The objective of this study was development and characterization of a halophilic bacterial consortium for rapid decolorization and degradation of a wide range of dyes and their mixtures. The 16S rRNA gene analysis of developed halophilic consortium VN.1 showed that the bacterial consortium contained six bacterial strains, which were identified as Pseudomonas fluorescens HM480360, Enterobacter aerogenes HM480361, Shewanella sp. HM589853, Arthrobacter nicotianae HM480363, Bacillus beijingensis HM480362 and Pseudomonas aeruginosa JQ659549. Halophilic consortium VN.1 was able to decolorize up to 2,500 mg/L RB220 with >85% chemical oxygen demand (COD) reduction under static condition at 30 °C and pH 8.0 in the presence of 7% NaCl. VN.1 also exhibited more than 85% COD reduction with >25 mg/(L h) rate of decolorization in the case of different reactive dye mixtures. We propose the symmetric cleavage of RB220 using Fourier transform infrared, high-performance liquid chromatography (HPLC), nuclear magnetic resonance and gas chromatography-mass spectrometry analysis, and confirmed the formation of sodium-4-aminobenzenesulfonate, sodium-6-aminonepthalenesulfonate, and sodiumbenzene/nepthalenesulfonate. Toxicity studies confirm that the biodegraded products of RB220 effluent stimulate the growth of plants as well as the bacterial community responsible for soil fertility.

INTRODUCTION

The serious environmental pollution caused by the release of large amounts of dyestuff-containing effluents into natural ecosystems has caused significant concern. With the development of industry, a number of dyes have been extensively produced and used in textile, printing, manufacture of paints and varnishes, manufacture of plastics, food industries, etc. Since the dyeing and printing processes were always inefficient, about 10–15% of unused dyes were discharged into the water bodies directly (Qu et al. 2010). Azo dyes constitute the largest group of mutagenic and carcinogenic xenobiotic pollutants (Oturkar et al. 2011) and disposal of these dyes as well as remediation of associated contaminated sites remains a problem. Also, these dyes are resistant to light, biological activity, ozone and other degradation conditions due to their complex aromatic structure (Joshi et al. 2004; Zhou et al. 2014,). In addition to this, the dyes can be absorbed on particulate matter such as sediments or suspended solids, causing rapid depletion of transparency of water, water quality and dissolved oxygen (DO) in the receiving water body (Asgher et al. 2007). Therefore, the conventional wastewater treatments such as chemical and physical methods were ineffective, costly and might cause secondary pollution problems (Galindo et al. 2001). In the current scenario, microbial or enzymatic treatment offers an indispensable, eco-friendly and cost-effective solution towards restoring ecosystems polluted with azo dyes and could help to reduce the enormous water consumption compared to physico-chemical methods (Moore et al. 1989). Nowadays recent research is focused on the treatment of dye-contaminated water by applying different biotechnological approaches (Gavrilescu et al. 2015). Varieties of micro-organisms, including bacteria, fungi, yeasts, actinomycetes and algae, are capable of degrading azo dyes, among which bacterial cells represent an inexpensive and promising tool for the removal of various azo dyes from textile dye effluents (Dafale et al. 2008). Bacteria capable of dye decolorization, either in pure cultures or in consortia, have been reported (Kalyani et al. 2009). It has been demonstrated that synergistic metabolic activities of a mixed microbial consortium can lead to complete mineralization of azo dyes (Tony et al. 2009). The mechanism of microbial degradation of azo dyes involves the reductive cleavage of azo bonds (–N = N–) with the help of azoreductase under anaerobic conditions, resulting in the formation of colorless solutions (Chang et al. 2001). For the reduction of azo dyes, reduction to the anion radical occurs by a fast one-electron transfer reaction, followed by a second, slower electron transfer event to produce the stable dianion. Thus, the functional group of azo dye with higher electron density might be unfavorable to this second electron transfer to form the dianion, leading to low or no capability for decolorization (Pearce et al. 2003). Owing to this reason, the sulfonated reactive group of azo dyes is normally considered to be more recalcitrant than carboxylated azo dyes. In addition, the rate-limiting step during bacterial decolorization of sulfonated azo dyes is the permeation through the bacterial cell membrane (Lourenco et al. 2000).

The aim of the study was to investigate a potential bacterial consortium for decolorization of various sulfonated reactive azo dyes commonly used in textiles, paint, garments and allied industries in India.

