Exploring bioremediation strategies to enhance the mineralization of textile industrial wastewater through sequential anaerobic-microaerophilic process

International Biodeterioration & Biodegradation 106 (2016) 97e105

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Exploring bioremediation strategies to enhance the mineralization of textile industrial wastewater through sequential anaerobic-microaerophilic process Kshama Balapure a, Kunal Jain b, Nikhil Bhatt a, Datta Madamwar b, * a

Post Graduate Department of Microbiology, Biogas Research and Extension Centre, Gujarat Vidyapith, Sadra, 382 320, Gujarat, India Environmental Genomics and Proteomics Lab, BRD School of Biosciences, Satellite Campus, Vadtal Road, Post Box No. 39, Sardar Patel University, Vallabh Vidyanagar, 388 120, Gujarat, India

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 September 2015 Received in revised form 12 October 2015 Accepted 12 October 2015 Available online xxx

The study exemplifies sequential anaerobic-microaerophilic bioremediation process for treatment of textile industrial wastewater having 10,000 mg l
1 of COD and 3330 mg l
1 of the BOD. The experimental results showed that, in an anaerobic phase, with cattle dung slurry as an initial feed, nearly 60% of COD and BOD was removed from textile wastewater at an optimum HRT of 2d and OLR of 5.0 kg COD m
3d
1. Further, COD and BOD removal efficiency of bacterial consortium BDN was enhanced upto 97% under microaerophilic phase, at HRT of 12 h. Moreover, optimum color removal (80%) was observed in anaerobic reactor. The combine treatment process removed 99% of color at combine HRT of 60 h. The activity of lignin peroxidase was higher as compared to other enzymes studied. The UVevis, FTIR, 1H NMR and GCeMS analyses of treated textile industrial wastewater revealed the degradation of dye compounds and formation of lower molecular weight intermediates. The toxicity of textile industrial wastewater decreased subsequently from anaerobic to microaerophilic treatment process. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Textile wastewater Anaerobic-microaerophilic Mineralization Phytotoxicity

1. Introduction The textile industry, apart from being an important contributor to the economy of the many countries, is also a major source of various liquid and solid wastes. These industrial wastes are considered as a harmful pollutant, which are released into the natural water resources or wastewater treatment systems (Rondon et al., 2015). It was estimated that more than 80,000 tons/year of dyes are consumed in textile dyeing processes, which requires 70e150 dm3 of water and 40 g of reactive dyes per kg of cotton (Mendez-Martineza et al., 2012). The amount of water consumed and released also varies depending on the type of fabrics used. Therefore, the composition of the dye wastewater varies with the type of textile produced (Mustafa and Delia, 2004). The entry of these pollutants into water streams poses a severe ecotoxic hazard and introduces the potential danger of bioaccumulation that may eventually affect human beings through the food chain (Mohana

* Corresponding author. E-mail addresses: [email protected] (K. Balapure), bhattnikhil2114@gmail. com (N. Bhatt), [email protected] (D. Madamwar). http://dx.doi.org/10.1016/j.ibiod.2015.10.008 0964-8305/© 2015 Elsevier Ltd. All rights reserved.

et al., 2008). Due to strict government legislation and regulation, textile wastewater remediation is a deeply studied topic worldwide. Moreover, several new and improved methods are also being developed continuously to remove one or more xenobiotic dyes from industrial textile wastewater. These methods include physical, chemical and advanced chemical oxidation treatment. However, considering the pitfalls of these techniques during practical implementation, the process itself needs further optimization in terms of quality, applicability and cost (Bhatt et al., 2005). Therefore, bioremediation using bacteria is gaining importance as it is cost effective, ecofriendly, and produce negligible sludge (Jain et al., 2012). Different taxonomic groups of bacteria have been reported for their ability to degrade azo dyes (Moosvi et al., 2007). Bacteria have sets of catabolic genes, capable of processing various metabolic pathways, which are integrated in such a manner that xenobiotic compounds (such as dyes) are converted to low molecular weight intermediates which can enter into central metabolic pathway leading to complete mineralization of those compounds including dyes. Azoreduction by sequential anaerobic-aerobic process is most commonly used worldwide, due to its simplicity and low cost

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(Wang et al., 2014). Anaerobic metabolic function facilitates reductive breakdown of azo dye molecules by cleaving the azo bond to the corresponding colorless aromatic amines, which, although resist further anaerobic degradation, are reported to be well amenable for aerobic degradation (Chan et al., 2009). In aerobic condition these amines could be mineralized by non-specific enzymes through hydroxylation and ring-fission of aromatic compounds (Wang et al., 2014). Earlier studies showed that many different types of fixed film and suspended growth systems have been used for degradation of textile wastewater through anaerobicaerobic systems (Kapdan and Oztekin, 2006; Chan et al., 2009; Senthilkumar et al., 2011). However, fixed film systems offer several other advantages in textile wastewater treatment when compared with suspended growth processes, such as handling convenience, increased process stability, little residual sludge, high biomass retention, ease of use in small scale treatment, more energy efficient and capacity to handle various shock loads (Balapure et al., 2015). Keeping this in mind, the present study was focused on the treatment of textile industrial wastewater using anaerobic treatment followed by microaerophilic fixed film reactor using pumice stone as a bedding material. Microaerophilic fixed film reactor combines advantages of the aerobic reactor (i.e. short hydraulic retention time (HRT), high biomass concentration, high specific surface area) and the anaerobic process (low quantities of waste, biological solids) (Laquidara et al., 1986). Thus, the sequential treatment ensures that, aromatic amines generated under anaerobic phase are further degraded and mineralized under microaerophilic phase which was seeded with enriched consortium BDN. The efficacy of microaerophilic fixed film reactor seeded with bacterial consortium BDN for mineralization of simulated wastewater containing mixture of six dyes (dye concentration of 300 mg l
1) have been already reported in our earlier study (Balapure et al., 2015). However, the microaerophilic fixed film reactor treatment was unable to provide complete degradation of industrial textile wastewater, due to the presence of high amount of COD and color concentration in textile industrial effluent as compared to simulated textile wastewater. Thus, anaerobic step was added before microaerophilic reactor for complete degradation of textile wastewater. The biodegradation of textile industrial wastewater having 10,000 mg l
1 COD and 3340 PteCo color was carried out through sequential anaerobic-microaerophilic reactor under different organic loading rate. The effect of organic loading rate on COD, BOD, TS, TDS, TSS, TVS, chloride reduction etc. were determined to verify treatment efficiency. The degradation of textile wastewater and its metabolites in both anaerobic and microaerophilic reactors were detected by UVevis, (UltravioleteVisible spectroscopy), FTIR (Fourier transformed infrared spectroscopy), 1H NMR (1H Nuclear magnetic resonance spectrometry) and GCeMS (Gas chromatography-mass spectrometry) analysis. The toxicity of the textile industrial wastewater before and after sequential treatment was also studied by assessing its phytotoxicity. 2. Material and methods 2.1. Sampling, characterization of industrial textile wastewater Textile industrial wastewater was collected from the local textile industry, near Naroda G.I.D.C, Ahmedabad, Gujarat, India and stored at 4  C till further use. It is worth to mention here that the dyeing process of this factory is continuous and utilizes many different types of azo dyes. The wastewater was characterized as per Standard Methods for The Examination of Water and Wastewater (APHA, 2012). Mean characteristics of the wastewater are

