Mechanism of Cr(VI) reduction by an indigenous Rhizobium pusense CR02 isolated from chromite mining quarry water (CMQW) at Sukinda Valley, India

Toxicological assessment of CMQW generated due to chromite mining activities at Sukinda Valley has revealed high chromium contamination along with Zn and Fe. The present study focused on the mechanism of chromate reduction by an indigenous multi-metal tolerant bacterium, Rhizobium pusense CR02, isolated from CMQW. The isolated strain has shown resistance up to 520 mg/L of Cr(VI) with an IC50 value of 385.4 mg/L. The highest reduction rate 8.6 × 10−2/h was recorded with 20 mg/L of initial concentration of Cr(VI). Extracellular (3.06 ± 0.012 U/mL), intracellular (3.60 ± 0.13 U/mL), and membrane (1.89 ± 0.01 U/mL) associated chromate reductases were found to be involved for reduction. The extracellular polymeric substances (EPS) produced by the isolate also enhanced reduction activity of 46.32 ± 1.69 mg/L after 72 h with an initial concentration of 50 mg/L. FTIR analysis revealed the involvement of functional groups –OH, –CO, and –NH for Cr(VI) biosorption whereas P=O, –CO–NH– and –COOH interacted with Cr(III). Zeta potential with less negative surface charge favored reduction of Cr(VI). Treatment of CMQW by bacterial isolate detoxified Cr(VI) minimizing chromosomal aberrations in root cells of Allium cepa L., suggesting the role of Rhizobium pusense CR02 as a promising bio-agent for Cr(VI) detoxification.


Introduction
Chromite mining activity is considered a 'necessary evil' because it is highly beneficial for human life; on the other hand, its improper management adversely affects health, the environment, and causes biodiversity loss (Jaishankar et al. 2014;Upadhyay et al. 2020). Among all toxic metals, chromium has a great impact because of its wide-scale application in industries for the manufacturing and processing of products (Vendruscolo et al. 2016). Chromium is extensively utilized in metallurgy, thermonuclear weapon manufacturing, leather tanning, electroplating, paint industries, textile dyeing, steel, fertilizer, petroleum refining, and pigment production units (Allegretti et al. 2006;Ilias et al. 2011;Ataabadi et al. 2015;Sahoo et al. 2021). According to reports from the Ministry of Mines, India (2017), about 0.14% of total geographical land (328 million hectares) has been utilized for mining activities. However, in India, about 99% of the total chromite ores are produced at Sukinda in Odisha (Indian Bureau of Mines 2016) and an estimate of about 183 million tons of raw chromium deposits spread over 200 km 2 at Sukinda Valley (Das et al. 2013b). However, the present scenario of mining sites in India has unequivocally put the question on the future of the environment and human safety. Land deterioration, a disorder in the topographic index of soil, air quality, and disturbance of water table occurs in the mining sites by erosion, leaching, weathering, and acid drainage activities (Saviour 2012).
Mining activities at chromite deposited sites lead to the release of Cr(VI) and other heavy metal pollutants. Sukinda chromite belt is considered the 4th worst polluted region in the world because of mining activities which generate about 160 million tons of overburden and subsequently, release about 11.73 tons of Cr(VI) to vicinity areas. Most of the chromite mine quarry water (CMQW) is discharged to Damasala Nala, which is severely polluted and channeled to the river Brahmani (Das and Singh 2011;Mishra and Sahu 2013;Upadhyay et al. 2020). Severe health complications in local people and loss of major biodiversity have been reported in the valley (Das and Singh 2011;Dhal et al. 2011;Mishra and Sahu 2013;Biswas et al. 2017;US Geological Survey [USGS] 2018; Nayak et al. 2020;Upadhyay et al. 2020). As per the report by Blacksmith Institute in 2007, about 70 and 60% of surface water and drinking water were contaminated with Cr(VI), respectively, in Sukinda Valley (BSI 2007).
