h–1). The growth yield (Y) can be expressed as

dx =Y ds

(3)

Eq. (3) can also be written as

x − x o = Y (So − S)

(4)

where So is the initial concentration of substrate expressed as COD(mg l  1), S is the final concentration of substrate expressed as COD(mg l  1), x is the concentration of biomass (mg l–1) and xo is the initial concentration of biomass (mg l  1) 2.6. Preparation of cell free extract The coculture and axenic strains were grown in their respective medium for 24 h at 37 °C and centrifuged at 10,000 g for 20 min. The biomass obtained was suspended in 50 mM potassium phosphate buffer (pH 7.4) and gently homogenized. Then the content was sonicated in a sonlyzer with an output of 60 amplitude and 12 strokes each of 30 s with one min interval at 4 °C. The extract obtained was used as enzyme source. The similar procedure was followed for the cells obtained after degradation (Parshetti et al., 2006). 2.6.1. Determination of enzyme activities The activity of oxido-reductive enzymes was analyzed for the axenic strain and coculture. The laccase, NADH dichlorophenolindophenol (NADH–DCIP) reductase and azoreductase activity were assayed spectrophotometrically at room temperature. The laccase activity was measured by observing the absorbance of a reaction mixture containing 10 mM guaiacol in 100 mM of acetate buffer with 0.1 ml enzyme at 470 nm and the enzyme activity was expressed in U ml–1 (Jadhav et al., 2008). NADH–DCIP reductase activity was determined by following the procedure reported in the literature (Jadhav et al., 2010). The reaction mixture containing 50 mM DCIP and NADH in 50 mM potassium phosphate buffer (pH 7.4) was added with 0.1 ml enzyme. DCIP reduction was monitored at 620 nm and calculated using the extinction coefficient of 19 mM/cm. For azoreductase assay, the reaction mixture containing 2 mM methyl red, 50 mM NADH in phosphate buffer (pH 7.0) and 0.1 ml enzyme was used (Jadhav et al., 2010). One unit of enzyme activity was defined as the change in absorbance unit per ml of enzyme. All enzyme assays were carried out in triplicate and the average values were reported. 2.7. Analytical studies 2.7.1. UV–vis spectrophotometric analysis About 5 ml of untreated and treated effluent was filtered through 0.22 mm filter and was subjected to UV–vis spectral analysis (Elico Ltd., India) in the wavelength range from 200 to 800 nm. 2.7.2. HPLC analysis The untreated and treated effluent were analyzed in HPLC (Shimatzu, Japan) equipped with UV–vis detector using C18 column (symmetry 4.6 mm  250 mm), diode array, a quaternary

S.R. Vijayalakshmidevi, K. Muthukumar / Ecotoxicology and Environmental Safety 114 (2015) 23–30

pump, and a degasser. Methanol was used as the mobile phase with a flow rate 1 ml min–1. 2.7.3. FTIR analysis The changes in the functional groups present in the effluent before and after treatment was analyzed using Fourier transformed infrared spectrometer (Perkin Elmer, Spectrum RXI FTIR), and the spectrum was recorded in the range from 400 to 4000 cm–1. 2.7.4. LCMS analysis LC–MS analysis of the untreated and treated samples was analyzed using LCMS-2020 (Shimadzu, North America) with C18 reverse phase column, at a wavelength of 265 nm. The flow rate maintained was 0.2 ml min  1 and the temperature was maintained at 35 °C. The total run time of 41 min was maintained. Two different solvents with different proportions, such as water with 0.1% formic acid and acetonitrile with 0.1% formic acid were used. The deuterium lamp (DL) temperature was set at 250 °C with m/z value 50–1000 runs in the positive ion mode. 2.8. XRD analysis X-ray diffraction pattern of the coculture after biodegradation was recorded using RIGAKU-MINIFLUXII, Japan diffractometer with monochromatic CuKα radiation (λ ¼ 1.5406) over the range of 10–80° (2θ). The metals were identified with powder diffraction standard file (JCPDS, Joint Committee on Powder Diffraction Standards Newtown Square, Pennsylvania, USA).