MATERIALS AND METHOD

Sample collection

All dyes used in this study were collected from Vim Dye Chem, Ganesh Dye Chem and Hue Laboratory, Vatva, Ahmedabad, Gujarat, India (Table S1, available online at http://www.iwaponline.com/wst/072/208.pdf). Oil-contaminated sludge samples were collected from the Oil and Natural Gas Corporation Limited (ONGC) area of Rajpura, Mansa, Gujarat, India (23°23′58.01″ latitude and 72°39′16.66″ longitude) and the dye-wastewater-contaminated soil samples were collected from the Gujarat Industrial Development Corporation (GIDC) area of Vatva, Ahmedabad, Gujarat, India (22°56′58.51″ latitude and 72°37′21.86″ longitude) for the isolation of potential dye-degrading bacterial strains.

Isolation, screening, development and identification of bacterial consortium

A total of 128 morphologically distinct bacteria strains were isolated on N-agar plates containing 100 mg/L RB220 dye and 4% NaCl concentration from sludge samples after enrichment in MSM (mineral salt medium) broth containing (g/L) (NH4)2SO4 1.0, K2HPO4 2.0, MgSO4.7H2O 0.5, NaCl 40.0, CaCl2.2H2O 0.04, peptone 1.0, and meat extract 1.0 amended with 250 mg/L Reactive Blue 220 (RB220) (structure of RB220 is given in Figure S1, available online at http://www.iwaponline.com/wst/072/208.pdf) at room temperature with pH 7.0. Bacterial strains that are capable of degrading more than 50% of the dyes were selected and mixed in different combinations for development of the bacterial consortium. Genomic DNA of all the isolates was extracted following Ausubel et al. (1997) using universal eubacterial primers 8F and 1492R (Desai et al. 2009). The purified amplified products were sequenced by the automated DNA Analyzer 3730 using ABI PRISM BigDye™ Terminator Cycler Sequencing v. 3.1 Chemistry (Applied Biosystem, USA). 16S rRNA gene sequence was analyzed using BLASTN programmed in the NCBI server to identify bacterial strains.

Optimization of nutritional and environmental parameters

The influence of various physico-chemical conditions, including inoculum size (1.0–25.0%), agitation speed (0–250 rpm), temperature (26–40 °C), NaCl concentration (10–100 gL−1), pH (5–10) and dye concentration (100–500 mg/L), were examined to achieve optimum decolorization of RB220. In addition, influence of co-substrates on decolorization and degradation of RB220 was optimized by supplementing the MSM broth with different carbon (glucose, lactose, sucrose, malt extract and maltose) and nitrogen (beef extract, yeast extract (YE), meat extract, casein, tryptose, tryptone, peptone, soyabean, malt extract and urea) sources at 1.0%. Also, 1 g of rice husk, rice straw, wood shavings and bagasse powder were mixed with 100 mL distilled water individually and autoclaved at 121 °C for 20 min. Five milliliters of extract of each agricultural waste was added in semi-synthetic medium and checked for the decolorization of RB220 by bacterial consortium VN.1 to make this process more economical. The effect of repeated dye addition on the decolorization performance of bacterial consortium VN.1 was studied with 300 mg/L dye concentration in batch culture. The percentage decolorization and time required were monitored after each cycle.

Decolorization and biodegradation analysis

Aliquots of 2.0 mL from experimental and control medium were withdrawn at regular intervals of 2 h up to 8 h, or until complete decolorization was observed, and centrifuged at 6,000 g for 15 min at 4 °C to obtain clear supernatant. RB220 decolorization was monitored at 610 nm (λ max) by a double-beam Specord® 210 BU UV–visible (UV–vis) spectrophotometer (Analytica Jena AG, Germany) against media blank, and growth was monitored turbidimetrically by resuspending the cell pellet in distilled water and measuring absorbance at 600 nm. All the experiments were performed in triplicate. Biodegradation was studied by extracting metabolites from effluent with equal volumes of ethyl acetate and evaporating it to dryness in a SpeedVac (Thermo Electron Corporation, Waltham, MA, USA). The extracted metabolites were mixed with HPLC (high-performance liquid chromatography) grade potassium bromide (KBr) in the ratio of 5:95 and analysed at the mid-infrared region (400–4,000 cm−1) by Fourier transform infrared (FTIR) spectroscopy using a PerkinElmer spectrum GX spectrophotometer (PerkinElmer, USA). HPLC analysis was performed in an isocratic Waters 2690 system equipped with a dual absorbance detector, using a C18 column (4.6 × 250 mm). Methanol:water (80:20) was used as mobile phase at a flow rate of 1.0 mL/min and column temperature was held at 25 °C. For proton nuclear magnetic resonance (1H NMR) spectrometry studies, extracted metabolites were dissolved in an appropriate volume of D2O and were analyzed by a Bruker 13C NMR-400 MHz (Bruker, USA). Gas chromatography–mass spectrometry (GC–MS) analysis of metabolites was performed using an Auto-System XL (PerkinElmer, USA). Gas chromatography was conducted using capillary column TES–MS (0.25 μm × 0.25 μm × 30 m) with helium as a carrier gas at a flow rate of 1 mL/min with a speed ratio of 1:40. The initial column temperature was maintained at 70 °C for 4 min and linearly increased at 10 °C/min to 290 °C and held for 4 min. The temperature of the injection port was 250 °C and the GC–MS interface was maintained at 220 °C. Compounds were identified on the basis of their mass spectra and NIST library. Copper concentration from treated and untreated RB220 wastewater was determined by an atomic absorption spectrophotometer (Elico, India, model SL 194), for assessing copper removal ability of the culture. To understand the degree of biodegradation of RB220, reduction in the chemical oxygen demand (COD) was measured before/after decolorization (APHA 1995). Total organic carbon (TOC) was measured by using Liqui TOC II apparatus (Elementar Analysen system GmbH Company, Germany) equipped with an infrared (IR) detector. To determine the residual TOC, the supernatant of decolorized broth was used with 1.5 and 2.5 min irradiation time and oxygen as a carrier gas (0.95–1.00 bar). TOC measurement was conducted under a maximum temperature of 850 °C and a flow rate of 200 mL/min. TOC removal ratio of before and after decolorization was calculated as follows: TOC removal ratio (%) = [initial TOC(0 h) − observed TOC(t)]/initial TOC(0 h) × 100.