The release of waste containing Cr(VI) leads to hazardous effects on humans, plants, aquatic organisms, and biomagnification. Workers employed in highly contaminated chromium sites have shown the symptoms of vomiting, skin irritation/allergies, diarrhea, bronchial asthma, nasal irritation, brain damage, premature death, and impair fetal development. Cr(VI) toxicity also causes perforation in the septum, ulcer in the lungs, eardrum perforation, ulceration, kidney necrosis, and lung carcinoma (Gibb et al. 2000;WHO 2012;Ertani et al. 2017;WHO 2020). Cr(VI) has been proven to be mutagenic, carcinogenic, and more toxic than Cr(III) (Kotas and Stasicka 2000;Jobby et al. 2018). Exposure to high concentration of Cr(VI) has been reported to cause alteration of enzyme specificity, oxidative damage, structural deformation in DNA [DNA-DNA crosslinks, DNA strand breaks, DNA-protein, Cr(III)-DNA adducts, and alkali-labile sites] in biological systems (Bruins et al. 2000). Furthermore, metabolic alternation, oxidative damage, morphological disorganization, chlorosis, senescence, abrasion, premature leaf fall, decrease in seed germination index, photosynthetic impairment, stunted vegetative growth, and death of the plant have also been reported due to the chromium toxicity (Mohanty et al. 2009;Sahoo et al. 2020).
Wastewater and industrial effluent of heavy metals, including Cr(VI) is treated through various conventional methods such as precipitation, reverse osmosis, ion exchange, solvent extraction, electro-coagulation, electrodialysis, membrane filter, cementation, photocatalytic reduction, etc. In the photocatalytic approach, sunlight-driven energy is used as a reaction-driven force for the reduction of Cr(VI). New research emphasizes synthesizing visiblelight-driven photocatalysts for the treatment of Cr(VI). Zhang et al. (2022) have reported on an efficient photocatalytic reduction of Cr(VI) by Zr 4+ doped and polyaniline coupled SnS 2 nanoflakes. Ge et al. (2021) have reported Fe 3 O 4 /FeWO 4 composites as a good source, nontoxic, low cost, visible light (λ > 420 nm), and driven photocatalyst for reduction of Cr(VI) under an aqueous environment. Similarly, Jiang et al. (2020) have also reported the application of MgFe 2 O 4 /CPVC nano-composite as a visible-light-driven photocatalyst for the treatment of Cr(VI) in the aqueous environment. But these physicochemical techniques have certain disadvantages like generation of secondary pollutants, reusability, high cost, difficulties in the separation process, and detoxification of toxic pollutants into nontoxic forms (Kathiravan et al. 2010). Applications of environmentfriendly biological methods have many advantages over conventional approaches. Microorganisms can biosorb, bioaccumulate, and metabolize the pollutants by intracellular and extracellular oxido-redox systems. Isolation, characterization, and application of indigenous chromium reducing bacteria (CRB) from chromium polluted environments have played a key role in chromium bioremediation. The indigenous microbes have diverse resistance mechanisms and are thus acclimatized to toxic environments (Dey and Paul 2012;Dey et al. 2014). Hence, biological reduction of Cr(VI) to Cr(III) by CRB is an ideal environment-friendly method for the treatment of CMQW and industrial effluents.
Keeping in view of hazardous nature of chromium, the present investigation focuses on the toxicological assessment and mitigation of CMQW by an indigenous novel CRB. The mechanism of chromium removal by novel bacteria strains using the oxido-redox system has also been investigated. Furthermore, reduced products were characterized by various methods to validate the conversion of Cr(VI) to a more stable and less toxic Cr(III) form. The Allium cepa L. root assay was used to test the genotoxic efficiency of collected CMQW and detoxification using indigenous potent CRB. The study would provide a detailed insight into mechanisms of chromium detoxification and application of isolated potent strain for in situ bioremediation of Cr(VI) contaminated water at Sukinda Valley.