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received as JN214485, JQ944794 and JQ944795, respectively. The RNA secondary structures predicted for Ochrobactrum sp., P. aeruginosa and P. vermicola showed energetically favorable secondary structure. Fig. 1a–c shows the RNA secondary structure and minimal free energy of the isolates. Each sequence showed several loop structures and their energies also differed among the isolates. The secondary structure of 16S rRNA was found to be formed due to strong local interactions by hydrogen bonding and base stacking. The addition of free energy of such interactions provides an approximation for the analysis of the stability of the given structure (Mathews, 2006). The free energy obtained for Ochrobactrum sp., P. vermicola and P. aeruginosa was  71.6 kcal mol  1,  34.2 kcal mol  1 and 133.9 kcal mol  1, respectively. Among these isolates, P. aeruginosa showed lowest free energy, which indicates its genetic stability. The gene structure stability and temperature resistance depend on GC content (Marmur and Doty, 1959; Wada and Suyama, 1986, Bernardi and Bernardi, 1986; Filipski, 1990) and The GC content of Ochrobactrum sp. was about 54.39%. Almost similar results were reported for O. intermedium (55%) (Teyssier et al. 2003). The GC content was observed almost alike in P. vermicola (52.53%) and P. aeruginosa (52.10%). This could be the reason for the better synergistic effect of the consortium. Fig. 1d shows the phylogenetic tree of Ochrobactrum sp., P. aeruginosa and P. vermicola with other related microorganisms found through BLAST results. The phylogenetic tree showed the evolutionary relationship, which gives a synergistic effect to stay as coculture for the degradation of effluent.

2.9. Toxicological studies The genotoxicity analysis was performed using Allium cepa bulbs. The roots were exposed to treated and untreated effluent after 10, 20 and 30% dilution. The growth after 48 h of incubation at room temperature and 12 h light/dark cycle was observed (Jadhav et al., 2010). The microscopic observations and photographs were obtained using light microscope (Nikon H600L Eclipse LV100) and the mean values of root length, mitotic index (MI) and chromosomal aberrations in the cells were observed. The experiments were performed in triplicate and mean values were reported. The cell death assay was performed by Evan's blue staining to check the cell viability of onion root bulbs before and after treatment as reported by Jadhav et al. (2011). The phytotoxicity analyzes were also performed to identify the lethal effect of untreated and treated effluent. The test was carried out using the seeds of Phaseolus mungo and Vigna mungo, which are commonly used in Indian agriculture. The test was carried out according to Parshetti et al. (2006). These experiments were also performed in triplicate and mean values were reported. 2.10. Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparison test. The data were considered significant when the P value was below 0.05.

3. Results and discussion 3.1. Isolation and identification of the isolates The bacterial culture composed of three bacterial species was differentiated by 16S rRNA sequence analysis and identified as Ochrobactrum sp., P. aeruginosa and P. vermicola. The retrieved sequences were submitted to Genebank and the accession ids

Fig. 1. Secondary structure and free energy prediction of the 16S rRNA sequences of the isolates isolates, (a) Ochrobactrum sp. (JN214485), (b) Providencia vermicola (JQ944794) and (c) Pseudomonas aeruginosa (JQ944795) and (d) Phylogenetic relationship between identified isolates.

