Introduction

Increased demand of dyestuffs and dye-based products has resulted in proliferation of large number of synthetic dye consuming and manufacturing industries (Pearce et al. 2003). Among different chemical colorants, azo dyes are extensively being used by various dyes and printing-based industries because of their ease of production, wide availability and occurrence in different colors (Zhao et al. 2014). This vast usage results in widespread distribution of these toxic dyes in the environment through industrial effluents (Pearce et al. 2003; Ayed et al. 2011; Solís et al. 2012).

The discharge of untreated or poorly treated colored dye-based effluent into the pristine water bodies like pond or river results in alteration of their various ecological functions. For instance, the presence of color in the water bodies reduces the light penetration efficiency thus inhibiting the photosynthetic activity of phytoplanktons (Asad et al. 2007; Amoozegar et al. 2011). It ultimately causes disturbance of entire aquatic ecosystem. In addition to that, azo dyes or their degraded products are considered as toxic, carcinogenic, and mutagenic in nature and hence possess serious health threat to aquatic and other life-forms (Cui et al. 2012; Yan et al. 2012). Acknowledging above facts, decolorization is an essential requirement before discharging colored dye-based effluent into natural environment (Prasad and Rao 2013).

Major environmental concern regarding azo dyes is their recalcitrant nature making it difficult to degrade easily because of the presence of one or more azo-groups (–N=N–) (Ayed et al. 2011; Zhou et al. 2007). This is the group which is also responsible for the color of the dye (Adedayo et al. 2004). This necessitates requirement of special physico-chemical methods like photooxidation, ozonation, and chemical oxidation for degradation of such dyes (Pearce et al. 2003). The degradation of dye will also lead to color removal thus solving the major environmental problem. However, these methods are un-sustainable in nature as they are less efficient, costly, and generate secondary pollutants (Pearce et al. 2003; Garg et al. 2012). Microbial degradation of dyes or bioremediation is one of the sustainable alternatives and has gain tremendous importance over physico-chemical methods (Prasad and Rao 2013).

Textile industries are one of the notable consumers of azo dyes and generators of contaminated effluents (Solís et al. 2012). Textile effluents are generally characterized by presence of high salt and alkaline pH along with high color, COD, and BOD content (Prasad and Rao 2013; Maier et al. 2004; Khalid et al. 2012). Alkaline pH and high salinity serve as major bottlenecks for successful application of microbes in textile wastewater treatment. High pH drastically affects the growth and metabolic functions of microbial cells, while presence of salt residues particularly sodium ions results in plasmolysis of cells (Amoozegar et al. 2011; Khalid et al. 2012; Yu et al. 2015). Hence, for effective bioremediation of aforementioned effluent, there is usually a prerequisite of specialized alkali and halophilic/halotolerant azo dye-degrading microorganisms. These extremophilic classes of microorganisms are metabolically diverse and have necessary adaptive features that help them to sustain and perform efficiently under harsh conditions of textile and other similar effluents.

In the present study, a newly isolated halotolerant and alkaliphilic Nesterenkonia lacusekhoensis EMLA3 was evaluated for an azo dye, methyl red (MR) degradation under alkaline conditions. Various researchers have reported degradation of azo dyes including MR in the presence of high salt content using different halotolerant or halophilic microbial strains (Asad et al. 2007; Amoozegar et al. 2011; Yu et al. 2015). However, there are limited reports on degradation of azo dyes under alkaline pH and in combination of both. The author could find only report of Prasad and Rao (2013), in which obligate alkaliphilic Bacillus cohnii MTCC 3616 was used for removal of azo dye in the presence of NaCl. To best of our knowledge, present study is the first report on degradation of azo dye using Nesterenkonia lacusekhoensis in the presence of high alkaline pH, high salinity, and toxic metals.

Materials and Methods

Material

Methyl red was obtained from Central Drug House (New Delhi, India). Media components were procured from Hi Media Laboratories Pvt. Ltd (Mumbai, India). All chemicals used were of high purity and analytical grade.

Preparation of methyl red (MR) solution

Stock solution of MR (5000 mg L−1) was prepared by dissolving 0.5 g MR in 100 mL of absolute ethanol and stored in amber glass bottle.

