Bioreduction of Cr(VI) by Viable Cells and Cell-Free Extract of a Moderate Halophile, Halomonas Smyrnensis KS802 and Evaluation of Their in Vitro Ecacy in Tannery Euent

The present study was aimed to characterize the chromate reducing ability of cells and cell-free extract (CFE) of Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) and evaluate their effectiveness in tannery euents. Viable cells of the strain reduced 200 µM Cr(VI) in basal medium for halophiles (MH) in 10 h and was inversely proportional to Cr(VI) concentrations. The rapid reduction by cells (10 (cid:0) cells/mL) was achieved with 7.5% NaCl, at pH 7 and 37°C which increased with increasing cell density (10¹° cells/mL). While acetate, Cu³ (cid:0) , Fe³ (cid:0) , SO (cid:0) ² (cid:0) , and CO (cid:0) ² (cid:0) were stimulating the reduction, the inhibitors retarded the process signicantly. The NADH-dependent chromate reduction of the CFE was found to be constitutive with K m and V max values of 56.58 µM and 3.37 µM/min/mg protein respectively. The optimal reductase activity of the CFE was evident at 200 µM Cr(VI), 10% NaCl, pH 8.0 and at 45°C. A higher concentration of CFE and electron donors increased the enzyme activity but was impacted negatively by toxic metals and anions. Both the cells and CFE were capable of reducing Cr(VI) remarkably from tannery euent. FTIR and XRD spectra of chromate reducing cells conrmed possible complexation of reduced Cr-species with functional groups on cell surface.


Introduction
Chromium (Cr) toxicity is one of the major causes of environmental pollution emanating from chrome tanning processes, electroplating, paint and pigment manufacturing industries (Shahid et al. 2017).
Among the different valence states of Cr, trivalent [Cr(III)] and hexavalent [Cr(VI)] chromium are stable and have environmental signi cance. The hexavalent chromium is highly soluble, mobile, toxic, mutagenic and carcinogenic (Sultan and Hasnain 2007) compared to less soluble and mobile Cr(III). In addition to Cr(VI), most of the industrial e uents also contain other co-contaminating heavy metals, dyes, salts, ions etc. Therefore, treatment of hypersaline e uents polluted with Cr(VI) has become a challenging problem to the industries.
Due to high cost, low e ciency and generation of toxic wastes, the conventional physico-chemical methods of Cr(VI) removal from polluted e uents have been found unsuitable and as an alternate, microorganisms like bacteria, fungi and algae were implemented for bioremediation of toxic Cr(VI) in an eco-friendly way (Jeyasingh and Philip 2005). The chromium-resistant as well as tolerant bacteria inhabiting such polluted environments can survive and grow by adopting the mechanisms of biosorption, bioaccumulation, e ux, and most frequently by reduction of hexavalent chromium. Over the years, extensive work has been carried out on the removal of Cr(VI) by several species of bacteria isolated primarily from Cr-polluted environments (Thatoi et al. 2014). Extracellular, intracellular or membrane bound reductases of these bacteria have been reported to reduce Cr(VI) either aerobically or anaerobically, or by both (Losi et al. 1994).
Halophiles are functionally and taxonomically diverse communities found in hypersaline environments which exhibit optimum growth at a salinity of approximately 15-20% but are often able to grow in saturated brine. They are classi ed as: slight-(2-5% NaCl), moderate-(5-20% NaCl) and extreme halophiles (20-30% NaCl) according their salt requirements (DasSarma and Aora 2002). In addition halophiles have the ability to adapt in high external osmotic pressure (NaCl) and maintain an osmotic balance between their cytoplasm and the hypersaline extracellular environment. This salt adaptation also makes the cellular structural components and both the intra-and extracellular enzymes to work under salt stress (Oren 2002). Moreover, environmental contamination due to industrial and municipal e uents discharged into saline and hypersaline entities make the halophiles tolerant to the toxic metals, dyes, hydrocarbons, pesticides etc (Gnanamani et al. 2010). Such adaptive features of halophilic and halotolerant microorganisms have directed the scienti c and technical attention towards their increasing application in hypersaline wastewater treatments and removal of toxic chemicals.
Some of the halophilic microorganisms have already been reported for bioremediation of oil (Al-Mailem et al. 2008), dyes (Asad et al. 2007), oxyanion (Amoozegar et al. 2008), organic pollutants (Le- Borgne et al. 2008;Castillo-Carvajal et al. 2014) and polyaromatic hydrocarbons (Nanca et al. 2018). In the perspective of heavy metal tolerance of halophilic bacteria, very few studies on potential removal or detoxi cation of Cr(VI) Mishra et al. 2012;Subramanian et al. 2012;Focardi et al. 2012;Mabrouk et al. 2014;Biswas et al. 2018) were conducted till date. Moreover, metal removal by halophiles in the presence of salt has become crucial as waste water generated by most of the industries including the tannery waste contains higher concentration (1-10%, w/v) of salt as NaCl (Sivaprakasam et al. 2008).
A chromium tolerant moderately halophilic bacterium, Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) isolated in this laboratory from a saline soil sediment collected from a multipond solar saltern of Gujarat, India has recently been established and documented as a potential candidate for reduction of Cr(VI) during growth in batch culture (Biswas et al. 2018). The present study was aimed to assess the e cacy of whole cells as well as cell-free extracts of Halomonas smyrnensis KS802 in reducing Cr(VI) and also to evaluate their potential for Cr(VI) reduction in tannery e uents under laboratory conditions.