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S.R. Vijayalakshmidevi, K. Muthukumar / Ecotoxicology and Environmental Safety 114 (2015) 23–30

3.2. Biodegradation analysis As an initial study, the influence of physicochemical parameters on the biodegradation using axenic strain and coculture was evaluated. The culture was grown at shaking and static conditions to identify the favorable condition for the growth. Aerobic organisms generally exhibit better growth at shaking conditions and hence, axenic culture showed lower COD reduction at static conditions. However, coculture showed 72 and 85% COD reduction at static and shaking conditions, respectively. This clearly indicates that the coculture could degrade the effluent at both the conditions. Since better biodegradation was observed at shaking conditions, further experiments were carried out under shaking conditions. Ochrobactrum sp. showed the maximum COD reduction of 52% at its optimum temperature (30 °C), while P. aeruginosa and P. vermicola showed 40 and 44% COD reduction, respectively, at their optimum temperature of 37 °C at the end of 16 h. The optimum temperature for the coculture containing Ochrobactrum sp, P. aeruginosa and P. vermicola was 37 °C and it showed 85% COD reduction at the end of 16 h. This is due to the fact that each strain present in the coculture simultaneously attacks the molecule and enables faster degradation. The other advantage is that the metabolites of the co-existing strains may be easily assimilated by other organisms present in the culture (Moosvi et al., 2007; Asgher et al., 2007; Jadhav et al., 2008; Tony et al., 2009). Therefore, the degradation efficiency of the coculture was better than that of axenic strain. The % COD reduction was also better (450%) in the temperature range from 30 to 45 °C. This is important due to the fact that the temperature of textile effluent tends to fluctuate and depends on the process. However, beyond 45 °C, the growth of microorganisms will be affected. The influence of pH on % COD reduction was studied by varying the pH from 3 to 10. Ochrobactrum sp. and P. vermicola showed the maximum COD reduction at pH 6 (59% and 45%, respectively) whereas P. aeruginosa showed better reduction at pH 7 (45%). On the other hand, coculture showed 85% COD reduction at pH 6 and it also showed better COD reduction ( 450%) in the pH range from 5 to 10. This indicates that the coculture has greater ability to degrade the textile effluent at a wide pH range compared to the axenic isolates. Ochrobactrum sp. at its optimal pH and temperature (pH 6 and 30 °C) showed the maximum COD reduction of 71%, while P. aeruginosa showed 68% COD reduction at its optimum temperature and pH (37 °C and pH 7) after 16 h of incubation. It has been reported that Pseudomonas sp. actively participate in the biodegradation of industrial textile dyes and also act as a part of the consortium involved in textile dye and simulated textile effluent treatment (Dafale et al., 2007; Kalyani et al., 2009; Silveira et al., 2009, Jadhav et al., 2010; Ayed et al., 2010; Phugare et al., 2011). In case of P. vermicola, the organism showed 65% COD

reduction at its optimal conditions (pH 7and 37 °C) after 16 h of incubation. The coculture showed 97% COD reduction at optimal conditions (pH 6 and temperature 37 °C) at the end of 16 h. The studies reported in the literature on the degradation of textile effluent by bacterial consortium are summarized in Table 1. Demirand et al. (2013) reported longer treatment time of 10 days for the treatment of textile effluent using Curvularia lunata and Phanerochaete chrysosporium. Table 1 shows that eight strains including Pseudomonas, and Bacillus cereus utilized for the treatment of textile effluent with lower COD concentration (4503 mg l  1) showed 84% degradation after 72 h of incubation (Ayed et al., 2012) whereas Pseudomonas DAS consortium showed 78% degradation (initial COD – 6760 mg l–1) at the end of 48 h (Jadhav et al., 2010). Though the favorable pH and temperature for the microbes were identified, the effluent treatment was carried out at its natural pH (10.2) and ambient temperature to reduce the operating cost of the process. The effect of incubation time on % COD and BOD reduction for coculture is shown in Fig.2. The results showed about 95% COD and 93% BOD reduction at the end of 16 h of incubation. Phugare et al. (2011) used Providencia sp. and Pseudomonas sp. for the textile effluent treatment and obtained 78% COD reduction after 20 h of treatment at optimal conditions. In the present study, more effective degradation was observed due to the effective isolates. The biodegradability index (BI) (BOD5/COD) is the important parameter in effluent treatment and the wastewater is considered to be biodegradable if the value of BI is above 0.5 (Al-Momani et al., 2002; Metcalf and Eddy, 1985). The initial BI value of the effluent was 0.53. The effect of axenic strains and coculture on BI was evaluated. The BI obtained for Ochrobactrum sp., Providentia vermicola and P. aeruginosa were 0.63, 0.55and 0.57, respectively, whereas the coculture showed the highest BI of 0.75. 3.3. Wastewater characteristics The characteristics of textile effluent treated with coculture are presented in Table 2. Intense changes in the characteristics of the effluent were observed after the treatment and about 95% COD

Fig. 2. Effect of incubation time on % COD and BOD reduction for coculture (conditions: pH:10.37 °C,150 rpm,16 h).