Phytotoxicity studies

The ethyl-acetate-extracted products of RB220 were dried and dissolved in sterile distilled water for phytotoxicity studies. The phytotoxicity study was carried out (at room temperature) on Phaseolus aureus, which has an importance in Indian agriculture (Kalyani et al. 2009). Eighty seeds of Phaseolus aureus were regularly supplied with distilled water as a control, RB220 wastewater and its degradation products for up to 1 month. Volumes for supplement of water, dye and degraded dye metabolites were kept the same, i.e. 5 mL per day. Toxicity effect was measured in terms of percent germination, lengths of the plumule and radical, wet weight number of fibrils, and microbial profiling of the rhizospheric zone of Phaseolus aureus after every 7 days for up to 1 month.

RESULTS AND DISCUSSION

Screening, development and identification of bacterial consortium

Many researchers have mentioned that a high degree of biodegradation and mineralization can be expected when co-metabolic activities within a microbial community complement each other. In such a consortium, the organisms can act synergistically on a variety of dyes and dye mixtures. One organism may be able to cause a biotransformation of the dye, which consequently renders it more accessible to another organism that otherwise is unable to attack the dye (Nigam et al. 1996).

The ONGC area of Rajpura, Mansa, has been receiving crude-oil-contaminated soil from various oil spills, and Vatva GIDC, Ahmedabad, has been receiving wastewater from various dye manufacturing industries, thus becoming potential sites for obtaining a well-acclimatized indigenous microbial community capable of degrading various dyes. Out of 128 bacterial isolates, 24 isolates found to be capable of degrading various reactive azo dyes (with more than 4% NaCl concentration and 4.0 to 15.0 mg/(L h) average rate of decolorization) were utilized for the development of the bacterial consortium (Figure S2, available online at http://www.iwaponline.com/wst/072/208.pdf). A total of 20 consortia were developed using 24 selected isolates in combinations of five to nine isolates with application of five reactive dyes. It was observed that the presence of isolates coded A, B, C, D, E and F, in some selected consortia improved the decolorization ability of the consortium (Table S2, available online at http://www.iwaponline.com/wst/072/208.pdf). Afterwards the combination of isolates A, B, C, D, E and F, labeled VN.1, was found to be capable of degrading all tested dyes efficiently compared to the other tested consortia (Figure S3, online at http://www.iwaponline.com/wst/072/208.pdf).

The bacterial community structure was analyzed on the basis of 16S rRNA gene sequencing, which showed that VN.1 was composed of six bacterial species, namely Pseudomonas aeruginosa JQ659549, Pseudomonas fluorescens HM480360, Enterobacter aerogenes HM480361, Bacillus beijingensis HM480362, Shewanella sp. HM589853 and Arthrobacter nicotianae HM480363, representing three bacterial phyla: Proteobacteria, Firmicutes and Actinobacteria. Their phylogenic position is presented in Figure S4 (online at http://www.iwaponline.com/wst/072/208.pdf).