Sample collection and CMQW characterization
CMQW sample was collected from Sukinda chromite mines (21.061561N, 85.829338E) during the pre-monsoon period (Fig. 1). Sampling was followed by estimation of redoxpotential and pH of the mine water. Physicochemical parameters such as dissolved oxygen (DO), total hardness (TH), total suspended solids (TSS), total dissolved solids (TDS), biological oxygen demand (BOD), alkalinity, sulfate, and chemical oxygen demand (COD) were analyzed (APHA 2012). About 10 mL of sample was acid digested and filtered through a 0.22-micron membrane filter and analyzed by ICP-OES (PerkinElmer Avio TM 200) equipped with Syngistix TM software for inductively coupled plasma optical emission spectrometry. Total carbon content and Cr(VI) in the collected sample were analyzed by TOC-analyzer (Shimadzu) and 1,5-diphenylcarbazide method (Zahoor and Rehman 2009), respectively.

Biochemical and molecular identification of potent chromate resistant bacteria
The spread plate method was employed for the enumeration of bacteria in diluted (10 −2 dilution) CMQW on a nutrient agar plate at 37 °C for 24 h (Sahoo et al. 2021). Colony morphology was observed followed by a screening of potent chromate resistant strain on 100 mg/L Cr(VI) [K 2 Cr 2 O 7 as source of chromium] amended in Luria Bertani (LB) broth (g/L: tryptone 10.0, NaCl 10.0, yeast extract 5.0, and pH 7.2) agar plates. Catalase, urease, citrate, oxidase, methyl red, indole, amylase, triple sugar iron (TSI), Voges-Proskauer (VP), nitrate reduction, H 2 S production, solubilization of tri-calcium phosphate, hydrolysis of chitin, starch, and gelatine were performed and findings compared with Bergey's Manual (Holt et al. 1994). The maximum tolerance limit (MTL) of strainCR02 was estimated with nutrient agar plates that are supplemented with a stepwise increasing concentration of individual metal solution [As 2 O 3 , CoSO 4 , CdSO 4 , CuSO 4 , HgCl 2 , K 2 Cr 2 O 7 , MoO 3 , NiCl 2 , Pb(NO 3 ) 2 , and ZnSO 4 ] aseptically, followed by incubation for 24 h at 35 °C. However, the minimum dose at which bacteria were able to grow was considered the MTL (Saranya et al. 2018). Biofilm capacity of the novel strain was characterized by the crystal violet staining method (O'Toole and Kolter 1998).
Furthermore, the bioremediation potential of the isolated strain was examined through biofilm assay, as the biofilmproducing bacteria are efficient to tolerate metal toxicity and could be the ideal agent for bioremediation of heavy metals. Bacteria were grown in LB broth (0.25% glucose); without Cr(VI) and with 100 mg/L Cr(VI) at 35 °C for 24 h as control and treatment, respectively. The culture was removed and rinsed with deionized water three times; adherent cells were stained with 0.5% crystal violet for 30 min and further rinsed with deionized water. Bacterial retained crystal violet was extracted with ethanol and acetone (80:20) for 15 min. Absorbance was recorded at 492 nm and classified according to Stepanovic et al. (2007): non-biofilm producer (OD ≤ ODc), weak biofilm producer (OD > ODc, but ≤ 2 × ODc), moderate biofilm producer (OD > 2 × ODc, but ≤ 4 × ODc), and strong biofilm producer (OD > 4 × ODc). Here, 'ODc' represents media without bacterial inoculants and 'OD' represents media with bacterial inoculants.
Genomic DNA was isolated from the potent chromateresistant strain CR02 by using the QIAamp DNA Mini Kit from Qiagen. The 16S rDNA was amplified with universal primer sets, forward primer E8f (5′-AGA GTT TGA TCC TGG CTC AG-3′), and reverse primer 1541r (5′-AGG GAG GTG ATC CANCCRCA-3′) (Mahbub et al. 2017). Corresponding neighbor sequences (based upon maximum similarity and zero E-value) were downloaded from the NCBI database and valid type strain sequences from the EzBio-Cloud database. A phylogenetic tree was constructed by aligning the sequences and bootstrapped neighbor-joining relationships (1000 replicates) with MEGA-X software (Tamura et al. 2013;Kumar et al. 2018;Sahoo et al. 2021).