Table 1 Biodegradation performance of different coculture in textile effluent treatment. Name of the bacterium in Coculture

Textile Effluent concentration (COD mg/L)

% Degradation and Time duration

Authors

Proteus vulgaris and Micrococcus glutamicus Pseudomonas sp. DAS consortium Providentia sp. and Pseudomonas sp. P. cepacia, P. vesicularis, S. epidermidis, S. paucimobilis, B. cereus,, filamentous bacteria Bacillus sp. Aspergillus ochraceus and Pseudomonas sp. B. laterosporus and G. geotrichum Curvularia lunata and Phanerochaete chrysosporium Coculture-Ochrobactrum sp. Providentia vermicola and Pseudomonas aerugiosa

18,600 6760 9860 4503

60, 78, 78, 84,

Saratale et al. (2010) Jadhav et al. (2010) Phugare et al. (2011) Ayed et al. (2012)

3920 3400 354 9333

96 h 48 h 20 h 72 h.

96.35 h 74,48 h 96,10days 95.16 h

Lade et al. (2012) Kurade et al. (2012) deMirand et al., (2013) The present study

S.R. Vijayalakshmidevi, K. Muthukumar / Ecotoxicology and Environmental Safety 114 (2015) 23–30

textile dye effluent degradation was studied for Ochrobactrum sp., P. vermicola, P. aeruginosa and coculture. The results obtained are presented in Table 3. The results showed better enzyme activities for coculture compared to axenic strains. However, further analysis of the results showed that the individual strains showed better activity for different enzymes. For example, better laccase activity was observed with P. aeruginosa whereas P. vermicola showed better NADH–DCIP activity. Ochrobactrum sp. showed better azoreductase activity compared to other microbes. Hence, microbial coculture deteriorates complex xenobiotics present in the effluent in a faster manner.

Table 2 Characteristics of textile effluent. Parameter

Before treatment

After treatment

COD BOD TSS TDS TS pH TOC E.C mS/cm Sulfate Chloride TKN Total hardness Ca hardness Cr þ (total) Pb þ Ni þ Cu þ Cd þ

9333 5000 24,000 51,000 75,000 10.25 8205 11.94 6666 7090 142 660 460 0.2 0.8 0.42 0.85 0.43

306 180 5900 4800 10,700 6.4 1750 3.01 120 992 91 124 101 BDL 0.3 BDLn BDL BDL

27

3.6. Analytical studies

All the values except pH and E.C are expressed in mg/l. BDL (below detection limit) ¼ o .2 mg/l.

n

reduction and 78% TOC reduction were observed. The BOD was found to be reduced from 5000 to 180 mg l  1. Hence, it is evident that the organic load of the effluent has been reduced by the robust coculture used. The total solids content (75,000 mg l  1) and TDS content (51,000 mg l  1) was much higher in the untreated effluent. The results showed a significant reduction in total solids, total dissolved solids, sulfate, chloride, TKN and total hardness. The pH of the effluent was reduced from 10.25 to 6.4 after the treatment. The presence of metals such as Cr, Cu, Ni and Cd were detected in the untreated effluent and these metals were almost removed after the treatment. 3.4. Growth kinetics The specific growth rate of 0.116 h–1 and yield coefficient of 1.22 mg of dry weight of biomass/mg COD were obtained for the biodegradation using coculture. The values reported in the literature were 0.76 mg MLSS/mg COD (Pala and Tokat, 2002) and 0.34 MLVSS/mg COD (Sahinkaya et al., 2008). The difference in the Y value may be due to the variations in the effluent characteristics and the nature of microbes involved in the effluent treatment. 3.5. Enzyme analysis The oxido-reductive enzymes were reported to be responsible for the degradation of dye containing effluent and these enzymes degrade the xenobiotics mostly through redox reactions (Telke et al., 2008; Kurade et al., 2012). The role of oxido-reductive enzymes, such as laccase, NADH–DCIP reductase and azoreductase in