Microorganism

The strain EMLA3 used in the present study was isolated from highly alkaline textile effluent sample (pH 13.0) collected from local textile industry, National Capital Region (NCR), Delhi, India. The strain EMLA3 was identified using phenotypic studies (physiological and biochemical characteristics) as well as using 16 S rRNA sequencing from Microbial Type Culture Collection Facility (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India.

Strain EMLA3 was maintained on nutrient agar slants containing MR (pH 11.5) at 4 °C and subcultured every 20 days.

MR degradation studies

Mother culture preparation

Mother culture was prepared by transferring a loop full of stock culture of strain EMLA3 in the nutrient media (pH 11.5), followed by incubation at 30 °C and 200 rpm in an orbital shaker (Innova, Burswick, USA). The overnight-grown culture of strain was used for dye removal studies.

Media and culture conditions

Two types of culture media (A) Nutrient media (NB) containing (g L−1): Peptone, 5.0; NaCl, 5.0; Yeast extract, 1.5; Beef extract, 1.5 and (B) Mineral salt media (MSM) containing (g L−1): NaCl, 0.5; (NH4)2SO4, 1.0; K2HPO4.3H2O, 1.5; KH2PO4, 0.5; MgSO4.7H20, 0.2 both adjusted to initial pH of 11.5 and supplemented with required amount of MR were used to study the dye removal efficiency of strain EMLA3.

One milliliter (v/v) of mother culture of strain EMLA3 was inoculated to 250-mL Erlenmeyer flasks containing 50 mL of NB media having initial 50 mg L− 1 of MR dye. In another set of experiment in which MSM was used as basal media, 3.0 mL (v/v) mother culture was first centrifuged at 4752×g for 10 min and the resultant pellet was subsequently suspended in saline (0.8%, w/v, NaCl) and vortexed for one min. One milliliter (v/v) of above cell suspension was next inoculated to 250-mL Erlenmeyer flasks containing 50 mL MSM media having initial 50 mg L−1 of MR dye. All the flasks were incubated at 30 °C and 200 rpm for 72 h (Innova, Burnswick, USA). Aliquots of samples were withdrawn after 0, 16, 36, 64, and 72 h followed by centrifugation at 4752×g for 10 min to pellet the cell mass. Resultant supernatants thus obtained were scanned in the region of 350 to 700 nm for dye decolorization/removal using UV–Vis spectrophotometer (TechComp, Shanghai, China). Un-inoculated MR containing media and media inoculated with autoclaved cells of the strain EMLA3 were set as control setups.

The degradation percentage of MR was calculated using the following formula (Jadhav et al. 2008):

$$\text{Degradation }\,\text{percentage }\,(\%)= \frac{\left( {{A}_{\text{i}}}-{{A}_{\text{f}}} \right)}{{{A}_{\text{i}}}}\times 100$$

where A i is initial absorbance, A f is the final absorbance.

Gas chromatography and mass spectrometry (GC–MS)

Metabolites formed after degradation of MR were identified using (GC–MS). After decolorization of initial 50 mg L−1 of MR by EMLA3, the sample was centrifuged (9503×g for 10 min) and resultant cell-free supernatant was extracted thrice with equal volume of ice-cold ethyl acetate. This was followed by air-drying of pooled organic layer and subsequent injection of this concentrated sample into GC-MS (Shimadzu QP-2010, Japan) under split mode. In GC, the column temperature was initially maintained at 60 °C for 3 min, and then finally increased to 280 °C at the rate of 10 °C min−1. The metabolites were identified using inbuilt standard mass spectra NIST (National Institute of Standards and Technology) and Wiley libraries.

Attenuated total reflectance: Fourier transform infrared spectroscopy (ATR-FTIR) analysis

For identification of functional groups of MR metabolites, FTIR spectrum (4000–400 cm−1) of cell-free aqueous samples before and after degradation of 50 mg L−1 MR was obtained using ATR-FTIR spectrometer (Perkin Elmer, USA).