MTT assay and DNA stability assessment
The effect of a high concentration of Cr(VI) on the growth and genomic content of bacteria was investigated through growth inhibition and DNA profiling study by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay (Jena et al. 2012;Grela et al. 2015) and gel-based approaches, respectively (Das et al. 2021). Strain was grown in LB broth with varying Cr(VI) concentrations (10,20,30,40,50,60,70,80,90,100,200,300,500, and 1000 mg/L) and bacterial culture without Cr(VI) was considered control (Grela et al. 2015). Inoculants were seeded as per 0.5 McFarland standard in 96 well-plate for both control and treatment [Cr(VI) concentrations], followed by incubation at 35 °C for 48 h. The incubated culture was mixed with 20 μL of 0.5 mg/ mL of MTT in PBS (pH 7.4), followed by 2 h of incubation at 35 °C. MTT gets converted to a purple insoluble formazan crystal in metabolically active cells. About 100 μL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals and absorbance was taken at 570 nm using a microplate reader (CYTATION5 imaging reader, BioTek). The percentage of cell inhibition and the IC50 value were calculated according to Jena et al. (2012). On the basis of the IC50 result, DNA was isolated from bacterial biomass cultured with 50, 100, 300, 500, and 1000 mg/L of Cr(VI), followed by an estimation of DNA band intensity through the Gel Doc system (Bio-Rad).

CMQW detoxification potency of strain-CR02 in batch culture
Bacteria were grown in LB broth for 18 h at 35 °C with constant shaking at 120 rpm, and when the bacterial culture at OD 600 reaches 0.5, the culture was drawn and centrifuged for 10 min at 8000 × g. Furthermore, the collected bacterial pellet was (1.0 g of biomass) inoculated to 1000 mL of sterilized CMQW for 24 h. After 24 h of incubation, residual Cr(VI), TCr, and removal efficiency were evaluated and expressed as mg/L. Residual Cr(VI) and TCr in CMQW after 24 h of incubation were analyzed by 1,5-diphenylcarbazide and ICP-OES method, respectively. The percentage of Cr(VI) removal from CMQW was estimated according to Sahoo et al. (2021). Bacterial Cr(VI) removal puissance was assessed, both in LB broth (pH 7.5) having 2% (w/v) sucrose and without sucrose for 72 h at 35 °C with continuous shaking for 120 rpm. In brief, LB media have different concentration of Cr(VI) (10, 20, 50, and 100 mg/L) without sucrose as LB10, LB20, LB50, and LB100, respectively. Similarly, LB medium having different concentration of Cr(VI) (10, 20, 50, and 100 mg/L) with (2%) sucrose coded as LBS10, LBS20, LBS50, and LBS100, respectively.

Extracellular polymeric substances (EPS) characterization and Cr(VI) removal assay
Biofloculant formation activity of isolated strain was evaluated according to Gomaa (2012). Bacterial culture was inoculated with fermentation media [(g/L): glucose, 2; KH 2 PO 4 , 5; peptone, 5; yeast extract, 5 at pH 7.2] at 30 °C for 72 h at 120 rpm (Subudhi et al. 2014). Different carbon sources like fructose, glucose, maltose, sucrose, and lactose were optimized for the flocculation activities. Similarly, flocculant activity of strain-CR02 was estimated in fermentation media along with 100 mg/L of Cr(VI) at 30 °C for 72 h at 120 rpm. The bacterial suspension was centrifuged at 10,000 × g for 30 min, and collected supernatant was used for the flocculating activity. For estimation of flocculating activity, 0.5% of Kaolin clay suspension (in deionized water) was used; five hundred mL of supernatant (deionized water used as blank) was mixed with about 45 mL of kaolin suspension and 4.5 mL of CaCl 2 solution (1%). Subsequently, mixed content was vortexed for 5 min and kept for 5 min at 30 °C without disturbing. Absorbance (OD 550 ) was recorded, and flocculating activity was calculated as (Gomaa 2012; Ayangbenro et al. 2019): where 'a' and 'b' are the OD values of control and experimental sample, respectively at 550 nm.