presented in Table 1. Physico-chemical analysis of the wastewater was carried out for two months to cover variations in wastewater characteristics. 2.2. Experimental set-up The laboratory scale sequential anaerobic e microaerophilic fixed film reactors were used in the experiment. Both the reactors were constructed using glass column. The anaerobic fixed film reactor with a working volume of 1.5 l, was used with following specifications: reactor inner diameter 5.3 cm; reactor height 122 cm; media height 93 cm; total volume (without bedding material) 3.0 l. Microaerophilic fixed film reactor with a working volume of 750 ml having following specifications: reactor inner diameter 2.5 cm; reactor height 60 cm; media height 45 cm; total volume (without bedding material) 1.5 l, was used. The reactors were packed with uniform pieces (~119 mm3) of pumice stone (1 kg in anaerobic reactor and 550 g in a microaerophilic fixed film reactor). Anaerobic reactor was completely restricted from air supply to avoid gaseous exchange to maintain anaerobic conditions. In microaerophilic reactor, dissolved oxygen concentration was maintained in the range of 0.06e0.08 mg l
1 (Keharia and Madamwar, 2003). The hydraulic retention times (HRT) of the bioreactor were varied by changing the flow rate of the feed to the bioreactor. Textile industrial wastewater was fed into the anaerobic fixed film reactor at the required rate using a peristaltic pump (Gilson Miniplus 3, France). The effluent of the anaerobic fixed film reactor was used as the influent of the microaerophilic fixed film reactor. The abiotic and biotic control experiments were simultaneously performed having same reactor dimension parameters under similar conditions. 2.3. Inoculum development for bioreactors 2.3.1. Seed inoculum for upflow anaerobic fixed film reactor Cattle dung slurry (3.2% w/v) was used as a source of anaerobic bacteria for the development of biofilm in the anaerobic fixed film reactor. Cattle dung is a cheap and abundant source, having good, abundant source of anaerobic microorganisms. Moreover, it is also known to degrade pollutants or transform pollutants into less toxic substances. Keharia and Madamwar (2003) had used cattle dung slurry as an initial inoculum for the enrichment of strict anaerobes in the anaerobic fixed film reactor. 2.3.2. Seed inoculum for microaerophilic fixed film reactor Bacterial consortium BDN was used as a seed culture for the development of biofilm in the microaerophilic fixed film reactor. Bacterial consortium BDN was developed by culture enrichment technique, using Bushnell Hass Medium (BHM) amended with 100 mg l
1 model dye (Reactive Blue 160) and 0.5% yeast extract

Table 1 Physico-chemical characterization of textile industrial wastewater. Parameters

Concentration

pH Color (PteCo) COD (mg l
1) BOD5 (mg l
1) Total alkalinity (mg l
1) Total solids (mg l
1) Total suspended solid (mg l
1) Total dissolved solids (mg l
1) Chloride (mg l
1) Phosphate (mg l
1) Sulphate (mg l
1)

7.5 3340 10,000 3330 3950 4220 1510 1960 2400 650 930

± ± ± ± ± ± ± ± ± ± ±

0.3 25.7 34.3 24.2 31.6 25.4 20.8 19.8 14.8 22.5 29.5

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(Balapure et al., 2014). The bacterial consortium BDN consist of eight different bacterial strains namely Alcaligenes sp. BAB3053 (BDN1, NCBI Accession No. KF500593), Bacillus sp. BAB2731 (BDN2, KF500594), Escherichia sp. BAB2734 (BDN3, KF500595), Pseudomonas sp. BAB3054 (BDN4, KF500596), Providencia sp. BAB2749 (BDN5, KF500597), Acinetobacter sp. BAB2750 (BDN6, KF500598), Bacillus sp. BAB2751 (BDN7, KF500599) and Bacillus sp. BAB3055 (BDN8, KF500600). 2.4. Reactor operation 2.4.1. Biofilm development in anaerobic fixed film reactor Anaerobic fixed film reactor initially charged with 3.2% (w/v) cattle dung slurry. Biofilm was allowed to develop by incubating at 37  C for 35e40 d. During the incubation period, facultative organisms utilized the organic matter present in the substrate and have created anaerobic conditions for the growth of strict anaerobes. Cattle dung slurry contains 32,000 mg l
1 COD, 28,000 mg l
1 total solids, 21,000 mg l
1 volatile solids, 500 mg l
1 potassium and 625 mg l
1 total nitrogen, pH 7.8. Biofilm development was assessed by visual observation through changes in color from greenish yellow to dark green. After biofilm development, the effluent was slowly replaced by industrial textile wastewater. Initially reactor was operated at HRT of 10 d for at least three retention cycles upon reaching steady state condition. Subsequently HRT was decreased to 8, 6, 4, 3, 2 and 1 d HRT, while organic loading rate (OLR) was increased gradually. Steady state condition was judged by constant COD, BOD and color reduction of the effluent. Textile industrial wastewater was fed into the reactor at a required rate using peristaltic pump and treated effluents was analyzed for color reduction, COD, BOD, alkalinity, biogas production, TS, TVS, TSS, TDS, chloride, phosphate, sulphate and pollutant degradation were studied using FTIR, NMR GCeMS. 2.4.2. Biofilm development in microaerophilic fixed film reactor Prior to starting up the process, the whole bioreactor was inoculated (750 ml) with active culture of consortium BDN and incubated at 37  C for 15d or till active biomass was established. Once an active biofilm was established, anaerobic effluent (optimum HRT) was further treated in a microaerophilic reactor at varying HRTs ranging from 4 to 24 h. The seeded bacterial consortium BDN supported the degradation of intermediates formed during anaerobic treatment. The reactor was operated until stable performance with respect to COD, BOD and color removal was achieved. 2.5. Enzyme preparation and assays of microaerophilic fixed film reactor At an optimum HRT (12 h) effluent of microaerophilic reactor was collected and used for preparation of cell free extracts as reported earlier (Balapure et al., 2015). The protein content of all the samples was estimated by Lowry's method (Lowry et al., 1951). Azoreductase, NADH-DCIP reductase, lignin peroxidase and tyrosinase activities were assayed spectrophotometrically from cell free extracts as well as in the control supernatant as mentioned earlier (Balapure et al., 2015). The detailed procedures for oxido-reductive enzyme assays were described in the supplementary text. 2.6. Analytical procedure Untreated and treated effluents collected from the anaerobic and microaerophilic fixed film reactor was centrifuged at 10,000 g for 20 min and the supernatant was used for further analysis.