Effect of varying dye concentrations on dye (MR) degradation

The effect of varying MR dye concentrations was studied to find out maximum dye degrading efficiency of strain. The flasks containing different concentrations of MR (50, 100, 200, 400 mg L−1) were inoculated with EMLA3 strain followed by incubation at 30 °C and 200 rpm for 72 h in an orbital shaker. Aliquots of samples were periodically withdrawn (0, 16, 24, 40, 48, 64, 72 h) and analyzed for growth at 660 nm and dye removal at 430 nm (ʎmax of MR) (Adedayo et al. 2004).

Effect of different culture conditions, salinity, and heavy metals on MR decolorization

To characterize the MR degrading efficiency of strain EMLA3, the effects of initial pH (8.0, 10.0, 11.0, 11.5, 12.0, 13.0), incubation temperature (15, 25, 30, 35, 45, 50 °C), agitation/shaking speed (0, 50, 100, 200, 250 rpm), salinity (5, 20, 40, 60, 80, 100, 120, 140, 160, 200; g L−1 NaCl), and heavy metals (1.0 mM of Cu (II), Cr (VI), Ni (II) and 0.2 mM of Hg (II)) were individually monitored on MR removal. The aforementioned parameters were studied in basal media containing 100 mg L−1 of MR. Culture samples were withdrawn after 24 and 48 h of incubation, while in case of effect of salinity 72 h samples were also analyzed for dye decolorization. All the withdrawn samples were first centrifuged at 4752×g for 10 min and resultant supernatants assessed for dye removal at 430 nm.

Application of strain EMLA3 in MR removal from textile wastewater

To show the potential of strain EMLA3 for textile effluents decolorization, the bacterium was first tested for degradation of MR in real wastewater. The real textile wastewater (pH 13.0; salinity: 365 mg L−1; COD: 850 mg L−1, Cu: 0.35 mg L−1; Fe 2.0 mg L−1; Ni and Cr (VI): below detection limit; color: light reddish) was collected from a local textile industry, National Capital Region (NCR), Delhi, India. Due to low color intensity or dye content, the real wastewater was extraneous amended with 50 mg L− 1 of MR and its degradation using indigenous strain EMLA3 was monitored with course of time.

For inoculation, 30 mL of overnight-grown mother culture of strain EMLA3 in NB (pH 11.5) was centrifuged at 4752×g for 20 min. The resultant pellet was dissolved in 1.0 mL of wastewater sample and used as inoculum for the treatment. Strain EMLA3 was then inoculated to 50 mL of above-described wastewater sample taken in 250-mL Erlenmeyer flask. Similar setup without any exogenous inoculation of strain EMLA3 was set as control. Both experimental and control flasks were kept in static condition at room temperature for 96 h. Samples were withdrawn at every 24-h time interval and centrifuged at 4752×g for 10 min and residual MR was determined as described earlier.

However, since the above wastewater was found to contain low salinity level, color intensity, and toxic metal content; final detailed studies were conducted with simulated wastewater laden with alkaline pH, high salinity, toxic metals, and dye content. The simulated wastewater having characteristics: pH 11.5; salinity (NaCl): 20 g L−1; COD: 16,300 ± 200 mg L−1; Cu (II);.05 mM; Ni (II) 0.05 mM; Cr (VI) 0.05 mM; MR 50 mg L−1 was prepared in the laboratory using un-sterilized tap water.

The inoculum was prepared as described above. The wastewater treatment was carried out under two sets of experiments. In first set, 1.0 mL of inoculum was seeded to 50 mL of wastewater sample taken in 250-mL Erlenmeyer flask followed by incubation at room temperature for 120 h under static condition. While in another set of experiment, after inoculation of sample as before, the flask was kept under shaking conditions at 30 °C and 200 rpm in an incubator shaker for 120 h. Respective control setups without any exogenous inoculation of strain EMLA3 were also run simultaneously with the test samples. Aliquots of samples were withdrawn at every 24-h time interval and centrifuged at 4752×g for 10 min for estimation of residual dye. Chemical oxygen demand (COD) of the wastewater was determined using standard APHA method (APHA 1998) after the treatment.

