The isolated Enterococcus faecalis YZ 66 was able to decolorize DR 81 within 1.5 h at a dye concentration of 50 mg/L. UV visible scan of the culture supernatant withdrawn at different time intervals indicated the decolorization and decrease in dye concentration from batch culture. Peak obtained at 511 nm disappeared after complete decolorization. The absorbance peak in the visible region disappeared indicating complete decolorization. In the UV spectra, the peak at 511 nm was replaced by new peak at 240 nm (Additional file 1: Figure S1). The absorbance peaks in the visible region disappeared indicating complete decolorization . Decolorization with respect to time showed complete decolorization of the dye in 1.5 hours. There was proportionate increase in wet weight indicating growth of E. faecalis in the presence of dye (Additional file 1: Figure S2). There was no abiotic loss of DR 81 within 24 h incubation indicating that the decolorization of DR 81 was due to biological mechanism rather than adsorption. To confirm whether this decolorization is due to the variation in pH, change in pH was recorded which is in the range of 7.0 ± 0.2.
Effect of physicochemical conditions on the decolorization performance
The effect of various physiochemical conditions such as pH, temperature, dye concentration, carbon and nitrogen sources on decolorization of DR 81 by E. faecalis was studied in detail. All parameters were studied at 37°C under static condition. 10% inoculum A600 1.0 was used at a dye concentration 50 mg/L.
Effect of pH
It was observed that pH of the media, affects the colour of the solution and the solubility of the dye and the enzymatic activity related to decolorization is also dependent on the pH. Generally bacterial cultures exhibit maximum decolorization at pH values near 7 or slightly alkaline pH values and the rate of colour removal tends to decrease rapidly at strongly acidic or slightly alkaline pH values ,. E. faecalis showed complete decolorization of DR 81 at pH 7.0 within 1.5 h. It showed decolorization in the pH range of 5–8 while at pH 3 and 4 (about 40%) and at pH 9 and 10 about (about 30%) decolorization was observed after 24 hour of incubation (Additional file 1: Figure S3). Similar results were observed in Micrococcus sp. in the decolorization of 300 mg/L of Orange MR .
Effect of temperature
Pearce et al., (2003)  reported that the rate of colour removal increases with increasing temperature within a defined range that depends upon the system. The temperature required to produce the maximum rate of colour removal tends to correspond with the optimum cell culture growth temperature which is in the range of 35-45°C. Temperature affects microbial growth, enzymes production and consequently, the percentage of decolouration. It was reported by Mathew and Madamwar, 2004  that various microorganisms showed their survival at various temperatures ranging from 25-50°C. The decline in colour removal activity at higher temperature can be attributed to the loss of cell viability or to the denaturation of azoreductase enzyme. However, it has been shown that with certain whole bacterial cell preparation, azo reductase enzyme is relatively thermostable and can remain active up to temperature of 60°C over short period of time . E. faecalis YZ 66 decolorized the dye under study in the range of 96-99% within a temperature of 30-40°C. At 30°C, 99.35% decolorization was observed while at 40°C, 98.54% decolorization was seen, thus showing negligible difference in percent decolorization at both the temperatures (Additional file 1: Figure S3). At 45°C and 50°C, 17.85 and 14.81% decolorization was observed, respectively (Additional file 1: Figure S3). Similar results was observed in Pseudomonas aeruginosa degrades 97% of Remazol Red (50 mg/L) at 40°C, 72% at 10°C and 82% at 30°C, respectively .
Effect of initial dye concentration
Decolorization of different initial concentrations of the dye from 50–700 mg/L was studied under static anoxic condition. Rate of decolorization of dye increased with increase in concentration of the dye up to 300 mg/L but the time required for decolorization was more. The E. faecalis showed faster decolorizing ability up to 300 mg/L after which the rate of decolorization falls decreasing (Figure 1). Fifty four hours are required to decolourize 85.74% of the dye at 500 mg/L concentration. The activity was lower at dye concentration 600 mg/L and above which decolorization was strongly inhibited at dye concentration at 700 mg/L (Figure 1). It has been proposed that dye concentration can influence the efficiency of microbial decolorization through combination of factors imposed by dye at high dye concentration . Similar results were observed in Lysinibacillus sp. (for Metanil Yellow) , Sphingomonas paucimobilis (for Methyl Red) , and in Lysinibacillus sp. RGS (for Remazol Red) .