99

2.6.1. Analysis of physicoechemical parameters Biogas production was measured by displacement of saturated salt solution. pH, chemical oxygen demand (COD), biochemical oxygen demand (BOD), total alkalinity (TA), total solids (TS), total dissolved solids (TDS) total volatile solids (TVS), total suspended solids (TSS), chloride, phosphate and sulphate were analyzed according to Standard Methods for Examination of Water and Wastewater (APHA, 2012). Volatile Fatty Acid (VFA) was measured with a titrimetric method proposed by Anderson and Yang (1992). COD was measured using Hach DR 2010 spectrophotometer and Hatch COD reactor. Aliquotes (3 ml) of the untreated and cell free supernatant of the anaerobic and microaerophilic effluents were scanned in the range of 200e800 nm to observe the transformation of the dyes after sequential treatment. 2.6.2. Detection of biodegraded products The feed solution (industrial textile wastewater) and effluents of both the reactors were centrifuged at 10,000 g for 15 min. Cell free extracts containing the dye degraded intermediates/metabolites were extracted using equal volumes of ethyl acetate and evaporating it to dryness in a Speedvac (Thermo Electron Corporation, Waltman, Ma). These extracted metabolites were used for Fourier Transform Infrared spectroscopy (FTIR), 1H Nuclear Magnetic Resonance (1H NMR) and Gas chromatography-Mass Spectrometry (GCeMS) analyses. FTIR analysis was carried out using Perkin Elmer, Spectrum GX spectrophotometer in the mid infrared region of 400e4000 cm
1 to 16-scan speed (Kalyani et al., 2009). Further 1 H NMR spectrometry analysis was performed using Bruker 13C NMR-400 MHz (Jain et al., 2012). GCeMS analysis of degraded metabolites was performed using Auto-system XL (Perkin Elmer, USA). Metabolites were identified on the basis of their mass spectra and NIST library (Jain et al., 2012). The detailed protocol for FTIR, 1H NMR and GCeMS analyses were described in supplementary text. 2.7. Phytotoxicity study of anaerobic-microaerophilic treated metabolites Phytotoxicity test was performed to assess the toxic effect of untreated and treated wastewater on different plants. Phytotoxicity study was carried out under ambient condition using Sorghum vulgare, Triticum aestivum and Phaseolus mungo, which have an importance in the Indian agriculture. The seeds of the above mentioned plants were collected from the local grain market of Sadra, Gandhinagar, Gujarat, India. Fifty seeds of each plant were irrigated separately by adding 10 ml samples of textile wastewater, anaerobic and microaerophilic effluents at an optimum HRT. Control set was carried out using distilled water at the same time. Percent germination, length of the plumule and radical was recorded after 15 days. 2.8. Statistical analysis All the experiments were carried out in triplicate. Data were analyzed by one-way analysis of variance (ANOVA) with the TukeyeKramer multiple comparison test. Readings were considered significant when p was 0.05. 3. Results and discussion Wastewater treatment plants (WWTP) are subject to variations in its composition and other parameters that affect or decline the reactor performance, viz. pH, COD, BOD, TS, TSS, TDS, TVS, chloride, phosphate and sulphate, as well as others. However, these variations can be predicted and controlled to increase the efficiency of the reactor. But this is not the case for all variations, because

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sometimes reactor's performance can deteriorate due to extreme transient conditions. Thus, in order to select optimal operating condition different aforementioned physicoechemical parameters were investigated at different OLR and HRTs. 3.1. Treatment performance of anaerobic fixed film reactor 3.1.1. Influence of OLR on COD and BOD reduction COD and BOD removal efficiency are considered as the sole parameters of wastewater treatment system, which must be stable to assume the steady state condition. COD and BOD removal efficiencies of anaerobic fixed film reactor treating textile industrial wastewater under different organic loading rate (1e10 kg COD m
3d
1) are shown in Fig. 1. The results showed that, the COD and BOD removal efficiencies were 67 and 64%, respectively, during OLR of 1.0 kg COD m
3d
1 and 10 d HRT. Moreover, at an OLR of 5.0 kg COD m
3d
1 and 2 d HRT, COD and BOD removal efficiencies were not considerably affected and found to be more than 60%, indicating adaptation of bacterial culture. Consequently, further increase in OLR (10.0 kg COD m
3d
1) reactor showed a marked drop in COD and BOD removal efficiency (~30%). Low COD and BOD removal efficiencies, particularly at low HRTs or high organic loading rate indicated that, the metabolites released through the degradation of textile wastewater were recalcitrant and could not be ultimately mineralized under anaerobic conditions as reported by Panswad et al. (2001). The results of the present study can also be compared with O'Neill et al. (2000). They worked on biotreatment of simulated textile effluent containing varied ratios of starch and azo dyes and found 72.9 and 84.1% of COD and BOD removal respectively from textile effluent within 209e217 days. Gnanapragasam et al. (2010) studied on upflow anaerobic sludge blanket reactor for the treatment of real textile dye effluent and found that as OLR increases the efficiency of COD removal was decreased. 3.1.2. Influence of OLR on color removal and biogas production Table 2 illustrates the color reduction and biogas production profiles of effluent from the anaerobic reactor. The color removal efficiency remains almost stable, ranging from 86 to 83% at an OLR of 1.0e2.5 kg COD m
3d
1 and 10 to 4 d of HRT. Steady state performance of reactor was observed till OLR of 5.0 kg COD m
3d
1 and 2 d HRT (80% color removal) beyond that the color removal was reduced by 40%. At high OLR, lower rate of color removal may be due to the inhibitory effects of high concentration of dyes or intermediate products on the microbes, or the blockage of active sites of azoreductase enzymes by dye molecules with different

BOD removal

70

70

60

60

50

50

40

40

30

30

20

20

10

10

0

% BOD removal

% COD removal

COD removal

0 0

1

1.25 1.6 2.5 OLR (kg COD m-3d-1)

5

10

Fig. 1. Evaluation of operational parameters in terms of COD and BOD removal during the degradation of textile industrial wastewater in anaerobic fixed film reactor at an increasing OLR from 1.0 to 10. kg COD m
3d
1.