Statistical analysis

Experiments were done at least two times and the mean values reported. The errors bars shown in figures represent standard deviations calculated using MS-Excel and the value is within ±4%.

Results

Identification of strain EMLA3

The EMLA3 is a Gram-positive, halotolerant, alkaliphilic bacterium and was identified as Nesterenkonia sp. and showed 99.21% similarity with N. lacusekhoensis based on 16 S rRNA sequencing. The strain was deposited at Microbial Type Culture Collection Facility (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India with an accession number of MTCC 12340. The 16 S rRNA sequence of the strain was also deposited at NCBI GenBank with accession number KY515296. The detailed morphological and genotypic characteristics of the bacterium are presented in Table 1(Supplementary). The bacterium was isolated from highly alkaline textile effluent (pH 13.0; salinity: 365 mg L−1; COD: 850 mg L−1; Cu: 0.35 mg L−1; Fe: 2.0 mg L− 1; Ni and Cr (VI): below detection limit).

Methyl red (MR) degradation by N. lacusekhoensis EMLA3

N. lacusekhoensis EMLA3 showed significant dye degradation in both types of culture medium (NB-nutrient broth and MSM—mineral salt medium—pH 11.5) containing initial 50 mg L−1 of MR. However, Fig. 1 shows that faster MR degradation was observed in NB media compared to MSM, since strain EMLA3 lowered the initial dye content of 50 mg L−1 to 1.2 mg L−1 in 16 h with NB, whereas, it took 72 h to reduce the dye amount to <7.5 mg L−1 in MSM (Fig. 1). No dye removal was observed in control setups (un-inoculated media and media inoculated with autoclaved cells). Henceforth, due to better dye degradation, NB media was selected as basal culture media for further studies.

Fig. 1
figure 1

Methyl red (MR) removal by strain EMLA3 from nutrient-rich and mineral salt medium. Strain EMLA3 was individually grown in nutrient medium (NB) and mineral salt medium (MSM) containing 50 mg L−1 MR, initial pH 11.5 and incubated at 30 °C and 200 rpm. Un-inoculated medium was used as control

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The dye degradation by N. lacusekhoensis EMLA3 was further supported by the formation of lower molecular weight intermediate metabolites (Benzeneacetamide, MW-179; Acetophenone, MW-120) as revealed through GC-MS (Supplementary Figures S1a, S1b, S1c). FTIR analysis of aqueous extract of MR before and after degradation with EMLA3 is presented in Fig. 2. Compared to control (un-degraded sample), the degraded sample showed appearance of new peaks at 2925 and 2854 cm−1. Another major peak at 1747 cm−1 was also evident in case of EMLA3-treated sample.

Fig. 2
figure 2

FTIR spectra of untreated (control) methyl red sample (a) and EMLA3-treated methyl red sample (b)

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Effect of varying dye concentrations

Figure 3a represents the effects of varying MR concentrations on MR degradation. N. lacusekhoensis EMLA3 was able to decolorize >97% of 50, 100, and 200 mg L−1 dye concentrations at 16, 48, and 72 h, respectively. Figure 3b shows the growth profile of the bacterium at aforementioned MR concentrations. With increase in concentrations of MR, growth inhibition was observed. At 400 mg L−1 MR concentration, no growth and MR degradation was detected up to one week of incubation tested.

Fig. 3
figure 3

Effect of varying dye concentrations on growth (a) and MR degradation (b). The strain EMLA3 was separately grown in nutrient medium (pH—11.5) containing varying concentration of MR dye and incubated at 30 °C and 200 rpm. Samples were withdrawn during incubation for determination of growth and residual dye

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Since isolate EMLA3 showed faster degradation with higher inoculum sizes (data not shown), henceforward, further studies were carried out with higher inoculum size of 8% (v/v).