Flocculating activity = (a − b)∕a × 100 EPS extraction was performed according to Bramhachari and Dubey (2006). About 5 mL of bacterial isolate (OD 0.5 at a wavelength of 600 nm) were grown on 100 mL of LB medium containing 100 mg/L Cr(VI) enriched with 2% (w/v) sucrose for 72 h at 35 °C. A total of 10 mL sample was centrifuged at 10,000 × g for 30 min at 20 °C. The resulting supernatant was boiled at 100 °C for 15 min, cooled, and precipitated with 4% (w/v) trichloroacetic acid, followed by centrifugation at 10,000 × g for 30 min at 4 °C. The supernatant was further precipitated overnight at 4 °C with two volumes of absolute ethanol followed by centrifugation at 10,000 × g for 30 min at 4 °C heat drying at 100 °C for 15 min. EPS dry weight was calculated and expressed in g/L. Protein estimation was done by taking bovine serum albumin (BSA) as standard, following the Bradford assay (Bradford 1976). Lipid content of isolated EPS was estimated by chloroform: methanol (2:1) extraction method. Phenol sulfuric acid assay was used for estimation of total carbohydrate contents for EPS according to Dubois et al. (1956). In brief, about 100 μL of EPS (1 mg/mL in 50 mM tris-HCl, pH 7.0) was added to 500 μL of 80% (w/v) phenol solution, followed by mixing with 2 mL of H 2 SO 4 at 30 °C for 10 min. Absorbance was recorded at 490 nm and sugar content was calculated from the standard glucose curve (Gupta et al. 2019). The chromium removal efficiency of EPS was estimated at the interval of 1 h up to 6 h of incubation (Zahoor and Rehman 2009). The percentage of Cr(VI) removal was estimated according to Sahoo et al. (2021) as follows: Removal (%) = (A − B)/ A × 100 [here, 'A' and 'B' represents initial and final residual Cr(VI) contents in sample, respectively]. Here K 2 Cr 2 O 7 was used as sole source of Cr(VI).

Chromate reductase genes and localization of chromate reductase enzymes
Both molecular and enzymatic approaches were employed for the validation of chromate reductase activities in the isolated potent strain-CR02. For validation of bacterial chromate reductase genes (ChR and ChrT) in strain-CR02, PCR-based screening was done with genomic DNA (Sahoo et al. 2021). PCR amplification of ChR and ChrT genes was carried out in thermal cycler (Veriti-96, Applied Biosystems, USA) by using primer sets of ChR (forward 5′TCA CGC CGG AAT ATA ACT AC3′, reverse 5′CGT ACC CTG ATC AAT CAC TT3′) (Patra et al. 2010) and ChrT (forward 5′ATC ATG TCA GAT ACC TTG AAA GTG G3′, reverse 5′TGC TTT AAC CCG CCG AAT ATA3′) (Zhou et al. 2017). Based on homology similarity of sequences, a phylogenetic tree was constructed by using MEGA-X with neighbor-joining method (Tamura et al. 2013;Sahoo et al. 2021).