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The isolate H. smyrnensis KS802 was grown in MH medium at 32 °C under continuous shaking for 24 h and cells were harvested by centrifugation (5,000 × g) for 10 min at 4 °C. The cell mass was washed thoroughly with sterile saline (0.9% NaCl) and re-suspended in phosphate buffer (pH 7.0). Chromate reduction by viable cells was conducted in Basal MH medium which contained (g/L) yeast extract 1.0; glucose, 1.0; NaCl, 25; MgCl 2 , 6H 2 O, 7; MgSO 4 , 7H 2 O, 9.6; CaCl 2 , 2H 2 O, 0.36; KCl, 2; NaHCO 3, 0.06 and NaBr 0.026 (pH 7.2). Basal MH medium (20 mL/100 mL ask) supplemented with 200 µM Cr(VI) and 10 9 cells/mL was maintained for the reduction process. Similar set of experiment was performed with autoclaved (at 15 p.s.i. for 15 min) cell mass. A set without chromium was also maintained as negative control. The asks were incubated at 32 °C under continuous shaking (120 rpm) and samples were withdrawn aseptically at regular interval and analyzed for residual Cr(VI) following standard S-diphenyl carbazide (DPC) method (Park et al. 2000). To one mL of cell-free culture supernatant, 3 mL of 0.166 M H 2 SO 4 and 1 mL of 0.05% DPC solution was added, mixed well and OD was read immediately at 540 nm.
Residual Cr(VI) was estimated from the calibration curve prepared in the same way. The amount of Cr(VI) reduced was determined from the difference and expressed as % Cr(VI) reduced.

Chromate reduction by cell-free extract
The cell-free extract (CFE) of H. smyrnensis KS802 was prepared according to Focardi et al. (2012) with minor modi cation. Cell mass from 36 h grown culture in MH medium supplemented with 200 µM Cr(VI) at 32 °C were harvested by centrifugation (5,000 × g), washed and sonicated in phosphate buffer (0.1 M; pH 7.0) at 5% of the original culture volume using an ultrasonic probe (Sonics, mod. 130) (5 cycles of 120 s on and 60 s off at 130 W). The sonicated cell suspension was centrifuged (12,000 × g, 4 °C, 20 min) and the supernatant was used as the cell-free extract. Total protein was estimated according to Lowry et al. (1951) using bovine serum albumin (Sigma, Milano) as standard.
Chromate reduction by the CFE was assayed using NADH (Sigma, Milano) as an electron donor. The reaction mixture (1 mL) contained 100 µM Cr(VI) and 5% NaCl in 0.8 mL of 25 mM potassium phosphate buffer (pH 7.0) and 100 µM NADH. An aliquot of 200 µL of CFE was added to the mixture to initiate Cr(VI) reduction. An assay mixture with similar composition but without CFE was used as the negative control.
Reduction of Cr(VI) was measured by estimating the decrease of Cr(VI) content in the reaction mixture after 30 min of incubation at 30 °C. Cr(VI) was quanti ed following standard S-diphenyl carbazide method (Park et al. 2000) as described above. One unit of chromate reductase was de ned as the amount of enzyme per mg of protein which reduced 1 µM Cr ( Rate kinetics of Cr(VI) reduction Reduction rates were described as the relationship between the decrease of chromium per unit time and the number of bacterial cells (Zhu et al. 2008) and expressed mathematically as: where V R is the speci c reduction rate in µM /number of cells/ h; ∆C, the difference in the reduced substrate concentrations in micromole; N, the cell concentration and t, the time in h. However, relative reduction rate, V RR =∆C/t, indicate the decrease in velocity of Cr(VI) ignoring the cell density in reduction system.
The kinetics of Cr(VI) reduction was calculated according to the following equation (Camargo et al. 2003): y = a.e − kt C/Co = a.e − kt Linearized form becomes: ln C/Co = lna − kt where a is constant, C/Co is the fraction of Cr(VI) reduction at time t, C is the concentration of Cr(VI) at time t, Co is the original Cr(VI) concentration, and k is the rate constant.