Effect of DO on decolorization

Bacterial decolorization is a reductive process; hence, the presence of molecular oxygen in a medium may diminish its reduction potential. We have studied the decolorization and the growth profile of the VN.1 at 30 °C in MSM amended with 300 mg/L of RB220 under shaking condition at 100 rpm and static condition (Figure 1(a)). There was a 7.1-fold increase in the decolorization potential of VN.1 as DO level dropped from 7.2 mg/L to nearly 0.1 mg/L under static condition. During this process it was observed that relatively faster growth and higher cell density was obtained (A600 1.1) in the oxygen-rich environment (shaking conditions) as compared to the static conditions (A600 0.7). Decolorization was much lower in the presence of oxygen during dye decolorization in an aerobic environment. Electrons released by oxidation of electron donors like NADH are preferentially utilized to reduce free oxygen rather than be used in azo bonds cleavage by azoreductase, normally inhibited by the presence of oxygen (Chang et al. 2001). Since VN.1 has shown decolorization under microaerophilic condition, the demand for oxygen is much less, which will save a great amount of energy required in the aeration system and will reduce operational cost.

Figure 1

Effect of co-metabolites on dye decolorization by bacterial consortium VN.1 under static condition at 34 °C, pH 8.0. (a) Decolorization of RB220 (300 mg/L) by bacterial consortium VN.1 in MSM under static and shaking condition (110 rpm) along with 0.1% YE and peptone. (b) Effect of inoculum volume (v/v) on RB220 decolorization in MSM along with 0.1% YE and peptone. (c) Effect of initial dye concentration on decolorization rate by bacterial consortium VN.1 in MSM along with 0.1% YE and peptone. (d) Decolorization of RB220 (300 mg/L) by bacterial consortium VN.1 at varying initial NaCl concentration.

Figure 1

Effect of co-metabolites on dye decolorization by bacterial consortium VN.1 under static condition at 34 °C, pH 8.0. (a) Decolorization of RB220 (300 mg/L) by bacterial consortium VN.1 in MSM under static and shaking condition (110 rpm) along with 0.1% YE and peptone. (b) Effect of inoculum volume (v/v) on RB220 decolorization in MSM along with 0.1% YE and peptone. (c) Effect of initial dye concentration on decolorization rate by bacterial consortium VN.1 in MSM along with 0.1% YE and peptone. (d) Decolorization of RB220 (300 mg/L) by bacterial consortium VN.1 at varying initial NaCl concentration.

Effect of pH, temperature and inoculum size

VN.1 exhibited more than 98% decolorization over a broad range of pH 6–10 with more than 16 mg/(L h) rate of decolorization, while VN.1 achieved the highest decolorization activity for RB220 at pH 8 (Table 1). It is thought that the pH effect may be more likely related to the transport of dye molecules across the cell membrane, which was considered as a rate-limiting step for the decolorization (Lourenco et al. 2000). Similar results were observed in the decolorization of RV5R by developed consortium JW2, which is a combination of Paenibacillus polymyxa, Micrococcus luteus and Micrococcus sp. (Moosvi et al. 2007).

Table 1

RB220 decolorization by bacterial consortium VN.1 at different temperature and varying initial pH of medium along with 0.1% YE and peptone under static condition at *P < 0.05 by one-way analysis of variance with Tukey–Kramer multiple comparisons test

  Physico-chemical conditions
 
 Temperature (°C)
 
pH
 
 26 30 34 37 40 5.0 6.0 7.0 8.0 9.0 
Average rate of decolorization (mg/(L h)) 38.78 ± 1.056* 40.34 ± 0.897* 42.41 ± 1.236* 38.54 ± 1.045* 24.61 ± 0.589* 8.17 ± 0.365* 14.31 ± 0.928* 35.38 ± 1.256* 39.58 ± 0.254* 32.76 ± 0.847* 
  Physico-chemical conditions
 
 Temperature (°C)
 
pH
 
 26 30 34 37 40 5.0 6.0 7.0 8.0 9.0 
Average rate of decolorization (mg/(L h)) 38.78 ± 1.056* 40.34 ± 0.897* 42.41 ± 1.236* 38.54 ± 1.045* 24.61 ± 0.589* 8.17 ± 0.365* 14.31 ± 0.928* 35.38 ± 1.256* 39.58 ± 0.254* 32.76 ± 0.847* 

Decolorization of RB220 (300 mg/L) was investigated at various temperatures (26–40 °C); the maximum average rate of decolorization was 42.41 ± 1.236 mg/(L h) at 34 °C with bacterial consortium VN.1. Further increase in the temperature resulted in the decolorization rate of 24.61± 0.589 mg/(L h) at 40 °C (Table 1). Further increase in the temperature decreased the rate of decolorization because of inactivation of azoreductases or, as the temperature increases, the atoms in the enzyme molecule have greater energies and a greater tendency to move. Eventually, they acquire sufficient energy to overcome the weak interactions holding the globular protein structure together, and the deactivation that follows might be due to denaturation of many proteins at 40–50 °C. At higher temperatures (beyond 40 °C), the death rate exceeds the growth rate, which causes a net decrease in the concentration of viable bacterial cells as well as enzyme activities. Inoculum volumes play an important role in the degradation of various pollutant compounds. The average rate of decolorization (50.00 mg/(L h)) was increased with an increase in the inoculum volume up to 15 v/v (optical density 1.0 @ 600 nm = 8 × 108cells/mL); beyond that the average rate of decolorization was not affected by inoculum size (Figure 1(b)). This could be due to the scarcity of available nutrients and dye for the increased inoculum volume.