Effect of supplementation of carbon and nitrogen sources on the decolorization performance
Dyes are deficient in carbon and thus biodegradation without supplying extra carbon or nitrogen source is very difficult . Carbon and nitrogen sources have an important influence on the extent of decolouration using microorganisms. In order to enhance the decolorization performance of the DR-81, an extra carbon and nitrogen source was supplied in semi synthetic medium. There was no decolorization observed in semi synthetic medium. In the presence of lactose 98.12% decolourization was observed followed by 96.16, 95.25, 95.61 and 93.76% in the presence of meat extract, peptone, glucose and starch, respectively while less decolourization with other supplements of carbon and nitrogen source within 24 h of incubation (Additional file 1: Figure S4). In addition, supplying urea as a nitrogen source did not enhance decolorizing ability. Different microbial metabolic characteristics lead to differences in the uptake of sources, thus affecting azo dye decolorization. Addition of carbon source found to be less effective to promote the decolorization performance probably due to the preference of the cells in assimilating the added carbon sources over using the dye compound as the carbon source ,. Nitrogen sources are found important for microbial decolouration since it was observed that this source is essential for the regeneration of NADH ,.
Decolorization with repeated addition of dye aliquots
An ability of E. faecalis YZ 66 to decolorize repeated addition of DR 81 dye aliquot (50 mg/L) was studied under static condition. The isolate have an ability to decolorize 100% dye up to seventh aliquot and after that subsequent cycle showed no decolorization (Additional file 1: Figure S5). The eventual cessation of decolorization was likely due to nutrient depletion . Thus E. faecalis YZ 66 showed the ability to decolorize repeated addition of the dye aliquots is noteworthy for its commercial application.
Enzymes involved in dye decolorization
The use of microbial techniques to deal with pollution is a key research area in the environmental sciences. In these processes microbes acclimatize themselves to the toxic wastes and resistant strains develop naturally, which then transform various toxic chemicals into less harmful forms. The mechanism behind the biodegradation of recalcitrant compounds (azo dyes) in the microbial system is based on the action of the biotransformation enzymes . Besides uptake, the presence and activity of a network of detoxification enzymes is crucial for the metabolism and eventually the degradation of chemicals. To understand the decolorization mechanism, enzyme activities of laccase, lignin peroxidase, NADH-DCIP reductase, and azo reductase were monitored over time. The enhanced activities of enzymes were noted in induced cells (after decolorization) (Figure 2). The enzymatic profile presumably indicates communal action of oxidoreductive enzymes for the degradation of DR 81 into simple metabolites by E. faecalis (Figure 2). No enzyme activities were observed in cell free supernatant. The role of oxidoreductive enzymes in the decolorization of azo dyes have been characterized in various bacteria are well documented in recent reviews ,.
Decolorization studies of dye wastewater by E. faecalis
Most of the microbial decolorization studies in several laboratories showed the ability of bacteria, fungi, and algae in removing the color of textile dyes, but they do not find much application in treatment system for industrial effluent because of heterogeneity of the components in effluent depending upon production schedule. However, it is very important to test decolorization in real textile effluents, which are complex systems having strong colors, large amounts of suspended solids, broadly fluctuating pHs, high temperatures, high COD and high salt concentrations that can be inhibitory to microorganisms . Considering this perspective we have checked the efficiency of E. faecalis to decolorize actual textile wastewater. The true color of textile wastewater measured by using ADMI 3WL suggesting that E. faecalis could achieve higher color removal value (52%) with moderate reduction in COD (about 42%) and BOD (about 48%) after 10 days of incubation (Figure 3). Decolorization performance of dye wastewater by E. faecalis is comparable with Citrobacter sp. strain KCTC 18061P strain removed 70% of effluent color within 5 days with 35% COD reduction . Untreated dye effluents cause serious environmental and health hazards whereas in aqueous ecosystems is aesthetically unpleasant and leads to a reduction in sunlight penetration, dissolved oxygen concentration and had acute toxic effects on aquatic flora and fauna. This study is of particular relevance since the Panchganga river and Ichalkaranji area near Kolhapur, India are heavily industrialized, with significant wastewater discharge from textile and dye manufacturing industries which causes the harmful impacts to the environment. Our strain E. faecalis showed better colour removal of actual dye wastewater with significant reduction in COD and could be a potential strain for the treatment of textile dyestuffs and textile and dye industry effluent via appropriate bioreactor operations and will be useful to small textile industries in an ecoefficient and economically feasible that could effectively decolorize and detoxify dye containing wastewater.