Chromium reduction in tannery e uent
Untreated tannery e uent containing 100 µM of Cr(VI) and 10% (w/v) NaCl collected from Kolkata based tanneries in Bantala, Kolkata, West Bengal, India was lter sterilized and used for the reduction studies. Both the raw e uent [100 µM, Cr(VI)], e uent supplemented with 500 and 1000 µM Cr(VI) were used to examine the e ciency of viable whole cells (10 and 10¹ cells/mL) and CFE (0.2 and 0.4 mg protein/mL). As usual, the hexavalent chromium reduction (%) was determined following the DPC method described above.
FTIR of used bacterial cell mass was recorded following the method of Chandra and Singh (2014). The cell mass harvested after treatment was washed thrice with 0.9% saline water and lyophilized at -56 °C in a lyophilizer (LSL Secfroid). The dried cell mass and potassium bromide (1:24) were mixed, grinded and pressed to form pellet by using a hydraulic press. The FTIR spectrum (400-4000 cm ¹) of the pellet was recorded in a Jasco-6300, (USA) IR spectrometer.
Powder X-ray diffraction (p XRD) was performed according to the method of Dhal et al. (2010). In brief, p XRD patterns of the treated cell mass was recorded in a Rigaku Ultima Diffractometer 1840 using Niltered CuKα (λ = 1.315 Å) radiation generated at 20-60 kV and 2-60 mA and the instrument was calibrated with standard silicon using a scintillation scan detector at slow scan in 2θ = 3-80°. The interplaner d-spacing at different θ values were calculated.
A control set (cell mass without exposer to tannery e uent) was also analyzed using FTIR and p-XRD.

Data analysis
All the experiments were done in triplicate sets and the results are represented as average of triplicate values ± S.D.

Results
Chromate reduction by whole cells Time course of Cr(VI) reduction by freshly grown viable whole cells (10 9 cells/mL) of H. smyrnensis KS802 as conducted under batch culture in basal MH medium showed a gradual increase with time leading to complete reduction of 200 µM Cr(VI) after 10 h of incubation under continuous shaking.
Concomitant with chromate reduction, the total number of cells/mL of the medium also showed an increase (Fig. 1a) keeping parity with percent Cr(VI) reduced.
With increasing concentration of Cr(VI) in the reaction medium, a decreased bio-reduction of Cr(VI) by cells of H. smyrnensis KS802 was noted. It was evident that with a variation of concentration ranging from 200-1000 µM of Cr(VI) under the experimental conditions, the reduction e ciency of the cell mass ranged from 100 − 23.5% in 10 h (Fig. 1b). Kinetics of Cr(VI) reduction at different substrate concentrations tted well (R 2 ≥ 0.99) with the linearized form of exponential rate of equation. The highest (5 × 10 ² h ¹, 200 µM/mL) and lowest (8 × 10 ³ h ¹, 1000 µM/mL) rates of bioreduction was correlated with the lowest and highest concentrations of Cr(VI) respectively (Fig. 1c).
Among the different electron donors used, cells of H. smyrnensis KS802 preferred acetate, propionate as well as glucose as the most promising electron donors totally reducing 200 µM Cr(VI) in 10 h. Other electron donors such as citrate, succinate and gluconate were also found effective for chromate reduction by H. smyrnensis cells ( Table 1). Most of the low molecular weight metal oxyanions tested so far were found to be highly stimulatory to Cr(VI) reduction by cells of H. smyrnensis KS802 while, TeO ² followed by AsO ² were to some extent inhibitory to chromate reduction (Table 1). All the tested inhibitors lowered chromate reduction by whole cells. As against control, 2, 4-dinitrophenol (DNP) appeared to be stimulatory, whereas carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was most inhibitory showing only 11.25% Cr(VI) reduction after 10 h ( Table 1). Presence of some of the metal ions were detrimental to Cr(VI) reduction by viable cells of H. smyrnensis KS802. The order of such reduction was Sn² (81.25%) > Ag² (61.25%) > Hg² (56%) > Cd² (52.25%) > Zn² (50%). However, presence of Cu³ and Fe³ was stimulatory to reduction of Cr(VI) while Mn² , Ni² , and Pb² mediated reduction of Cr(VI) was almost equivalent to the control (Table 1).