structures (Senthilkumar et al., 2011). Mustafa and Delia (2004) observed ~ 55% of color removal at 55 h HRT in the treatment of textile industrial wastewater using UASB reactor. In contrast, Somasiri et al. (2008) found 92% color removal in all experimental stages during the treatment of textile industrial wastewater in the laboratory scale UASB reactor. The main reason for variation in color removal rates in different studies may be due to the type of dyes, microbes used and the process parameters used in the reactor. The biogas generation is directly related to the type of substrate, given as feed to the reactor. It was observed that the biogas production was increased exponentially with an increase in OLR and then remained constant near OLR of 5.0 kg COD m
3d
1 (Table 2). Further, increase in OLR from 5.0 to 10.0 kg COD m
3d
1, biogas production was also increased and reached up to 3.0 L d
1. The increased biogas production with an increasing OLR indicated that, a significant part of the mixed dye present in textile wastewater was used as carbon and energy source by the methanogens (Isik and Sponza, 2008). 3.1.3. Influence of OLR on pH and VFA/alkalinity ratio The pH is an integral expression of the acid base condition of anaerobic treatment process, while VFA to alkalinity ratio is the best option to monitor the process stability of anaerobic reactor (Venkata Mohan et al., 2005). The changes in VFA to alkalinity ratio at increasing OLR are presented in Table 2. pH and VFA/alkalinity ratios of anaerobic effluent were ranging from 7.5 to 7.0 and 0.07 to 0.1 respectively up to an OLR of 5.0 kg COD m
3d
1 and 2 d HRT. So the VFA concentration was very low and pH was stable. At lower HRTs (1 d) and higher OLR (10.0 kg COD m
3d
1), pH dropped down to 6.3, the VFA concentrations were higher than 450 mg l
1 which exceeded the critical value of VFA/alkalinity ratio (0.40) indicating the reactor instability. According to Ori et al. (2005) at increased OLR, the concentration of toxicants or inhibitory substances in the reactor was also increased, thus the activity of methanogenic and acetogenic populations might have decreased, causing an accumulation of the VFA, which in turn, increases the acidity and decrease in pH. Isik and Sponza (2008) reported that the VFA/alkalinity ratio must be of very low range for the stable anaerobic digester. 3.1.4. Influence of OLR on solids removal The effect of the OLR on the concentration of different types of solids in the process effluents are shown in Table 2. The concentration of TS and TVS were between 1500 and 1560 mg l
1 and 560e630 mg l
1, respectively, during an OLR of 1.0e5.0 kg COD m
3d
1 and HRT of 10 to 2 d. But the major disturbance was observed at the increased OLR of 10.0 kg COD m
3d
1, having 3230 and 1350 mg l
1 of TS and TVS concentration respectively. Similar behavior was observed in the case of TDS and TSS concentrations. The relation between OLR and solids removal have been reported in several studies (O'Neill et al., 2000; Moosvi et al., 2007; Balapure et al., 2015). All aforementioned parameters indicated that the anaerobic reactor was stable up to OLR of 5.0 kg COD m
3d
1 and 2 d HRT. Moreover, as observed from Table 2, at 2 d HRT in anaerobic phase, the concentration of chloride, sulphate and phosphate from textile industrial wastewater gradually decreased. Beyond 2 d HRT, the performance of the reactor was drastically affected. Thus, it was selected as an optimum HRT for further study. 3.2. Performance of a microaerophilic fixed film reactor at increasing HRTs It was commonly observed and well established that the cleavage of chromophore groups of dye molecules, essentially

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101

Table 2 Performance of an anaerobic fixed film reactor at increasing OLR under optimal operating conditions at 37  C. Hydraulic retention time (HRT) (d) 10
3
1

OLR (kg COD m d ) pH VFA/alkalinity ratio TS (mg l
1) TDS (mg l
1) TVS (mg l
1) TSS (mg l
1) Chloride (mg l
1) Sulphate (mg l
1) Phosphate (mg l
1) COD (mg l
1) BOD (mg l
1) Biogas production (L d
1) Color removal (%)

8

1.0 7.5 0.07 1500 760 560 700 1220 280 180 3290 1192 1.1 86

± ± ± ± ± ± ± ± ± ± ± ± ±

6

1.25 7.5 0.07 1517 771 571 718 1227 294 192 3357 1230 1.15 85

0.004 0.002 36.5 20.4 22.6 25.8 26.2 18.2 12.4 28.3 21.8 10.4 12.3

± ± ± ± ± ± ± ± ± ± ± ± ±

0.002 0.002 34.2 18.6 24.4 23.2 22.5 15.4 20.9 26.6 20.2 15.6 15.2

4

1.6 7.3 0.08 1528 779 579 730 1232 310 216 3500 1261 1.3 84

required anaerobic conditions, while for complete mineralization of corresponding intermediates from textile wastewater requires microarophilic/aerobic conditions. It was observed that the COD and BOD removal efficiency was nearly 73% at HRT of 4 h. As the HRT went up to 12 h, COD and BOD removal efficiency was increased and reached up to 97%. According to the results observed from Table 3, the COD and BOD removal efficiency of anaerobic effluent was enhanced with increasing HRT, however, after 12 h HRT there was a negligible effect on COD/BOD reduction. Fig. 2 displays an increase in HRT from 12 to 24 h, where it can be observed that COD removal efficiency was unaltered. Higher efficiency of COD removal at 12 h HRT could be attributed to the catabolic potential of acclimatized enriched consortium BDN which was enhanced under microaerophilic conditions. Furthermore, the COD originated during decolorization of dye compounds in anaerobic condition (63%) was further successfully decreased under microaerophilic conditions (97%). Thus, the above results suggested the complete mineralization of dye compounds in textile wastewater via COD reduction and leading to the formation of CO2 and H2O under microaerophilic conditions (Isik and Sponza, 2008). Moreover, mineralization of textile industrial wastewater was also confirmed by FTIR, 1H NMR and GCeMS analyses. Apart from COD, the microaerophilic fixed film reactor was also able to reduce BOD, total alkalinity, TS, TDS, TSS, TVS, chloride, sulphate and phosphate concentrations from textile industrial wastewater within 12 h of HRT (Table 3). Thus, 12 h HRT was considered as best HRT for further experiment. Several researchers worked on anaerobic-aerobic and microaerophilic-aerobic reactor configurations for decolorization

± ± ± ± ± ± ± ± ± ± ± ± ±

2

2.5 7.2 0.09 1545 785 600 738 1240 333 238 3564 1265 1.4 83

0.006 0.002 31.1 26.3 18.1 21.8 28.5 20.3 17.4 32.4 24.3 13.5 11.1

± ± ± ± ± ± ± ± ± ± ± ± ±

0.005 0.002 39.8 18.5 20.2 20.8 21.3 16.5 18.4 30.3 19.6 17.2 16.8

5.0 7.0 0.1 1560 800 630 750 1260 350 250 3668 1296 1.5 80

1 ± ± ± ± ± ± ± ± ± ± ± ± ±

COD removal

0.007 0.002 29.6 25.3 21.4 26.5 20.9 14.4 20.1 23.4 22.7 9.8 10.5

10.0 6.3 0.4 3230 1630 1350 610 2010 525 410 6780 2325 3.0 40

± ± ± ± ± ± ± ± ± ± ± ± ±

0.008 0.002 33.3 29.1 28.7 24.6 32.5 21.6 24.4 32.2 24.2 15.2 17.6

BOD removal 100

80

80

60

60

40

40

20

20

% COD removal

100

0

% BOD removal

Parameters

0 0

4

8

12 16 HRT (h)