Effect of initial dye concentration

Decolorization of various RB220 concentrations exhibited distinct patterns of dye decolorization: whereas 2,500 mg/L of dye was completely mineralized within 30 h, less than 10 h was required by the VN.1 to degrade 600 mg/L of RB220 as observed from (Figure 1(c)). Moreover it was observed that cell density (A600 0.70 to A600 0.32) and rate of decolorization simultaneously decreased with an increase in dye concentration (100 mg/L to 2,500 mg/L) (Figure 1(c)). Thus, decline in decolorization efficiency at higher concentration of dye may be due to accumulation of sulfonic acid groups, Cu metal complex present in dye molecule, inadequate concentration of biomass for the uptake of higher dye concentrations, and blockage of active sites of azoreductase by dye molecules.

Effect of NaCl concentration

One of the interesting findings useful in the dye decolorization study was the resistance of the potent culture to NaCl concentration in the medium. VN.1 showed a slight decrease in decolorization with an increase in salt concentration above 70 gL−1 (Figure 1(d)). This might be possible because of the thriving effect under the elevated osmolarity; it is essential for cells to measure external osmolarity and to adjust their metabolism accordingly by maintaining osmolarity or inducing biosynthesis (Markus et al. 2003). Such kinds of acclimating experiments most often result in reduction or disappearance of the lag phase. In addition, the lower activities under high salt conditions limit cells in generating sufficient NADH, which is required as an electron donor for microbial decolorization. Controls with sterilized cells gave no significant reduction. There was no visual adsorption of dyes by the bacterial consortium.

Effect of co-substrate supplementation on the decolorization

On providing different salts of nitrate, ammonium sulfate exhibited only 21% decolorization of the dye, whereas sodium and potassium nitrate decolorized 12% and 15% of RB220 respectively within 24 h (Figure 2(a)). Likewise, in the presence of urea, tryptone, tryptose, malt extract, casein, and soyabean 4%, 65%, 75%, 52%, 56% and 50% of RB220 was decolorized, respectively. VN.1 was able to decolorize only 2% of 300 mg/L of RB220 within 24 h without any carbon or nitrogen sources, whereas on supplementing peptone and YE in a medium, decolorization was enhanced 50-fold (Figure 2(a)) within 7 h at 34 °C under static condition. However, in the absence of YE, the culture showed 100% decolorization of RB220 with 28.56 mg/(L h) average rate of decolorization; whereas VN.1 without peptone exhibited 100% decolorization with 42.86 mg/(L h) average rate of decolorization. Hence, preliminary studies revealed the obligatory requirement for a nitrogen source for metabolism of RB220. Kapdan et al. (2000) reported YE as a dual source of carbon and nitrogen in the decolorization of various dyes by mixed bacterial consortium PDW.

Figure 2

Effect of co-metabolites on dye decolorization by bacterial consortium VN.1 under static condition at 34 °C, pH 8.0. (a) Effect of various carbon sources (1 g/L) on RB220 (300 mg/L) decolorization in MSM. (b) Effect of various inorganic and organic nitrogen sources (1 g/L) on RB220 decolorization in MSM. (c) RB220 decolorization by bacterial consortium VN.1 at increasing concentration of YE in MSM. (d) RB220 decolorization by bacterial consortium VN.1 at increasing concentration of peptone in MSM.

Figure 2

Effect of co-metabolites on dye decolorization by bacterial consortium VN.1 under static condition at 34 °C, pH 8.0. (a) Effect of various carbon sources (1 g/L) on RB220 (300 mg/L) decolorization in MSM. (b) Effect of various inorganic and organic nitrogen sources (1 g/L) on RB220 decolorization in MSM. (c) RB220 decolorization by bacterial consortium VN.1 at increasing concentration of YE in MSM. (d) RB220 decolorization by bacterial consortium VN.1 at increasing concentration of peptone in MSM.