Values of are presented as mean of three experiments ± SD
Supplementation of NaCl (5-10%, w/v) in the reduction medium enhanced the chromate bioreduction and the fastest reduction rate (35.5 µM/10 cells/h) was recorded with 7.5% NaCl in 8 h of incubation (Fig. 2a). A remarkable inhibition of chromate reduction (41.25%), however was recorded at 20% NaCl. Reduction of Cr(VI) was signi cantly affected on increasing the cell density from 10 to 10¹ cells/mL (Fig. 2b) and the fastest complete reduction of 200 µM Cr(VI) was achieved in 6 h with 10¹ cells/mL with maximum reduction rate of 53.5 µM/10 cells/mL/h. At low cell density (< 10 cells/mL), only 18.5-56.75% reduction of 200 µM Cr(VI) was recorded and was accompanied with reduced rate of bioreduction. So far the in uence of pH and temperature is concerned, the complete Cr(VI) reduction was observed at pH 7.0 in 10 h (at the rate of 33.5 µM/10 cells /h) (Fig. 2c) and at 37 °C in 8 h (at a rate of 37.4 µM/10 cells/h) (Fig. 2d).
Chromate reductase activity of cell-free extract Time course of chromate reductase activity of the cell-free extracts of induced and non-induced cells of H. smyrnensis KS802 followed an almost similar pattern although CFE of induced cells showed higher % of Cr(VI) reduction (Fig. 3a) compared to that of non-induced cells. An almost complete loss of enzyme activity of the CFE was detected when treated with trichloroacetic acid (TCA) and moist heat. While metabolic inhibitors like N,N,-di-cyclohexyl carboiimide (DCC) and CCCP showed pronounced retardation of enzymatic activities (8.08 and 4.14 U/mg protein), EDTA, DNP and β marcapto-ethanol were found to stimulate the reductase activity with speci c activity of 20, 22.5 and 24.25 U/ mg protein respectively (Fig. 3b).
Speci c activity of the chromate reducing enzyme in CFE was proportional with Cr(VI) up to a concentration of 200 µM Cr(VI) showing maximum activity of 24.95 U/mg protein (Fig. 4a). The linearized Lineweaver-Burk plot as drawn for saturation kinetics of the reductase activity was also effectively justi ed. The apparent Michelis-Menten constant (K m ) and the maximum velocity (V max ) were calculated as 56.58 µM and 3.37 U/mg protein respectively (Fig. 4b).
The optimum pH, temperature and NaCl concentration for reductase activity of the CFE of the present strain was found to be pH 8.0, 45 °C and 10% NaCl with speci c activity of 28.25, 29.5 and 29.42 U/ mg protein respectively (Fig. 5a, 5b and 5c) whereby the enzyme showed a broad range of tolerance of these factors.
An increased enzyme activity was found with increasing enzyme concentration (01-0.4 mg protein/ mL) and a saturation of the activity (33 U/mg protein) was noted when the reaction mixture was supplemented with CFE equivalent to 0.4 mg protein/mL (Fig. 6a). Similarly, increase in concentration of acetate and NADH gradually increased the speci c activity of the enzyme (Fig. 6b). Both the electron donor showed their e ciency towards Cr(VI) reduction but presence of NADH had a superior enzyme activity over acetate.
Considering the in uence of oxyanions on Cr(VI) reduction by cells of H. smyrnensis KS802, effect of different concentrations of CO ² , IO , SO ² , PO ³ , NO on reduction of Cr(VI) with CFE was tested. It was noted that at low concentration (200 µM), oxyanions like CO ² , IO , SO ² , PO ³ , NO stimulated the enzymatic activity and the highest reductase activity was found in presence of SO ² (33 U/mg protein) followed by CO ² (31.25 U/mg protein) and IO (28.75 U/mg protein). However, at increased concentration (400 µM) of oxyanions, only CO ² , IO and SO ² were still stimulatory while, PO ³ and NO at identical concentration was having negative effect on chromate reduction (Fig. 6c).
Toxic as well as heavy metals have versatile effect on enzyme activity. Stimulation of the reductase activity (33 U/ mg protein) was observed in presence of 200 µM Fe³ and Cu³ while at higher concentration (400 µM), the speci c activity was lowered to 28.25 and 25 U/ mg protein. The highest inhibitory metal was Zn² followed by Ni² and Pb² (Fig. 6d).