Analysis of metabolites resulting from decolorization
To understand and confirm the possible mechanism of dye decolorization, analysis of products of biodegradation of DR 81 were studied by TLC, HPLC, FTIR and GC-MS. TLC analysis showed the appearance of one spot in the sample containing the extracted metabolites of completely decolorized medium with Rf value 0.71 where as Rf value of DR 81 was noted as 0.97 confirming the biodegradation of DR 81 by E.faecalis YZ 66. HPLC elution profile of DR81 showed a distinct single peak at retention time of 1.71. min. Three peaks at retention time of 3.008, 3.861 and 4.021 min were showed that the degradation of DR 81 into different products by E. faecalis YZ 66. Disappearance of a distinct peak of DR81 confirmed the degradation of the dye. HPLC analysis of metabolites formed after biodegradation of DR 81 showed the peaks with different retention times than the original dye which indicates the biodegradation of DR 81 into different metabolites (Figure 4A and B).
The FTIR spectrum of a control dye and metabolites was compared. The spectrum of the control dye displayed a peak at 3789.44 cm−1 and 3491.49 cm−1 N-H stretching. The peak at 1658.48 cm−1 represents N = N symmetric stretch. A peak at 1563.99 cm−1 represents N-H bending. A peak at 1224.58 cm−1 represents C-O stretch band of phenol. The peaks at 1121.4 and 1057.76 cm−1 represents C-N stretch along with O = S = O symmetric stretch. The peaks at 616, 712 and 852.382 cm−1 represents C-H of substituted aromatics. FTIR spectrum of metabolites obtained after decolorization showed peaks at 3994.35 cm−1 and 3690.54 cm−1 represents phenolic –OH group, 3054.53 cm−1 represents = C-H stretch, 2987.28 cm−1 showed –C-H stretch and 1265.37 cm−1 represented –C-O stretching vibrations (Figure 5 A and B).
To verify the degradation products formed during dye decolorization by E. faecalis, GC–MS analysis was carried out. The low molecular weight aromatic compounds were produced from the degradation of Direct Red 81 by E. faecalis. Accordingly, the pathway for the degradation of Direct Red 81 is proposed as depicted in Figure 6, showing various steps involved in the degradation mechanism. However, very little is known about the nature of the degradation products formed in these reactions (Table 1) and the reaction mechanism about oxidoreductive enzymes. We propose that initially primary reductive cleavage in azo bond of Direct Red 81 results in the product such as, sodium-4-aminobenzenesulfonate, 1,4-benzenediamine and 7-benzylamino-3-dibenzyl-1-4-hydroxy naphthalene-2-sulfonic acid. Further deamination of sodium-4-aminobenzenesulfonate results into sodium benzenesulfonate with a mass peak of 178. Whereas the asymmetric cleavage of product 7-benzylamino-3-dibenzyl-1-4-hydroxy naphthalene-2-sulfonic acid by oxidative enzymes (laccase) resulted in the formation of 1-phenylmethanamine-ethene and 8-aminonaphthol as a products. Further deamination reaction resulting in the formation of low molecular weight compound such as naphthalene as a final product (Figure 6). Therefore, analytical studies confirmed the biodegradation of Direct Red 81 dye, in which the smaller molecular weight intermediates are formed by the consecutive action of oxidoreductive enzymes present in E. faecalis.
Untreated or partially treated effluent may be disposed off in the water bodies and this water can be used for irrigation purpose. Thus it was found necessary to study phytotoxicity of the dye before and after degradation. The relative sensitivities towards the dye DR 81 and its degradation products in relation to Sorghum vulgare and Phaseolus mungo seeds were represented in the Table 2. There was no significant difference in the root and shoot length in case of the selected plants irrigated with the dye but in case of metabolites irrigated selected plants root and shoot length was significantly increased (P ≤ 0.05) as compared to control. Phytotoxicity study showed good germination rate as well as significant growth in the plumule and radical for both the plants (P ≤ 0.05) in the metabolites extracted after decolorization as compared to dye sample. This indicates the detoxification of DR 81 by E. faecalis. Hence this indigenous bacterial strain could be a good biocatalyst for the treatment of textile dyes and effluent containing dyes.