3.6.1. UV–vis spectrophotometric analysis The UV–vis spectral analysis was performed to identify the level of degradation before and after treatment (data not shown). The results showed significant reduction in absorbance values for the treated effluent. It indicates structural changes in the molecular structure of dyes, which leads to the change in their color intensity. The intense peaks observed at 260, 391 and 573 nm with an absorbance value around 3 for the untreated effluent were found to be reduced slowly to below 0.5 after the treatment. This confirms the biodegradability nature of the microorganisms used. 3.6.2. HPLC analysis The untreated and treated effluent was analyzed using HPLC (Fig. S1). HPLC analysis of the untreated effluent showed peaks at 2.942, 3.392 and 3.758.The treated effluent exhibited entirely different chromatogram and the peak present in the untreated effluent at 3.392 disappeared and several small peaks in the retention time ranging from 5.692 to 6.933 were observed. This indicates the formation of metabolites and confirms the biotransformation of the textile effluent after treatment. 3.6.3. FTIR analysis The FTIR analysis of untreated and treated effluent was done (Fig. S2). The untreated effluent showed a peak at 2923 cm  1for – C–H stretching of alkenes which was found to be deformed to 2919 cm  1 in the treated effluent. The broad peak observed at 3426 cm  1 and 3753 cm  1 attributed to the –NH and –OH stretching vibrations were found to be narrowed and their intensity reduced for the treated effluent. The peak at 1630 cm  1 for the untreated effluent indicated N ¼N stretching and the disappearance of this peak in the treated effluent confirmed the active role of azoreductase in azo bond cleavage during the degradation process and it is deformed to 1384 cm  1 in the treated effluent. The peak at 1100 cm  1 indicates C–O stretching of alcohol in the untreated effluent, which has been reduced to 1067 cm  1 in the treated effluent. Thus the significant difference

Table 3 Enzyme assay. Enzyme

a

Laccase Azoreductaseb NADH–DCIP reductasec

Ochrobactrum sp.

Providentia vermicola

Pseudomonas aeruginosa

Coculture

Control

After treatment

Control

After treatment

Control

After treatment

Control

After treatment

0.117 0.21 3.42 7 0.02 7.03 7 0.04

0.296 7 0.03 7.3 7 0.046 8.337 0.63

0.16 7 0.4 0.72 7 0.2 8.03 7 0.3

0.43 7 0.21 1.88 7 0.03 11.03 7 0.032

0.117 0.2 0.5 7 0.03 1.03 7 0.0

0.83 70.07 3.88 70.18 4.5 70.08

0.117 0.2 8.727 0.3 7.03 7 0.5

1.13 70.03 11.64 70.21 19.36 71.82

Values are Mean 7 SD. Significantly different from control cells at nPo 0.01, a

Enzyme units/min/mg protein. b μM of Methyl red reduced/min/mg protein. c mg DCIP reduced min_1 mg protein_1.

nn

Po 0.001 by one-way analysis of variance (ANOVA) with Turkey Kramer comparison test.