20

24

Fig. 2. Variations in COD and BOD removal efficiencies at different HRTs in microaerophilic fixed film reactor. (The influent for microaerophilic fixed film reactor was the effluent of the anaerobic fixed film reactor after HRT of 2 d).

of textile wastewater. Franciscon et al. (2015), who studied sequential anaerobic-aerobic treatment of Disperse Red 1 dye containing wastewater, found ~80% azoreduction in an anaerobic reactor after 72 h, which was reached up to 100% after aerobic treatment. Lourenc et al. (2001) also used a combination of anaerobic-aerobic sequencing batch reactor for the removal of color from simulated textile effluents. They achieved 90% color removal with a retention time of 15 days in anaerobic stage and 10 h under aerobic phase. Lin et al. (2010) studied a continuous two-stage anaerobic/aerobic biological fluidized bed system to decolorize

Table 3 The results of microaerophilic reactor treating anaerobic effluent (2 d HRT) at different HRTs. Parameters

Hydraulic retention time (HRT) (d) 4

8

12

16

20

24

OLR (kg COD m
3d
1) pH VFA/alkalinity ratio TS (mg l
1) TDS (mg l
1) TVS (mg l
1) TSS (mg l
1) Chloride (mg l
1) Sulphate (mg l
1) Phosphate (mg l
1) COD (mg l
1) BOD (mg l
1) Color removal (%)

0.91 6.8 0.08 415 305 89 148 120 84 62 990 320 88

0.45 7.0 0.06 360 220 71 123 94 52 48 550 195 92

0.30 7.3 0.04 210 110 55 90 75 39 25 80 30 99

0.22 7.3 0.04 213 108 55 88 77 42 23 82 30 99

0.18 7.3 0.04 210 105 53 87 78 43 23 82 30 99

0.15 7.3 0.03 208 104 52 85 78 44 20 83 30 99

± ± ± ± ± ± ± ± ± ± ± ±

0.003 0.005 20.5 24.4 12.4 22.2 24.3 12.2 11.6 23.7 19.5 10.5

± ± ± ± ± ± ± ± ± ± ± ±

0.005 0.004 24.4 28.6 14.3 26.5 20.2 16.2 18.9 15.1 18.8 17.4

± ± ± ± ± ± ± ± ± ± ± ±

0.007 0.003 21.2 20.3 19.1 11.8 18.7 22.4 16.2 9.5 8.8 14.6

± ± ± ± ± ± ± ± ± ± ± ±

0.004 0.006 19.8 15.3 11.2 20.0 20.9 17.2 14.6 10.6 8.2 18.5

± ± ± ± ± ± ± ± ± ± ± ±

0.005 0.005 20.0 21.1 14.4 16.4 17.5 12.7 16.7 9.3 8.0 13.9

± ± ± ± ± ± ± ± ± ± ± ±

0.007 0.008 26.1 19.6 13.5 13.7 15.7 20.2 17.4 8.5 9.2 14.5

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and mineralize Reactive blue 13 and found 83.2% of color and 90.7% of COD removal at 70 h of HRT. Furthermore, Sandhya et al. (2005) used sequential microaerophilic-aerobic fixed film bed reactor for treatment of simulated wastewater having a COD of 721 mg l
1. They observed 74.67% COD reduction with OLR of 2.239 kg m
3d
1 within 7.72 h of HRT of microaerophilic reactor, while the complete COD reduction was observed in aerobic reactor at 7.79 h of HRT. 3.3. Overall performances of anaerobic-microaerophilic fixed film reactor system Dye decolorization and degradation is generally considered to have a higher efficiency under sequential anaerobicmicroaerophilic conditions. As depicted in Fig. 3, COD and color removal efficiencies were 63 and 80% respectively in an anaerobic fixed film reactor at a HRT of 2 d (48 h). The treatment efficiency was further increased in a microaerophilic reactor at 12 h of HRT. Thus, maximum color and COD removal efficiencies of 99 and 98% were achieved at total HRT of 60 h in the whole system, treating industrial textile wastewater. The results of this study showed that the color was mainly removed under anaerobic conditions while the COD was mainly removed under microaerophilic conditions. Similarly, Kapdan and Oztekin (2006) observed around 50% COD removal under anaerobic conditions and it reached up to 80% under aerobic phase. The anaerobic effluent of 2 d HRT and microaerophilic effluent of 12 h HRT were used in further biodegradation and toxicity analysis. 3.4. Oxido-reductive enzyme activities of microaerophilic reactor at an optimum HRT (12 h) The azo dye reduction in anaerobic condition is a non-specific and extracellular process, in which reducing equivalents from either biological or chemical sources are transferred to the dye molecule (Kalme et al., 2008). In case of aerobic or microaerophilic condition, a multistep conversion process of oxygenases has been employed for biodegradation of xenobiotics. The microaerophilic process is more beneficial over anaerobic treatment due to the involvement of molecular oxygen, which is beneficial for removal of toxic amines and converted them into non-toxic metabolites (Kodam et al., 2005). Study of enzymatic behavior during treatment of textile industrial wastewater under anaerobic is quite difficult, because of the limitation to maintain anaerobic conditions throughout the enzyme assays. Therefore, the present study monitored the activities of oxido-reductive enzymes during mineralization of

% COD and color removal efficiency

COD removal

Color removal

100 80

Anaerobic

60

Microaerophilic

40 20 0 0

12

24

36

48

60

HRT (h) Fig. 3. Treatment efficiency of sequential anaerobic e microaerophilic reactors in terms of COD and BOD removal at a total HRT of 60 h.