Effect of culture conditions on MR degradation

Effect of initial pH of the medium

The optimum pH range for N. lacusekhoensis EMLA3 strain was found to be 8.0–11.5 for MR removal (Fig. 4a), as it showed >97% degradation at an initial pH of 8.0, 10, 11.0, and 11.5 after 24 h. However, good degradation efficiency of 30 and 72% was detected at pH 12.0 also after 24 and 48 h of incubation, respectively, while no prominent dye removal was observed at an initial pH of 13.0 up to 48 h of incubation, tested. Unless otherwise mentioned, no shifting of ʎmax of MR and dye removal was observed at tested pH values in control set ups. A decrease in initial pH of the media was noticed after MR degradation with a drop in initial pH of 8.0 to 5.0, 10.0 to 7.75, pH 11.0 to 8.36, and pH 12.0 to 10.68. No change in initial pH was observed in case of media adjusted to initial pH 13.0.

Fig. 4
figure 4

Effect of different culture conditions on MR degradation by Nesterenkonia lacusekhoensis EMLA3. a Effect of initial pH. EMLA3 cells (8%, v/v) were inoculated to nutrient medium containing 100 mg L−1 MR of varying initial pH followed by incubation at 30 °C and 200 rpm. b Effect of incubation temperature. Different flasks having 100 mg L−1 MR containing nutrient medium (pH 11.5) were inoculated with 8% (v/v) of EMLA3 cells and incubated at different incubation temperatures under constant shaking of 200 rpm. c Effect of agitation speed. Nutrient medium (pH 11.5) containing initial 100 mg L−1 MR were inoculated with 8% (v/v) of EMLA3 cells and incubated at 30 °C under static condition and different agitation speeds

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Effect of temperature

Figure 4b shows the effect of different incubation temperatures on MR removal by N. lacusekhoensis EMLA3. Optimum temperature for MR degradation by EMLA3 strain was observed in the range of 30–35 °C. The bacterium showed 97 and 96% reduction of initial dye content at 30 and 35 °C, respectively, at 24 h of incubation. At 25 and 45 °C, initially 63 and 78% dye reduction was determined, respectively, at 24 h which further increased to 96 and 82% after 48 h of incubation, respectively. On the other hand, no change in initial dye content could be detected at 15 and 50 °C temperature up to 48 h of incubation tested.

Effect of agitation speed

Maximum MR decolorization reaching 97% was obtained at 100 and 200 rpm agitation speed at 24 h (Fig. 4c). However, static condition also allowed considerable level of dye reduction by the strain, where, 85 and 98% removal was observed after 24 and 48 h incubation, respectively.

Effect of salinity and heavy metals on MR decolorization

Effects of salinity and heavy metals on MR removal were also monitored since these are usually present in real textile wastewater (Khalid et al. 2012; Zhao et al. 2014).

Effect of salinity

Figure 5a shows the effect of salinity on dye removal by N. lacusekhoensis EMLA3. Presence of salinity in textile effluent is one of the major problems encountered during microbial application, as salts hamper the growth and activity of microbes (Solís et al. 2012). EMLA3 strain was capable of degrading up to 96% MR at 48 h in the presence of 5.0, 20, 40, and 60 g L−1 NaCl. Whereas, at higher salt concentration of 80 and 100 g L−1, 60 and 48% degradation was detected, respectively at 48 h, which further increased to 78 and 64%, respectively, at 72 h of incubation. In addition, isolate EMLA3 showed 45, 32, 27, and 11% reduction of dye content at 72 h in the presence of 120, 140, 160, and 200 g L−1 NaCl, in that order (Supplementary Figure S2).

Fig. 5
figure 5

a Effect of salinity on MR degradation by Nesterenkonia lacusekhoensis EMLA3. EMLA3 cells (8% v/v) were grown in nutrient medium (pH 11.5) in the presence of different salinity level and incubated at 30 °C and 200 rpm. MR concentration, 100 mg L−1. b Effect of heavy metals on MR degradation by Nesterenkonia lacusekhoensis EMLA3. EMLA3 cells (8% v/v) were grown in nutrient medium (pH 11.5) in the presence of different heavy metals and incubated at 30 °C and 200 rpm. MR concentration, 100 mg L−1. Control is in the absence of heavy metals

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Effect of heavy metal

Since some textile effluents are known to be harbored with heavy metals due to use of various metal-based dyes as coloring agents (Halimoon and Yin 2010), it was worthwhile to see the effects of various commonly occurring heavy metals on dye removal efficiency of strain EMLA3. In the presence of Ni (II), Cr (VI), and Hg (II), the strain exhibited 86, 91, and 96% MR removal, respectively at 48 h (Fig. 5b). On the other hand, in the presence of Cu (II), only 26% removal of MR was observed at 48 h. Control setup grown in the absence of any heavy metals showed 97% degradation at 24 and 48 h.