Characterization of functional sites for chromate reductase in strain-CR02, cultured bacterial suspensions (LB broth for 24 h at 37 °C, OD 600 = 0.6) were centrifuged at 14,000 × g for 15 min at 4 °C and collected supernatant was passed through 0.22 μm filter as cell-free extracellular contents (CFECs). The collected bacterial pellets were washed three times and resuspended with 10 mL of 50 mM phosphate buffer (pH 7.0). Furthermore, these cells were broken by ultrasonication at 35 W (Cole-Parmer, USA) for 20 cycles of 10 s on with a gap of 10 s off between each cycle in cold conditions. The bacterial lysate was centrifuged for 30 min at 15,000 × g at 4 °C to remove unbroken cells, followed by ultracentrifugation of collected supernatant at 90,000 × g for 1 h at 4 °C. Subsequently, these supernatants were used as intracellular contents (ICs) and the pellet was dissolved in membrane solubilization buffer (150 mM potassium acetate, 30 mM HEPES, 10% (v/v) glycerol, 2% digitonin, pH 7.4 at 4 °C) for 2 h at 4 °C. Solubilized membrane contents were further centrifuged at 15,000 × g for 30 min at 4 °C and collected supernatants were used as membrane components (MCs). Chromate reductase activity of CFECs, ICs, and MCs in strain-CR02 was evaluated according to Ontanon et al. (2018) with some modifications. Protein concentrations in collected CFECs, ICs, and MCs were estimated by the Bradford method (Bradford 1976). For chromate reductase assay, a total volume of 3 mL reaction mixture contained different components of bacteria (CFEs, ICs, and MCs) (1 mg/mL of protein), Cr(VI) [10 mg/L], and 50 mM phosphate buffer (pH 7.0) along with 6.5 mg/L NADH, followed by incubation in agitation at 30 °C for 60 min. Residual Cr(VI) was estimated every 15 min by the 1,5-diphenylcarbazide method. Control experiment [non-enzymatic Cr(VI) reduction] was performed as follows: reaction mixture containing 10 mg/L Cr(VI), 6.5 mg/L NADH along with 50 mM phosphate buffer with pre-heated (100 °C for 5 min) CFECs, ICs, and MCs, respectively. However, one unit (U) of Cr reductase activity was specified as the amount of enzyme that reduced 1 μM of Cr(VI) per min under the defined assay conditions. Specific activity for chromate reductase was defined as the unit activity per mg of protein (Ontanon et al. 2018).

Antioxidant assay
Ten (10) mL of control and 100 mg/L of Cr(VI) treated bacteria culture were centrifuged at 10,000 × g for 10 min at 4 °C, pellet was resuspended with 1 mL of lysis buffer [2 mM MgCl 2 , 0.1 mM EDTANa 2 , 100 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1% Triton X-100]. In the next phase sonication, (Cole-Parmer, USA) at a 35 kHz frequency rate with 10 s on and 10 s off for 20 cycles at 4 °C was carried out, and the lysate was centrifuged for 15 min at 20,000 × g and 4 °C (Zhang et al. 2013). Protein content in the crude extract was estimated by Bradford assay (Bradford 1976). Antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase were carried out to assess the mechanism of the isolated strain-CR02 against the toxic effect of Cr(VI). Peroxidase activity was measured according to the method of Chance and Maehly (1955). Similarly, catalase and SOD activity were measured by using the standard methods of Aebi (1984) and Ewing and Janero (1995), respectively. All the detailed enzymatic assay protocols were followed as stated in Sahoo et al. (2021).

Morphology and surface interactions assessment
Bacterial cells [control, CMQW, and Cr(VI) treated] were fixed in 2% (w/v) para-formaldehyde buffered with 0.1 M sodium phosphate buffer saline (pH 7.0) at 4 °C for 18 h. Cells were fixed with 1% of osmium tetraoxide at 4 °C for 2 h, washed and dehydrated with gradient ethanol-water solution (15%, 30%, 50%, 70%, and 90% ethanol) for 2 min each. Further slides were kept for drying under a CO 2 atmosphere for 15 min and mounted with aluminum stubs. Mounted glass slides were coated with 90 Å thick gold palladium (VG Microtech, East Sussex, TN22, England) and observed at 5 kV with field emission scanning electron microscopy (Model-Zeiss EVO40). Biosorption of chromium by isolated strain was analyzed by an energy-dispersive X-ray spectrometer (EDAX, USA) at 5 kV (Sahoo et al. 2020).