anaerobic effluent (2 d HRT) in the microaerophilic fixed film reactor. The results showed that, there was relatively less azoreductase activity (10.2 ± 1.3 mg Methyl red reduced min
1 mg protein
1) as compared to lignin peroxidase (12.4 ± U min
1 mg protein
1). The degradation of a broad range of substrates via a mechanism involving the formation of carbocation by peroxidases enzymes into non-toxic compounds have also been reported earlier (Saratale et al., 2011). Consequently, at the same HRT, NADH-reductase, laccase and tyrosinase activities were also detected (Table 4), which has ensured that the mechanism of textile wastewater mineralization was not physico-chemical. But bacterial consortium BDN seeded in microaerophilic reactor produces various oxidases and reductases which completely degrade azo dyes and detoxify contaminated water from the textile industry. 3.5. Detection of biodegraded products To study the mechanism of textile industrial wastewater degradation, different metabolites formed during sequential anaerobic (2 d HRT) and microaerophilic (12 h HRT) treatment were analyzed using UVevis and FTIR spectroscopy, 1H NMR and GCeMS. UVevis spectral (200e800 nm) analysis of influent showed four intense peaks in the UV region and a single peak in the visible region (630 nm). In UV region, peak between 220 and 230 nm indicated the presence of sulphonate groups in textile wastewater (Pinheiro et al., 2004), while the peak near 250 and 350 nm corresponds to phenyl and naphthyl moieties of dye molecules. After anaerobic treatment (2 d HRT) conjugated chromophores were broken down and peak at 630 nm was decreased without any peak shift, whereas new peaks were observed at 265 and 450 nm indicating the formation of possible aromatic metabolites (Oturkar et al., 2011). After the final treatment in microaerophilic reactor, seeded with consortium BDN (12 h HRT), peak at 630 nm completely disappeared and no significant peaks were observed, thereby possibly signifying complete degradation of the dyes (Fig. S1). FTIR spectrum of textile wastewater and its degraded product after anaerobic (2 d HRT) and microaerophilic (12 h HRT) treatment were analyzed at mid-IR fingerprinting region (4000e400 cm
1). FTIR analysis of textile wastewater (influent) showed the broad and strong peaks between 3405 and 3615 cm
1 represents eOH stretching vibrations. These eOH groups may form hydrogen bonds with organic molecules in textile wastewater (Fig. S2a). The presence of color in textile wastewater is due to the chromophore structure and the dyeing capacity is due to auxochrome groups. The auxochrome generally constitutes the aromatic structure based on rings of benzene and naphthalene (Saratale et al., 2011). Significantly, in the present study, chromophore of azo groups (eN]Ne) gave the absorption band near 1500e1600 cm
1 while, different polynuclear aromatic rings absorb strongly in the low frequency range between 900 and 675 cm
1 (Balapure et al., 2015). Auxochrome groups (i.e. eNH2, eCOOH, eSO3H, eOH etc.) present in textile wastewater is ionizable groups, that confers the binding capacity of dyes onto the textile material. In the present study, FTIR spectrum of textile wastewater confirms the presence of some auxochrome groups. The peaks at 3099 and 3471 cm
1 which showed eNH stretching of amides and eCH stretching of asymmetric alkanes respectively. A peak displayed at 2179 and 2110 cm
1 corresponds to eNHþ 3 vibrations of charged amine derivatives (Kagalkar et al., 2010), whereas, peak at 1150 cm
1 for eS]O stretching as in sulfonic acid and 1718 cm
1 for eC]O stretch of carboxylic acid. Furthermore, metabolites obtained after anaerobic treatment of textile wastewater showed disappearance of major peaks between 1500 and 1600 cm
1 indicated decolorization of azo dyes present in wastewater, but the formation of new

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103

Table 4 Enzymatic status of microaerophilic reactor at an optimum HRT (12 h) during mineralization of anaerobic effluent. Enzyme activity

Cell free extract of 12 h HRT treated effluent

Lignin peroxidase (U min
1 mg protein
1) Laccase (U min
1 mg protein
1) Tyrosinase (U min
1 mg protein
1) Azoreductase (mg Methyl red reduced min
1 mg protein
1) NADH-DCIP reductase (mg DCIP reduced min
1 mg protein
1)

12.4 3.1 0.2 10.2 15.9

± ± ± ± ±

0.6* 0.07* 0.04* 1.3** 3.4**

± Indicate the standard deviation of mean of three repeated activities. Significantly different from the control cells at *P > 0.05, **P < 0.001 by one-way ANOVA with TukeyeKramer multiple comparisons test.

peak at 1333 cm
1 suggested formation of primary aromatic amines which was additionally confirmed by NMR and GCeMS analyses. The formation of different peaks at 3236, 1650, 1265 and 1461 cm
1 showed eNO stretching of nitrites, acyclic eC]N stretching, eCeO stretching and eCeH deformation of alkanes respectively (Lade et al., 2012) (Fig. S2b). Moreover, peaks at 1626 and 1275 cm
1 also corresponds to the formation of amines, while 1599 cm
1 represents eNHþ 3 deformation indicating a new product formation with the amine group (Khandare et al., 2012). These results indicated that anaerobic process is often capable of decolorizing dye molecule; however, it cannot able to give the complete mineralization of the dye molecule. But as shown in Fig. S2c after treatment of anaerobic effluent in microaerophilic reactor having bacterial consortium BDN, no peaks were observed in the range of 900 to 675 cm
1 region indicating loss of the aromaticity of dye molecules, whereas, mineralization was confirmed by the formation of new peak at 1215 cm
1 probably associated with eCeH aliphatic stretching (Fig. S2c). This interpretation is corroborated by 1H NMR studies. 1H NMR spectrum of textile wastewater showed a broad singlet and complex multiple peaks between d 6.5 and 8.5 low field zone confirmed the presence of alkyl substituted aromatic rings in wastewater (Fig. S3a). After anaerobic treatment (2 d HRT), the signals between d 6.5 and 8.5 deshielded, while two resonance were appeared at d 6.95 and 7.64 belongs to 4-hydroxybenzenesulfonate (Lopez et al., 2004). The presence of 4-hydroxybenzenesulfonate was also confirmed by GCeMS analysis. Moreover, several singlet, doublet and triplet signals were formed in the range of d 5.3e6.2 corresponds to protons from alkenes formed during the breakdown of benzene and naphthalene rings of dye molecule (Fig. S3b) (Jain et al., 2012). Conversely, the absence of the corresponding peaks for aromatic protons in low field zone/higher frequency indicated the complete mineralization of dyes present in textile wastewater with microaerophilic strategy. However, few signals at lower frequencies between d 1.0 and 3.0 still remained, which indicated the presence of lower molecular weight aliphatic hydrocarbons. Thus, high molecular weight carcinogenic dye molecules are being converted into low molecular weight compounds due to the synthrophic action of bacterial consortium BDN seeded in the microaerophilic reactor (Fig. S3c). GCeMS analysis supported biodegradation of dye molecules present in textile industrial wastewater using sequential anaerobicmicroaerophilic process. During GCeMS analysis, the components eluted having different retention time were subjected to mass spectrometry and identified by interpretation of their molecular weight and mass spectra (data not shown). GCeMS analysis of anaerobic effluent (2 d HRT) showed formation of nine aromatic amines namely, (2-methyl-4 nitrophenyl) diazene, 2-methyl 4nitroaniline, sodium benzenesulfonate, ethyl sulfonyl benzene, 2amino 5-chlorotriazine, benzene acetic acid, 4-hydroxybenzenesulfonate, catechol, naphthalene, aniline and benzene with a mass peak at 166, 154, 177, 170, 132, 136, 232, 111, 128, 93 and 78 respectively. Consequently, as degradation proceeds, bacterial