The effect of various carbon sources on RB220 decolorization was studied in MSM. When sucrose and lactose were used as carbon sources, decolorization was 15.32% and 19.36% respectively within 24 h (Figure 2(b)). Decolorization in the presence of glucose, malt extract, maltose and starch was only 16.32, 86.32, 27.23 and 14.23%, which might be due to the presence of various carbon and nitrogen sources in the medium having stimulatory or inhibitory effect on enzyme systems involved in the decolorization (Jadhav et al. 2010) and related to metabolic regulation known as catabolic repression. Further, these authors added that during such repression there is a high possibility of inhibiting the transcription of cyclic-AMP-dependent genes (due to the presence or higher concentration of glucose); a few of them might be involved in dye decolorization, encoding for azoreductase (Chang et al. 2001). VN.1 utilized RB220 as a sole source of carbon and achieved >80% decolorization up to the seventh cycle of repetitive addition of dye (Figure S5, online at http://www.iwaponline.com/wst/072/208.pdf). Rice husk, rice straw and bagasse extract, being the simplest carbohydrate utilized by VN.1, achieved complete decolorization of RB220 within 18 h (Figure 2(b)). It is thought that in the case of complex substrates such as extract of rice husk and rice straw, bacterial consortium VN.1 could convert and degrade them, producing some volatile organic acids or alcohols (such as acetic acid and ethanol), which act as electron donors and apparently induces the reductive cleavage of azo bonds.

The use of agricultural by-products (rice husk and rice straw) instead of pure substrates (peptone and beef extract) for the enhancement of the decolorization of RB220 becomes an ecofriendly and economically feasible process. Utilizing agricultural lignocellulosic waste as a supplement to assist or stimulate the degradation of industrial effluents could be a promising technology and may resolve the problem of the disposal of agro-residues (Saratale et al. 2010). In contrast, addition of carbon sources seemed to be less effective in promoting the decolorization process (Figure 2(b)), probably due to the preference of the cells in assimilating added carbon sources over using the dye compound as the carbon source.

With the increase in YE and peptone concentration (0.25–15 gL−1) efficiency of decolorization remained unaltered as observed from Figure 2(c) and 2(d). Therefore, 1 gL−1 YE and peptone concentration was used as optimal nitrogen/carbon source in MSM for further characterization of VN.1 because all carbon sources adversely affected decolorization of RB220. Organic nitrogen sources such as YE and peptone are considered essential for regeneration of NADH, which acts as electron donor in azo bond reduction (Moosvi et al. 2007; Patel et al. 2013).

Spectrum of dyes

Textile manufacturing and processing industries widely use several different synthetic dyes, each of which are structurally diverse in their composition. Thus, VN.1 was assessed for its ability to decolorize 45 different dyes. Based on obtained results it was apparent that complete decolorization was observed within 24 h, for all the dyes used in this study except reactive (Green HE8B, Blue H5G, Turkoish Blue, Blue MR), acidic (Brown 165, Brown 241), direct (Blue FF, Turkoish Blue) and dispersed (Black, Navy Blue) (Figure S6, online at http://www.iwaponline.com/wst/072/208.pdf). RB220 and acid Mehndi took less than 7 h for decolorization, which might be due to the fact that dyes act as suitable substrate for the azoreductase of VN.1. Differences found in the decolorization characteristics for the individual dyes are attributed to the dissimilarity in specificities and structures of various dyes. The presence of a hydroxyl group in the para position of the aromatic ring leads to a faster cleavage of the bond by organisms (Chizuko et al. 1981).

Decolorization of mixtures of various reactive dyes and dye wastewater

Removal of dyes from wastewater economically is an important issue for industrial effluent discharge. In India an average discharge of about 1.5 million liters of dye-contaminated effluent per day leads to chronic and acute toxicity. Synthetic dyes with diverse structures are often used in the textile processing industry, and therefore effluents from industries are markedly variable in composition. Therefore decolorization ability of mixtures of reactive dyes, direct dyes, disperse dyes and acid dyes were evaluated at final concentration of 500 mg/L. VN.1 exhibited >85% COD reduction (from ∼ 10,500 to 13,200 mg/L COD of mixture of reactive dyes) with >25 mg/(L h) rate of decolorization in the case of the reactive dye mixture. Decolorization and rate of decolorization were higher in the mixtures of reactive dyes A, B and C compared to the mixtures of acidic, direct and disperse dyes, D, E and F respectively (Figure S7, online at http://www.iwaponline.com/wst/072/208.pdf). This might be due to the molecular weight, structure and presence of an inhibitory group like –NO2 and –SO3Na in a dye molecule of acidic, direct and disperse dyes.

Toxicity assay

Improper disposal of sulfonated azo dyes and their metabolic intermediates cause serious environmental and health hazards, if they are being disposed of in water bodies and the same water is being used for an agricultural purpose. Therefore, the relative sensitivity of RB220 effluent and its degradation products was analyzed in relation to Phaseolus aureus, one of the important crops of Indian agriculture. In addition to this, microbial communities of the rhizosphere of Phaseolus aureus were also analyzed. Both phytotoxicity and microbial toxicity studies showed good germination as well as significant growth in the plumule and radical for Phaseolus aureus and growth of soil microorganisms on dye metabolites (Table 2; Figure S8, online at http://www.iwaponline.com/wst/072/208.pdf), indicating that the metabolites generated after biodegradation of RB220 are less toxic when compared to the original dye. The feature of detoxification reveals the additional advantage of using bacterial consortium VN.1 for the decolorization and biodegradation of the target dye compound.