Application of Nesterenkonia lacusekhoensis EMLA3 in decolorization of wastewater

Applicability of N. lacusekhoensis EMLA3 strain in removal of dye from real and simulated wastewater was tested to prove its relevance in textile wastewater treatment. In real wastewater, strain EMLA3 showed 34, 68, 85, and 87% removal of dye at 24, 48, 72, and 96 h of treatment, respectively. Control setup on the other hand showed no significant decolorization at the same time period compared to test strain (Fig. 6a). The final pH of EMLA3 treated real wastewater drop-down to 8.80 from initial pH of 13.0 after 96 h of treatment.

Fig. 6
figure 6

a Application of Nesterenkonia lacusekhoensis EMLA3 in decolorization of real textile effluent. EMLA3 cells were inoculated to real textile effluent (pH 13.0; salinity: 365 mg L−1; COD: 850 mg L−1, Cu: 0.35 mg L−1; Fe 2.0 mg L−1; Ni and Cr (VI): below detection limit; MR 50 mg L−1) followed by treatment at room temperature under static condition. Samples were withdrawn periodically during treatment for determination of residual dye. b Application of Nesterenkonia lacusekhoensis EMLA3 in decolorization of simulated textile effluent. EMLA3 cells were inoculated to effluent (pH 11.5; salinity: 20 g L−1; COD: 16,300 ± 200 mg L−1; Cu (II);.05 mM; Ni (II) 0.05 mM; Cr (VI) 0.05 mM; MR 50 mg L−1) followed by incubation at room temperature under static and shaking (200 rpm) conditions. During treatment samples were withdrawn periodically for determination of remaining dye

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Since N. lacusekhoensis EMLA3 is capable of growing in alkaline and NaCl-rich environment, thus it was also used for removal of MR in alkaline simulated wastewater containing high salt and toxic metal contents under static as well as shaking conditions. With increasing treatment time, the decrease in peak intensity (ʎ max = 430 nm) was observed in case of test sample inoculated with strain EMLA3 under static condition. At respective time period of 24, 48, 72, 96, and 120 h degradation percentages of 34, 60, 68, 80, and 83% were observed based on decrease in peak intensity or absorbance (Fig. 6b). On the other hand, the test sample which was kept under shaking, only 30% degradation of MR could be observed after 120 h of wastewater treatment (Fig. 6b). In this case, initially no significant removal was found during first 48 h of treatment; however, further increment of time resulted in 5, 18, and 30% dye removal at 72, 96, and 120 h, respectively. Control setups (un-inoculated samples) placed under both the conditions showed no prominent change in initial dye content as observed up to 120 h of treatment.

Considerable dye removal from wastewater by strain EML3 under static mode encouraged us to further test the sample for initial COD reduction. Overall, 49% reduction of initial COD content (16,300 mg L−1) was found after 120 h treatment of the sample under static condition. However, the same sample when placed further for sequential treatment under shaking (200 rpm) for three days, COD reduction value increased to 76%. Un-inoculated control setup showed no reduction of initial COD after 120 h of static treatment, while 27% reduction of initial COD content was observed when the same sample was set up for sequential treatment under shaking condition. Change in initial pH from 11.50 to 8.60 was observed after final treatment of wastewater sample with EMLA3. Control setup on the other hand also showed decrease in initial pH value to 9.20.

Discussion

To get a microbial isolate endowed with intrinsic property of high pH tolerance and dye degradation capability, strain EMLA3 identified as Nesterenkonia lacusekhoensis was isolated from highly alkaline textile effluent. Collins et al. (2002) first time reported the identification of Nesterenkonia lacusekhoensis. After reporting, this bacterium was not explored much for different industrial and environmental applications as observed through extensive literature survey. The present study thus is an attempt to fill this lacuna by showing application of this halotolerant and alkaliphilic bacterium in degrading toxic azo dye.