consortium BDN present in the microaerophilic fixed film reactor converts aforementioned higher molecular weight aromatic amines into lower molecular weight aliphatic compound with a mass peak of 44, 52 and 56. Moreover, the detection of oxidoreductive enzyme activities at 12 h HRT clearly indicates their involvement in degradation of complex azo dye molecules into simple metabolizable structures. However, the detailed mechanism/pathway of the textile wastewater cleavage remains to be explored. 3.6. Phytotoxicity evaluation of anaerobic-microaerophilic treated metabolites The metabolites produced from dye degradation were reported to be more toxic than parent dye. Therefore, it becomes very important for dye bioremediation technology to assess the toxicity of the pollutants and metabolites formed after dye degradation in order to study the feasibility of the method. In present study, the relative toxicity of untreated and sequentially treated wastewater in relation to S. vulgare, T. aestivum and P. mungo was studied. The toxic nature of the dyes in untreated textile wastewater was confirmed when 20, 28 and 35% germination inhibition of S. vulgare, T. aestivum and P. mungo was observed. After anaerobic treatment of textile wastewater percent germination was enhanced by 60e65%, while subsequent to final treatment in the microaerophilic reactor (seeded with bacterial consortium BDN) more than 98% germination of each plant were appeared. In addition, significant improvement in shoot and root length (Table 5) of the plants were also observed in the microaerophically treated effluent. This concludes that consortium BDN was found to be efficient not only for degradation of textile wastewater but also for its detoxification. 4. Conclusions Sequential anaerobic-microaerophilic process is promising green technology for treatment of industrial textile wastewater. Experimental data depicted that, dye molecules were cleaved under anaerobic condition and mineralization was occurred in microaerophilic reactor through synthrophic interactions of consortium BDN. The system can efficiently remove 97% of COD, BOD and color from textile wastewater and meet the criteria of pollution control standards. The present study also indicated that dye mineralization was completely dependent on enzyme regulation by bacteria in response to mutagenic intermediates formed during anaerobic treatment. Phyotoxicity study demonstrated the success of sequential treatment in reducing the toxicity of the textile wastewater. Looking at the above results, further scale up studies at pilot plant is going on and we have observed some interesting results. During this study, metabolite concentration, mass balancing, kinetics pertaining to the bioreactor design and energy consumption are monitored and the process is being improved to meet the

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Table 5 Phytotoxicity of textile industrial wastewater and its degraded products for Sorghum vulgare, Triticum asetivum and Phaseolus mungo. Plants

Sorghum vulgare

Triticum asetivum

Phaseolus mungo

Observation

Germination (%) Plumule (cm) Radical (cm) Germination (%) Plumule (cm) Radical (cm) Germination (%) Plumule (cm) Radical (cm)

Treatments Distilled water

Untreated wastewater

Anaerobic effluent (2 d HRT)

Microaerophilic effluent (12 h HRT)

100 16.8 7.6 100 25.7 10.7 100 22.5 9.2

25 3.9 0.9 21 6.6 1.5 18 2.9 0.7

62 7.2 2.0 54 11.2 5.7 49 9.0 2.3

100 17.0 7.5 100 25.4 10.6 99 21.4 8.7

± 2.2 ± 1.4 ± 3.8 ± 1.5 ± 4.0 ± 2.3

± 3.4* ± 1.3* ± 4.2* ± 3.2* ± 3.7* ± 1.0*

± 4.6* ± 2.8* ± 2.5* ± 2.8* ± 2.2* ± 1.2*

± 3.2* ± 1.9* ± 1.2* ± 1.6* ± 2.6* ± 1.7*

Values are mean of three experiments, SEM (±), significantly different from the control (seeds germinated in distilled water) at *p < 0.05, **< 0.001, by the one way analysis of variance (ANOVA) with TukeyeKramer multiple comparison test.

economic feasibility during in situ bioremediation. Acknowledgments The authors are grateful to Gujarat State Biotechnology Mission (GSBTM), Gujarat, India and Department of Biotechnology (DBT), Ministry of Science and Technology New Delhi, India (BT/01/CEIB/ 09/V/05) for providing financial assistance. Authors are also thankful to Sophisticated Instrumentation Centre of Applied Research and Training (SICART), Vallabh Vidyanagar, Gujarat, India (997(a)) for providing facilities for FTIR and GCeMS. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibiod.2015.10.008. References Anderson, G.K., Yang, G., 1992. Determination of bicarbonate and total volatile acid concentration in anaerobic digesters using a simple titration. Water Environ. Res. 64, 53e59. APHA- American Public Health Association, 2012. Standard Methods for the Examination of Water and Wastewater, twentieth ed. APHA, Washington, DC. Balapure, K., Bhatt, N., Madamwar, D., 2015. Mineralization of reactive azo dyes present in simulated textile wastewater using down flow microaerophilic fixed film bioreactor. Bioresour. Technol. 175, 1e7. Balapure, K.H., Jain, K., Chattraj, S., Bhatt, N., Madamwar, D., 2014. Co-metabolic degradation of diazo dye e reactive Blue 160 by enriched mixed culture BDN. J. Hazard. Mater. 279, 85e95. Bhatt, N., Patel, K.C., Keharia, H., Madmwar, D., 2005. Decolorization of diazo-dye Reactive Blue 172 by Pseudomonas aeruginosa NBAR12. J. Basic Microbiol. 45, 407e418. Chan, Y., Chong, M., Lim law, C., Hassell, D.G., 2009. A review on anaerobic-aerobic treatment of industrial municipal wastewater. Chem. Eng. J. 155, 1e18. Franciscon, E., Mendonca, D., Seber, S., Morales, D.A., Zocolo, G.J., Zanoni, M.B., Grossman, M.J., Durrant, L.R., Freeman, H.S., Umbuzeiro, G.A., 2015. The potential of a bacterial consortium to degrade azo dye Disperse Red 1 in a pilot scale anaerobiceaerobic reactor. Process Biochem. 50, 816e825. Gnanapragasam, G., Senthilkumar, M., Arutchelvan, V., Sivarajan, P., Nagarajan, S., 2010. Recycle in upflow anaerobic sludge blanket reactor on treatment of real textile dye effluent. World J. Microbiol. Biotechnol. 6, 1093e1098. Isik, M., Sponza, D.T., 2008. Anaerobic/aerobic treatment of a simulated textile wastewater. Sep. Purif. Technol. 60, 64e72. Jain, K., Shah, V., Chapla, D., Madamwar, D., 2012. Decolorization and degradation of azo dye e reactive Violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil. J. Hazard. Mater. 213e214, 378e386. Kagalkar, A.N., Jagtap, U.B., Jadhav, J.P., Govindwar, S.P., 2010. Studies on phytoremediation potentiality of Typhonium flagelliforme for the degradation of Brilliant Blue R. Planta 232, 271e285. Kalme, S., Ghodake, G., Govindwar, S., 2008. Red HE7B degradation using desulfonication by Pseudomonas desmolyticum NCIM2112. Int. J. Biodeterior. Biodegr. 60, 327e333. Kalyani, D.C., Telke, A.A., Dhanve, R.S., Jadhav, J.P., 2009. Ecofriendly biodegradation and detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp. SUK1. J. Hazard. Mater. 163, 735e742. Kapdan, I.K., Oztekin, R., 2006. The effect of hydraulic residence time and initial COD