Table 2

Toxicity towards Phaseolus aureus. Values are mean of three experiments, ± standard error of the mean, significantly different from the control (seeds germinated in distilled water) at *P < 0.05 or **P < 0.01, by one-way analysis of variance with Tukey–Kramer multiple comparisons test

  Phaseolus aureus
 
Parameter Distilled water Reactive Blue 220 Extracted metabolites 
Germination (%) 90.0 41.0 99.0 
Pumule (cm) 15.40 ± 0.73* 7.50 ± 0.16* 21.60 ± 0.39* 
Radical (cm) 7.56 ± 0.55** 2.45 ± 0.39** 10.80 ± 0.37** 
Fibrils 16.0 ± 1.47* 9.0 ± 2.10* 25.0 ± 1.18* 
  Phaseolus aureus
 
Parameter Distilled water Reactive Blue 220 Extracted metabolites 
Germination (%) 90.0 41.0 99.0 
Pumule (cm) 15.40 ± 0.73* 7.50 ± 0.16* 21.60 ± 0.39* 
Radical (cm) 7.56 ± 0.55** 2.45 ± 0.39** 10.80 ± 0.37** 
Fibrils 16.0 ± 1.47* 9.0 ± 2.10* 25.0 ± 1.18* 

*Concentration of RB220 = 1,400 ppm.

Evaluation of decolorization and biodegradation of RB220

Decolorization and biodegradation pattern of RB220 by the VN.1 under static condition was studied using UV–vis, FTIR, NMR and GC–MS. UV–vis spectral analysis (250–750 nm) showed a single peak in the visible region at 600 nm, corresponding to its λ-max, and intense peaks in the UV region near 250–325 nm, corresponding to phenyl and naphthyl rings of RB220 (Figure 3(a)) (He et al. 2004). During decolorization the π bond of conjugated chromophore in RB220 was broken down due to azoreductase activity and the peak at 600 nm gradually decreased and completely disappeared within 8 h, without any shift in λ-max. Since the azo bonds were cleaved, corresponding intermediates with phenyl and naphthyl rings having absorbance in the UV region were accumulated in the medium, supported by the FTIR, HPLC and GC-MS results clearly indicating the formation of phenyl and naphthyl rings during degradation possesses. Further, with increase in time, such compounds with naphthyl/phenyl rings were completely cleaved to aliphatic hydrocarbons; thus, absorbance/concentration decreased in the UV region. As will be discussed in subsequent results, the end products without any conjugated bonds were of lower molecular weight aliphatic hydrocarbons, free amides, and alcohols, or even completely mineralized to CO2 and H2O, which indicated the opening of all aromatic nuclei.

Figure 3

UV–vis (a) and HPLC (b) over-lay spectra of RB220 and ethyl-acetate-extracted degraded products of RB220 at varied time intervals under static condition at 34 °C by bacterial consortium VN.1.

Figure 3

UV–vis (a) and HPLC (b) over-lay spectra of RB220 and ethyl-acetate-extracted degraded products of RB220 at varied time intervals under static condition at 34 °C by bacterial consortium VN.1.

The HPLC elution profile shows the presence of a new peak with marked decrease in intensity at a different retention time when compared to the control (2.85 min) and test (major peaks at 5.14 and 5.95 min) (Figure 3(b)). HPLC fractions obtained after 5 min of elution were further analyzed with standard benzene and naphthalene, resulting in the formation/accumulation of benzyl/naphthyl derivative after 8 h of incubation, which was subsequently degraded with increasing time to a simple molecule which was utilized as a precursor of the tricarboxylic acid cycle.

The FTIR scan of RB220 effluent showed characteristic bands at 1,143.09, 1,398.88, and 1,190.14 cm−1 corresponding to the presence of two meta-substituted SO3 groups and symmetric SO2 group, respectively in RB220. N = N stretching vibration of a symmetrical trans azo group of RB220 gave an absorption band near 1,599 cm−1, whereas para-substituted azobenzene (phenyl ring) exhibits a band near 1,454.40 cm−1. RB220 is a metal-containing azo dye where carbonic ion is bonded with a central copper metal ion, giving rise to asymmetrical stretching which was observed near 6,51,626.48 cm−1. The bands near 1,261, 889 and 831 cm−1 indicate the aromatic nature of the dye whereas the band at 1,045 cm−1 indicates S = O stretching of sulfonic acids. The bands appearing at 893 cm−1 and 632 cm−1 clearly indicate the presence of 1,2,4 trisubstituted benzene, benzene ring and aromatic compound.