Chromate reduction in tannery e uent
Raw tannery e uent containing 100 µM Cr(VI) and tannery e uent supplemented with additional Cr(VI) (500 and 1000 µM) was treated with both the viable cells and CFE of H. smyrnensis KS802. An increase in % reduction of Cr(VI) in tannery e uents was found with increased number of viable whole cells as well as concentration of CFE. Raw e uent when treated with 10 and 10¹ cells/mL showed complete reduction of Cr(VI) after 10 h (Fig. 7a). Supplementation of Cr(VI) in the e uent, however has retarded the chromate reducing e ciency. Only 68.25% of 500 µM and 35% of 1000 µM Cr(VI) in tannery e uents was reduced by 10¹ cells/mL in 10 h. The CFE showed 100 and 68% reduction of Cr(VI) from tannery e uent with 0.2 and 0.4 mg protein/mL. Increased Cr(VI) in tannery e uent (500 and 1000 µM) have affected the reduction process strikingly. Only 48% reduction of 500 µM Cr(VI), and 20% reduction of 1000 µM Cr(VI) in tannery e uent was observed with highest concentration of CFE (0.4 mg protein/mL) (Fig. 7b).
Comparative FTIR analysis of bacterial biomass used for reduction of Cr(VI) from tannery e uents ( Fig. 8b) with that of untreated cell mass (Fig. 8a) showed shifting of peaks as well as appearance of new peaks in the treated biomass. New peaks were visualized at 1744.45, 1240.76, 1179, 1103 and 976 cm ¹ while shifting of peaks at 3290. 39, 3077.50, 1646.88, 1532.84, 1394.72, 1229 and 1660 cm ¹ were also evident (Fig. 8b).