concentration on color and COD removal performance of the anaerobiceaerobic SBR system. J. Hazard. Mater. 136, 896e901. Keharia, H., Madamwar, D., 2003. Bioremediation concepts for treatment of dye containing wastewater: a review. Ind. J. Exp. Biol. 41, 1068e1075. Khandare, R.V., Rane, N.R., Wagjmode, T.R., Govindwar, S.P., 2012. Bacterial assisted phytoremediation for enhanced degradation of highly sulfonated diazo reactive dye degradation of highly sulfonated diazo reactive dye. Environ. Sci. Pollut. Res. 19, 1709e1718. Kodam, K.M., Soojhawon, I., Lokhande, P.D., Gawai, K.R., 2005. Microbial decolorization of reactive azo dye under aerobic conditions. World J. Microbiol. Biotechnol. 21, 367e370. Lade, H.S., Waghmode, T.R., Kadam, A.A., Govindwar, S.P., 2012. Enhanced biodegradation and detoxification of disperse azo dye Rubine GFL and textile industry effluent by defined fungal-bacterial consortium. Int. J. Biodeterior. Biodegr. 72, 94e107. Laquidara, M.J., Blanc, F.C., O'Shaughnessy, J.C., 1986. Development of biofilm, operating characteristics and operational control in the anaerobic rotating biological contactor process. J. Water Pollut. Control Fed. 58, 107e114. Lin, J., Zhang, X., Li, Z., Lei, L., 2010. Biodegradation of Reactive Blue 13 in a two stage anaerobic/aerobic fluidized beds system with a Pseudomonas sp. Isol. Bioresour. Technol. 101, 34e40. Lopez, C., Valade, A.G., Combourieu, B., Mielgo, I., Bouchon, B., Lema, J.M., 2004. Mechanism of enzymatic degradation of the azo dye Orange II determined by ex situ 1H nuclear magnetic resonance and electrospray ionization-ion trap mass spectrometry. Anal. Biochem. 335, 135e149. Lourenc, N.D., Novais, J.M., Pinheiro, H.M., 2001. Effect of some operational parameters on textile dye biodegradation in a sequential batch reactor. J. Biotechnol. 89, 163e174. Lowry, O.H., Rosebrough, N.J., Randall, A.L., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265e275. Mendez-Martineza, A.J., Davila-Jimeneza, A., Ornelas-Davilab, M., ElizaldeGonzalezb, O., Arroyo-Abadb, U., Sires, I., Brillas, E., 2012. Electrochemical reduction and oxidation pathways for Reactive Black 5 dye using nickel electrodes in divided and undivided cells. Electrochim. Acta 59, 140e149. Mohana, S., Shrivastava, S., Divecha, J., Madamwar, D., 2008. Response surface methodology for optimization of medium for decolorization of textile dye direct black 22 by a novel bacterial consortium. Bioresour. Technol. 99, 562e569. Moosvi, S., Kher, X., Madamwar, D., 2007. Isolation, characterization and decolorization of textile dyes by a mixed bacterial consortium JW-2. Dyes Pigments 74, 723e729. Mustafa, I., Delia, T.S., 2004. Anaerobic/aerobic sequential treatment of a cotton textile mill wastewater. J. Chem. Technol. Biotechnol. 79, 1268e1274. O'Neill, C., Hawkes, F.R., Hawkes, D.L., Esteves, S., Wilcox, S.J., 2000. Anaerobiceaerobic biotreatment of simulated textile effluent containing varied ratios of starch and azo dye. Water Res. 34, 2355e2361. Ori, L., Barak, E.M., Richard, E.L., 2005. Rapid, simple, and accurate method for measurement of VFA and carbonate alkalinity in anaerobic reactors. Environ. Sci. Technol. 36, 2736e2741. Oturkar, C.C., Nemade, H.N., Mulik, P.M., Patole, M.S., Hawaldar, R.R., Gawai, K.R., 2011. Mechanistic investigation of decolorization and degradation of Reactive Red 120 by Bacillus lentus BI377. Bioresour. Technol. 102, 758e764. Panswad, T., Iamsamer, K., Anotai, J., 2001. Decolorization of azo-reactive dye by polyphosphate and glycogen-accumulating organisms in an anaerobiceaerobic sequencing batch reactor. Bioresour. Technol. 76, 151e159. Pinheiro, H.M., Touraud, E., Thomas, O., 2004. Aromatic amines from azo dye reduction: status review with emphasis on direct UV spectrophotometric detection in textile industry wastewaters. Dyes Pigments 61, 121e139. Rondon, H., El-Cheikh, W., Boluarte, I.R., Chang, C., Bagshaw, S., Farago, L.J., Shu, L., 2015. Application of enhanced membrane bioreactor (EMBR) to treat dye wastewater. Bioresour. Technol. 183, 78e85. Sandhya, S., Padmavathy, S., Swaminathan, K., Subrahmanyam, Y.V., Kaul, S.N., 2005. Microaerophilic-aerobic sequential batch reactor for treatment of azo dyes containing simulated wastewater. Process Biochem. 40, 885e890. Saratale, R.G., Saratale, G.D., Chang, J.S., Govindwar, S.P., 2011. Bacterial

K. Balapure et al. / International Biodeterioration & Biodegradation 106 (2016) 97e105 decolorization and degradation of azo dyes: a review. J. Taiwan Inst. Chem. Eng. 42, 138e157. Senthilkumar, M., Gnanapragasam, G., Arutchelvan, V., Nagarajan, S., 2011. Treatment of textile dyeing wastewater using two-phase pilot plant UASB reactor with sago wastewater as co-substrate. Chem. Eng. J. 166, 10e14. Somasiri, W., Ruan, W., Xiufen, L., Jian, C., 2008. Evaluation of the efficacy of upflow anaerobic sludge blanket reactor in removal of colour and reduction of COD in real textile wastewater. Bioresour. Technol. 99, 3692e3699.

105

Venkata Mohan, S., Chandarasekararao, N., Krishnaprasa, K., Muralikrishna, P., Sreenivasrao, R., Sharma, P.N., 2005. Anaerobic treatment of complex chemical wastewater in sequencing batch biofilm reactor (AnSBBR): process optimization and evaluation of factor interactions using Taguchi Dynamic DOE methodology. Biotechnol. Bioeng. 90, 732e745. Wang, X., Cheng, X., Sun, D., Ren, Y., Xu, G., 2014. Fate and transformation of naphthylaminesulfonic azo dye reactive black 5 during wastewater treatment process. Environ. Sci. Pollut. Res. 21, 5713e5723.