Cleavage of the azo bond was evidently observed by disappearance of the specific band near 1,500–1,650 cm−1. Formation of free NH group was evident as the bands at 1,689.36 and 1,113.08 cm−1 show NH stretching of amides after complete decolorization of RB220, indicating the production of primary or secondary amides. The S = O bonds of SO2 and SO3 groups were cleaved from the native structure of the dye, which was clearly observed by the absence of the bands near 1,140, 1,180 and 1,330 cm−1. Mononuclear and polynuclear aromatic rings generally absorb strongly in the low frequency range between 900 and 675 cm−1, which was apparent in IR spectra, representing the presence of phenyl and naphthyl rings of RB220 (Figure 4).

Figure 4

FTIR (left) and 1H NMR spectra (right) of (a) RB220, (b) RB220 after complete decolorization (8 h) and (c) RB220 after 48 h treatment by bacterial consortium VN.1.

Figure 4

FTIR (left) and 1H NMR spectra (right) of (a) RB220, (b) RB220 after complete decolorization (8 h) and (c) RB220 after 48 h treatment by bacterial consortium VN.1.

During degradation, decrease in intensity of bands between 900 and 675 cm−1 suggested the breakdown of aromatic rings. Correspondingly, absence of bands at 817, 763, 720 and 671 cm−1 after complete degradation of RB220 clearly demonstrated the loss of aromaticity or fission of benzene rings of the dye. Bands at 2,925 and 2,929 cm−1 in the RV5 spectrum and degraded products respectively indicated the asymmetrical stretching of C— H in CH3.

The 1H NMR spectra of metabolites extracted from RB220 effluent showed the downshift signals between 6.9 and 8.2 ppm are from hydrogen of the naphthalene and benzene rings of the dye molecule (Figure 4). After 8 h, intensity of peaks/signals gradually decreased and a couple of new peaks appeared at 7.3–7.4 and 7.8–7.9 ppm. These signals might have appeared from the protons of benzyl/naphthyl derivatives. Conversely, absence of corresponding peaks for aromatic protons in low field zone/higher frequency between 6.0 and 9.0 ppm indicated the complete degradation and mineralization of RB220. A spin network in the high field zone observed as singlet, triplet and multiple signals were from saturated/unsaturated aliphatic compounds (Figure 4). Signals in this field were almost doubled after 8 h due to unstable alkenes resulting from ring opening of naphthalene and benzene nucleus (Figure 4). But after complete decolorization, appearance of the signal intensity in the high field zone 1.0–3.0 ppm was due to formation of lower molecular weight aliphatic hydrocarbons such as free CH3, and the signal intensity increased up to 16 h. Afterwards the signal intensity of high field zone 1.0–3.0 ppm decreased and completely disappeared after 30 h. Simultaneously the signal intensity in low field zone 7.3 to 7.9 ppm increased because of the formation/accumulation of protons of benzyl/naphthyl derivatives up to 8 h, probably due to breakage of N = N bond by azoreductases of VN.1. After 16 h, intensity of peaks/signals gradually decreased and a couple of new peaks appeared. These signals might have appeared from the protons of cresol, salicylate, and other naphthyl/phenyl derivative. All signals of low/high field zone completely disappeared after 30 h (Figure 4).

To get further insight into the RB220 degradation mechanism, we have studied the GC–MS behavior of degraded products at different time intervals. RB220 being a multisulfonated, non-volatile, aromatic compound, it is difficult to obtain mass spectra of the dye. Nevertheless based on the mass spectra of degraded products, and molecular weight and chemical structure of the dye we could identify four intermediary products. The base peaks at m/z 172 and 222 were identified as sodium 4-aminobenzenesulfonate and sodium 6-aminonephthelenesulfonate. Both theses intermediates were further broken down into sodium naphthelene-2-sulfonate and sodium benzenesulfonate respectively to give corresponding peaks at m/z 207 and 157 (Figure S9, online at http://www.iwaponline.com/wst/072/208.pdf).

CONCLUSION

Even after several decades of research, we are still in search of eco-friendly bioremediation approaches. In that view, the developed VN.1 possesses great efficiency to degrade a wide array of dyes under ambient conditions with minimal nutritional requirement. The efficiency of VN.1 to degrade mixtures of various dyes indicated its useful application in ex situ bioremediation of colored wastewater. Azoreductase would be good material for further research on the enzymological mechanism of dye decolorization. The synergy of results obtained has forced us to study further the treatment of industrial wastewaters using appropriate bioreactor systems by VN.1.

ACKNOWLEDGEMENT

The authors thank Ganesh Dye Chem Industry, Vatva, Ahmedabad, India, for providing the dyes and wastewater used in the study.

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Supplementary data