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S.R. Vijayalakshmidevi, K. Muthukumar / Ecotoxicology and Environmental Safety 114 (2015) 23–30

in FTIR peak pattern confirms the biodegradation of the textile effluent. 3.6.4. LCMS analysis The textile dye effluent was analyzed in positive ion and full scan mode from 100 to 1000 m/z. For the untreated effluent, several mass peaks with different values were obtained (Table S1). The mass peak at 878 m/z (m.w. 875) observed for the untreated effluent shows similarity to remezol red dye. The metabolites, formed after the degradation of textile effluent were identified and the possible degradation pathway was predicted based on the earlier reports. The degradation of dye by azo reductase leads to the formation of two reactive intermediates such as 3-amino [4.5 (6-chloro-1.3,5 triazine-2yl) amino]naphthalene 2.4,7 benzene trisulfonicacid (m.w. 638, m/z 635) and 2[(3-aminophenyl) sulfonyl]ethane sulfonicacid (m.w. 265, m/z 263). The 2[(3-aminophenyl) sulfonyl] ethane sulfonic acid undergoes further deamination to produce thiol sulfonyl-benzene having a mass peak (m.w. 246, m/z. 246) followed by a desulfonation reaction resulting in the formation of 2-ethylphenyl sulfone with a mass peak of 168 (m/z) (m.w. 169). Laccase cleaved the first intermediate, which resulted in the formation of 2-amino naphthalene (m/z 141, m.w. 143) and N-phenyl-1.3,5 triazine (m.w. 170, m/z 170). Furthermore, 2-amino naphthalene formed undergoes deamination to form naphthalene (m.w. ,128, m/z 125). The degradation pathway was identified (Fig. S3) and the similar trend was reported by Waghmode et al. (2012). Aniline was detected as a low molecular weight aromatic compound (m/z 94) in the treated effluent. 3.7. Heavy metals accumulation Heavy metal ions may be present in the textile effluent due to the metal associated dyes used or other constituents used during the dyeing process. These metal ions act as a co-factor to improve the activity of bacterial enzymes, which in turn influence the biodegradation (Kurade et al., 2012). Hence, the microorganisms accumulate the metals during the biodegradation. The metals were detected using XRD analysis of powdered bacterial biomass. The X-ray spectra obtained for the dried cells exposed to textile effluent were done (data not shown). This showed distinct peaks, which clearly indicate the accumulation of metals. The 2θ value of 27.46°, 32.28°, 45.94°, 57°, 66.74° and 75.8° showed the presence of chromium, lead, nickel, magnesium and carbon (data not shown). The presence of all these metals was confirmed using standard JCPDS reference code (04-0686(Pb), 65-3316(Cr), 882326(Ni), 35-0821(Mg), 50-1084(C)). Therefore, the influence of the above mentioned metal ions during the biodegradation is possible and may enhance the rate of biodegradation.

3.8. Toxicity analysis 3.8.1. Genotoxicity test The A. cepa test is a routine test used for identifying the genotoxicity of any toxic compounds. The test was carried out to identify the % cell viability, MI and chromosomal aberrations in the root cells. In the cell viability test, the number of viable cells was analyzed before and after treatment by Evan's blue staining (Table 4a). The viable cells exclude the Evan's blue stain, while the damaged cells retain the stain (Taylor and West, 1980). For the control, the cell viability was found to be 95.6% (95.6 71.5). The root cells grown in untreated textile effluent at different concentrations (10–30%) showed 66.6–70.6% viability and for the cells grown in treated effluent at different concentrations (10–30%), the value was 89.3–93.3%. The value obtained was high for the treated effluent and this confirms the less toxic nature of the treated effluent. The cytotoxicity of the toxic compound was determined based on the MI value, which can be used as a biosensor for environmental pollutants (Carita and Morales, 2008). The genotoxic nature of the textile dye effluent before and after treatment is shown in Table 4a. The decrease in MI indicates a decrease in cytotoxic nature of the effluent. The textile effluent in general has adverse effects on chromosomal cell division and this kind of aberrations in mitotic cell divisions may be due to malfunctioning of the spindle apparatus proteins (Jadhav et al., 2010) or may be due to reduced ATP synthesis during cell division (Sik et al., 2009). The treated sample MI was in the range similar to that of control. The chromosomal aberrations were more with 10% concentration of the untreated effluent compared to the control and treated effluent. This indicates a strong genotoxic nature of the effluent. The chromosomal aberrations observed include laggards, chromosome breaks and anaphase bridges. After the treatment, the decrease in COD level might be the reason for the reduction in the number of aberrant mitotic cells. The frequency value of total alterations (TA) for control was 0.344 whereas for the treated effluent the values are ranging from 0.35 to 0.67 and for the untreated effluent the range was from 3.06 to 5.17. The root length was drastically affected with untreated sample (1.5–2.4 cm), while a better growth was observed with treated sample (6.5–7.3 cm). The results obtained were similar to that of results reported in the literature (Jadhav et al., 2010; Carita and Morales, 2008). 3.8.2. Phytotoxicity analysis The test with untreated effluent showed 45% seed germination for Vigna mungo, whereas Phaseolus mungo showed 40% germination. In contrast, % germination increased to 90% and 80%, for P. mungo and V. mungo, respectively, in the presence of treated effluent. The length of plumule and radical also increased rapidly