Discussion
Bioreduction of Cr(VI) in industrial wastes specially in tannery e uents which also contain toxic metals, oxyanions and high amount of NaCl could be achieved exploiting selective halophilic bacterial strains with unique adaptive features such as tolerance to high salinity and osmotic pressure, low oxygen and nutrient availability, and toxic substances including heavy metals (Oren, 2002). Therefore, studies on Cr(VI) reduction by halophiles and their enzymes could be of great importance in bioremediation of Cr(VI) contaminated industrial e uents. In this study, we have initially evaluated the chromate reduction potential of the previously reported moderately halophilic bacterium, H. smyrnensis KS802 (Biswas et al. 2018) using its viable whole cells and cell-free extracts.
Freshly grown cells of H. smyrnensis KS802 restored its chromate reducing activity and effectively reduced the Cr(VI) in the synthetic Basal MH medium with limited cellular growth possibly due to poor nutritional composition of the basal medium used (Fig. 1a). Acetate a suitable electron donor for reduction of Cr(VI) by cells of H. smyrnensis (Table 1) might have favored the process by protecting the concerned enzyme from inactivation (Soni et al. 2012). The role of oxyanions as competing electron acceptors in Cr(VI) reduction process is well known and SO 4 2− in particular is likely to facilitate Cr(VI) uptake via the sulphate uptake pathway (Cervantes et al. 2001) and thereby promoting the process in H. smyrnensis KS802 cells (Table 1). The inhibitory effect of CCC, NaN and NaF at higher level has been explained due to retardation of cytochrome oxidase, enolase and disruption of chemiosmotic gradient (Opperman and Heerden 2007). The inhibitory activity of Hg² as in most cases appeared to be due to its binding to thiol (SH) group and inactivation of enzyme (Xu et al. 2012). But the Cu ² and Fe ² mediated improvement of Cr(VI) reduction by cells of H. smyrnensis KS802 could be attributed to their functions as cofactors for many enzymes in the cellular metabolism (Dogan et al. 2011).
It is a commanding feature of the KS802 cells to reduce Cr(VI) under high salt concentrations (Fig. 2a) compared to other Halomonas spp. so far reported (Focardi et al. 2012;Murugavelh and Mohanty 2012). The increase in Cr(VI) reduction with increased viable cells (Fig. 2b) is the general trend of bioreduction of Cr(VI) as reported previously (Dey and Paul 2014). Reduction of Cr(VI) at wide pH range indicated the conformational change of active site of enzyme with availability of CrO ² (Karthik et al. 2017) (Fig. 2c) while, its thermotoletant feature (Fig. 2d) corroborated several previous studies Subramanian et al. 2012;Chandra and Singh 2014).
A higher speci c activity of the CFE of induced cells compared to non-induced cells indicated the constitutive nature of the enzyme which was promoted to a higher degree under chromium stressed condition (Fig. 3a) (Mangaiyarkarasi et al. 2011). Loss of the chromate reduction ability of CFE with TCA and moist heat treatment established the involvement of proteinaceous enzymes in the process. The enzyme mediated reduction by the CFE was further con rmed by the inhibition of the process following treatment with CCCP and DCC which functions as an uncoupler of proton gradient and ionophore in addition to inhibition of oxidative phosphorylation of ETS (Takamiya 1983). The stimulation of Cr(VI) reduction activity of CFE in presence of DNP suggested the involvement of other alternative intracellular Cr(VI) reduction mechanisms (Shen and Wang 1993).
Increase in Cr(VI) concentration have an inverse effect on reductase activity of CFE (Fig. 4a) and chemical kinetics of the reduction in relation with substrate concentration tted well with linearized Lineweaver-Burk plot with low K m value (56.58 µM) (Fig. 4b). This results indicated that the reduction of Cr(VI) was dependent on the substrate and low K m value as found in Michelis-Menten equation suggested very high a nity of the substrate to the enzyme and therefore, at very low Cr(VI) concentration, the present enzyme could reach with maximal velocity (V max ). Moreover, the variations of optimum pH (8.0), temperature (45 °C) and NaCl conc. of (10%) of the CFE (Fig. 5a, 5b and 5c) CFE of H. smyrnensis KS802 may offer suitability for bioremediation industrial e uents (Kiran et al. 2007).
With increasing concentrations of CFE, acetate and NADH, the reductase activity was increased gradually ( Fig. 6a, 6b). It was pointed out that a Cr(V) intermediate is formed in presence of NADH which decreased reactive oxygen species (ROS) formation as well as chromium toxicity and thus highly stimulated the reductase activity (Bae et al. 2005). Reductase activity of CFE was positively in uenced with tested oxyanions (Fig. 6c) which might be the resultant of non-competitive association of oxyanions and CrO ² and facilitation of CrO ² entry through respective oxyanions uptake pathways (Cervantes et al. 2001;Qian et al. 2016). Improvement of Cr(VI) reduction as noted with toxic metal ions (Fig. 6d) though contradicted the ndings with non-halophilic strains (Sarangi and Krishnan 2008;Elangovan et al. 2010), promoting effect of Fe³ on Cr(VI) reduction by Halomonas sp. has already been reported (Focardi et al. 2012).
Whole cells as well as CFE of H. smyrnensis KS802 was targeted for their application in bioreduction of Cr(VI) in tannery e uent which also contains a high concentration of NaCl, diverse toxic metals and oxyanions. The cells (10 -10¹ cells/mL) (Fig. 7a) as well as crude enzyme (0.2-0.4 mg protein/mL) ( Fig. 7b) exhibited remarkable e cacy in reducing Cr(VI) from tannery e uent indicating their potential for bioremediation.
Such potentials were re ected by possible interaction of chromium compounds with bacterial cell surface functional groups (Pradhan et al. 2007) as indicated by the shifting as well as appearance of new peaks in the FTIR analysis (Yee et al. 2004;Park et al. 2005) (Fig. 8a and 8b). Moreover, results of powder XRD analysis of the treated cells showed that a number of sharp peaks with a crystallinity index of 0.83 ( Fig. 9a and 9b) indicated possible combination of crystalline reduced Cr-compounds on the cell surfaces during the reduction process. The nature of such complex and speciation of chromium, however, could not be determined during the present study.

Conclusion
Page 13/18 Characterization of the chromate reduction activities of viable whole cells and CFE from H. smyrnensis KS802 showed e cient bioreduction of Cr(VI) under saline stressed environment co-contaminated with toxic metals and oxyanions. E cacy of both cells and CFE in the anthropogenic toxic tannery e uents also proved promising at the laboratory level batch culture studies. An improvement of the Cr(VI) reductase activity under optimized conditions appeared to be fruitful for development of an well organized bioremediation process of Cr(VI) reduction for toxic e uents in large-scale bioreactor systems.

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Availability of data and materials
All data represented or analyzed during this study are included in this article.

Competing interests
The authors declare no con ict of interest

Authors' contributions
Dr. Jhuma Biswas designed and performed the experiments and prepared the draft manuscript: Prof. A. K. Paul supervised the work and analysis of experimental data and nalized the manuscript.