Initially the strain was checked for degradation of MR in MSM and NB to see the nutritional requirements. Although the faster degradation was observed in NB, there was considerable color removal in MSM also. The rapid dye removal in NB could be due to easy availability of electron donors or redox mediators in the form of yeast extract/peptone in NB, which helped in reducing the azo bond of MR through azo reductase enzyme (Telke et al. 2008; Silva et al. 2014). While in case of MSM, metabolites of strain EMLA3 might have played the role of electron donors. Since MSM is a minimal salt media having lesser amount of nutrients; this might be the reason for slower growth of bacterium (A 660 MSM: 0.180; A 660 NB: 0.935 at 16 h of incubation) and further low degradation due to late availability of metabolites as electron donors. Similar findings are also reported by Vijaya and Sandhya, (2003) and Telke et al. (2008), who also found faster degradation of azo dye in nutrient media compared to basal salt medium using mixed microbial culture and Rhizobium radiobacter MTCC 8161, respectively.

No reduction in initial dye content in un-inoculated media and media inoculated with autoclaved cells ascertained the role of live Nesterenkonia lacusekhoensis EMLA3 cells in removal of MR and not that of media components and other abiotic factors. The degradation of dye in present case is corroborated by GC-MS and FTIR studies. GC-MS showed the presence of low molecular weight metabolites viz. Benzeneacetamide and Acetophenone in EMLA3 treated sample. Likewise in FTIR, the presence of CH3 stretch at 2925 and 2854 cm−1 along with C=O stretch at 1747 cm−1 indicates the presence of possible metabolites N,N′dimethyl-p-phenyle-nediamine (DMPD) or its derivatives (Jadhav et al. 2008; Gomare and Govindwar 2009). During degradation, MR is first transformed into DMPD and aminobenzoic acid (ABA) through cleavage of azo bond (Zhao et al. 2014). The resultant metabolites were then further metabolized into lower molecular weight products. Similar MR degradation pathway is proposed for N. lacusekhoensis EMLA3 strain on the basis of GC-MS and FTIR analysis. However, further studies need to be undertaken to understand the complete biodegradation pathway of MR by EMLA3 strain.

N. lacusekhoensis EMLA3 was found to decolorize 97% of initial 50 mg L−1 MR after 16 h of incubation. However, with increase in dye concentrations, decolorization ability of the strain was found to decrease. This is being attributed to the toxicity of dye on the bacterium. Similar trend in growth and MR degradation pattern with increasing dye concentrations was also reported by Ayed et al. (2011) and Garg et al. (2012). In the present study, significant removal of dye was observed during the early stationary phase of growth at all the concentrations studied. This showed that high cell mass is prerequisite for significant removal of MR by strain EMLA3. This is also evident through inoculum study, as faster and complete removal of MR was observed with higher inoculum size of N. lacusekhoensis EMLA3.

The efficiency of biodegradation of azo dyes depends upon number of environmental and operational factors, which need to be studied individually to maximize the decolorization process (Pearce et al. 2003; Seesuriyachan et al. 2007). The study will also help in finding the limiting range of particular microbial strain towards different factors. Knowing working range of microbes could be beneficial in increasing the functioning efficiency of microbes in wastewater treatment by providing the requisite optimum conditions (Pearce et al. 2003). With these notions, the effects of various factors on MR degradation by EMLA3 strain were investigated in the present study also.

The strain EMLA3 favored wide alkaline pH range (8.0–11.5) for degradation of MR and this proved to be desirable characteristics since real wastewaters or effluents often have varied alkaline pH range. In the literature, we could not find any report stating textile wastewater treatment at such high pH using microbes. Contrary to our results, an alkaliphillic bacterium (Bacillus cohnii MTCC 3616) reported by Prasad and Rao (2013) showed optimum degradation of azo dye at pH 9.0, and further increase in initial pH to 10 and 11.0, resulted in reduced degradation rates.