Table 4a Toxicity analysis for the untreated and treated effluent. A. cepa in genotoxicity test Analysis

%Cell viability RL MI MN CB TA TCA Frequency of TA

Control

95.6 7 1.5 4.5 70.1 8.03 70.035 0 1 1 300 0.3447 0.01

Untreated effluent concentration (%)

Treated effluent concentration (%)

10

20

30

10

20

30

69.667 1.5 2.4 7 0.20 13.617 0.02 1 7 8 250 3.067 0.23

70.66 7.19 2.17 0.1 15.95 7 0.20 2 8 10 280 0.727 0.02

66.66 71.5 1.53 7 0.15 19.03 7 0.3 1 14 15 290 5.17 70.01

93.3 7 1.5 6.5 7 0.2 10.247 0.20 0 1 1 290 0.357 0.01

917 1 7.737 .44 8.99 7 0.15 1 1 2 300 0.677 0.01

89.3 7 1.5 7.17 0.81 7.357 0.18 0 1 1 280 0.36 7 0.02

RL, root length; MI, mitotic index; MN, micronuclei; CB, chromosome breaks; TCA, total number of cells analyzed; TA, total number of alterations. Values are Mean 7SD.

S.R. Vijayalakshmidevi, K. Muthukumar / Ecotoxicology and Environmental Safety 114 (2015) 23–30

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Table 4b Toxicity analysis for the untreated and treated effluent. Phytotoxicity analysis Parameters

Germination (%) Plumule (cm) Radicel(cm)

Vigna mungo

Phaselous mungo

Distilled Water

Untreated effluent

Treated effluent

Distilled water

Untreated effluent

Treated Effluent

100 3.03 7.11 3.03 7.15

45 0.20 70 0.23 70

90 1.22 7 0.13 2.46 7 0.01

100 10.69 7 0.51 6.247 0.08

40 1.38 70.22 0.86 70.3.

80 8.0 7 0.01 3.98 7 0.01

Values are Mean 7SD.

for Phaseolus mungo and V. mungo when treated effluent was used (Table 4b). This indicates that the metabolites produced after biodegradation are less toxic than that of untreated effluent. This concludes that the treated water can be used in agriculture field or recycled.

4. Conclusion The competent coculture used in the present study degraded the textile effluent within 16 h of incubation. The effective decolorization was confirmed by UV–vis spectroscopy and biodegradation of effluent was confirmed by HPLC and FTIR analyzes. The possible metabolic pathway and metabolites formed were identified by LCMS. The phytotoxicity and genotoxicity studies proved the toxic nature of the untreated effluent and less toxic nature of the treated effluent. The lethal effect of the effluent was drastically reduced after biological treatment with coculture.

Acknowledgment The author wishes to thank the Department of Science and Technology (DST), New Delhi, India for providing grant under WOS-A project (Grant No. SR/WOS-A/LS-11/2011).

Appendix A. Suplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.09.039.

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