The decrease in initial pH of the media after dye degradation, observed in the current study is probably due to combined action of some metabolites (organic acids) produced during growth of N. lacusekhoensis EMLA3 in NB along with acidic metabolites such as derivatives of aminobenzoic acid produced during degradation of MR. Ayed et al. (2011) and Vijaya and Sandhya (2003) have also found similar decrease in initial alkaline pH of the media during decolorization of MR by Sphingomonas paucimobilis and mixed culture, respectively. This property of decreasing pH of the alkaline medium by the Nesterenkonia lacusekhoensis EMLA3 may find application in lowering pH of highly alkaline wastewaters which are generally neutralized using large quantity of acids, a costly and non-environmental friendly method (Kumar et al. 2011).

Maximum MR removal by strain EMLA3 was observed in the temperature range of 30–35 °C. The decline in removal activity at lower (<25 °C) and higher temperature range (>45 °C) can be ascribed to slow growth and lower metabolism of strain EMLA3 at these temperatures. Likewise higher dye removal rates at high agitation speeds compared to static condition is due to faster growth because of efficient oxygen and nutrient transfer from media to cells in case of agitated medium (Saini et al. 2013).

Salinity and heavy metals usually limits successful bioremediation of textile wastewater (Yan et al. 2012; Seesuriyachan et al. 2007). Hence, the choice of microbe should be such as that it not only tolerates but is also effective under these tough conditions. The ability of Nesterenkonia lacusekhoensis EMLA3 to remove dye in the presence of high salinity level (>100 g L−1) and in the presence of different heavy metals shows the potential of halotolerant, alkaliphilic EMLA3 strain in treatment of such harsh textile wastewaters. Though the bacterium was able to degrade the dye in the presence of heavy metals, the decolorizing ability was observed to be reduced and the extent of reduction varied according to the toxicity and inhibiting effect of individual heavy metal tested. Among the metals tested, Cu (II) had maximum inhibitory effect on decolorization of MR which could be due to inhibition of enzymatic activity responsible for dye degradation. This assumption can be supported by literature where Nachiyar and Rajakumar (2005) and Yan et al. (2004) have also reported the inhibition of azoreductase activity due to the presence of copper ions.

Degradation of MR by alkaliphillic EMLA3 in the presence of NaCl could be compared to only reported study on alkaliphilic bacteria Bacillus cohnii, where the strain was able to degrade azo dye in the presence of up to 7% NaCl and further increase in NaCl concentrations resulted in growth inhibition of the microbial strain (Prasad and Rao 2013). However, alkaliphilic bacterium EMLA3 used in the present study was able to grow and degrade dye in the presence of up to 20% (200 g L−1) of NaCl. This halotolerant feature of the present bacterium hence provides the scope for its potential applications in treatment of highly saline textile effluents.

Since Nesterenkonia lacusekhoensis EMLA3 exhibited good removal of dye in simple synthetic medium, thus it was worthwhile to explore the potential of this bacterium towards decolorization of heterogenous and complex textile wastewater. The strain was efficient in decolorizing dye from highly alkaline real textile wastewater. Nevertheless, due to low salinity level and metal content in real wastewater further studies were done with simulated wastewater. Application of EMLA3 to oligotrophic simulated wastewater having the presence of high alkaline pH, salinity, and heavy metals too showed efficient dye removal and reduction of COD under sequential static and shaking treatment. On the other hand, sole shaking treatment of the sample resulted in no prominent dye degradation and COD reduction. The reason for this is the transfer of electrons liberated from oxidation of electron donors to oxygen in preference to azo dye. In view of the fact that oxygen has high redox potential; it causes reduction of oxygen over azo dye leading to lower dye degradation (Pearce et al. 2003). However, under static conditions, the presence of lower oxygen content resulted in reduction of azo bond and hence efficient decolorization of sample (Pearce et al. 2003).

Overall good MR degrading potential of N. lacusekhoensis EMLA3 shows its prospective in large-scale treatment of alkaline and salty industrial effluents. Studies related to elucidating the complete mechanism of MR decolorization by EMLA3 will further help in understanding the adaptive features of this unexplored and novel microorganism towards toxic azo dyes.