The electrokinetics based on Zeta potential of bacterial cells in both control and Cr(VI) conditions were calculated according to Lage et al. (2012) to find out the changes in the surface charges of the bacterium as changes in surface charge play an important role in the interaction of different ions onto the cell wall. The zeta potential of strain-CR02 in the presence and absence of Cr(VI) was measured using a Litesizer500 equipped with a universal dip cell omega cuvette (Mat. no. 155765). In brief, control bacterial cultures and culture with Cr(VI) 100 mg/L at 35 °C for 48 h were harvested at 10,000 × g for 10 min followed by resuspended with PBS (pH 7.4) to minimize the reign of pH .
Bacterial surface chemistry and involvement of different functional groups on the cell wall can be confirmed by Fourier transform infrared spectroscopy (FTIR). In the present investigation, FTIR was used to identify the changes in functional groups of novel isolated strain and extracted EPS under different stress conditions. Approximately 5 mg of dried biomass and EPS were finely powdered with 50 mg of potassium bromide. Powdered samples were pressed and KBr pellets were used for the IR study (Sahoo et al. 2020). The infrared spectra (%T) were recorded within a range of 400 to 4000 cm −1 by using a Spectrum RX I PerkinElmer, FTIR Spectrometer (Thermo Scientific).

Characterization of bacterial reduced products
Batch cultured bacterial sample was recovered from 100 mg/L Cr(VI) amended with 2% (w/v) sucrose in LB media after 72 h followed by three times washing with deionized water. Powdered biomass was subject to XRD and XPS analysis. XRD was done by using Rigaku Ultima-IV X-ray diffractometer (Rigaku, Tokyo, Japan), having Cu Kα radiations (40 kV, 40 mA) with a scan rate of 8° and 2θ/min ranging from 20 to 80° in a continuous scan mode (Srivastava and Thakur 2012; Mishra et al. 2021). The surface chemistry of potent chromium-resistant strain-CR02 was analyzed by XPS (XPS, PHI 5000 VersaProbe II, ULVAC-PHI Inc., USA) equipped with micro-focused (200 μm, 15 kV) and monochromatic Al-Kα X-ray source (hν = 1486.6 eV). Both survey spectra scan (with an X-ray source power of 50 W and pass energy of 187.85 eV) and narrow scans (high-resolution spectra of the major elements were recorded at 46.95 eV pass energy) results were recorded. PHI's Multipak software was used for processing XPS data, and the C 1s peak at 284.8 eV binding energy was used as a reference.

Sub-chronic toxicity efficacy on Allium cepa L.
Allium cepa L. (2n = 16) bulbs were collected from the local market (morphologically similar and healthy bulbs were selected for this study). Dry roots and leaves were removed from onion bulbs followed by rinsing with sterilized water. These bulbs were allowed to germinate under distilled water for 48 h and nearly about 0.5 cm of grown root bulbs was selected. Autoclaved distilled water was used as control, CMQW as a positive control, and detoxified CMQW by strain-CR02 for experimental purposes. For each treatment, five onion bulbs were taken and grown in 50 mL of solution each for 144 h at room temperature in dark conditions, followed by daily replacement of solution. Each treatment was carried out in triplicate for statistical validation. Genotoxicity assay for each group, 5 numbers roots (2-3 cm) was randomly selected followed by fixation with Carnoy's solution (3 volume of ethanol: 1 volume of glacial acetic acid) and stored for 2 h at 4 °C. After fixation, the root tips were transferred to 70% ethanol (preservation purpose) for mitotic aberration (MA) and chromosomal aberration (CA) study (Chauhan et al. 1999). In brief, root tips were hydrolyzed in 1 M HCl at 60 °C for 5 min, washed with distilled water, and stained with 2% (w/v) acetocarmine solution for 3 min. Glass slide was prepared by putting slight pressure to make cells squash on the surface of the slide and randomly examined under a light microscope at ×400. About 1000 numbers of cells were taken into consideration for scoring; the occurrence of micronuclei, nuclear buds, binuclei, and chromosomal aberrations in diving metaphase, anaphase, and telophase were examined. The mitotic index of respective treatments was calculated as the percentage of cells undergoing mitotic stages (including prophase) per 1000 cells examined per onion bulb. However, chromosomal aberrations were calculated as a number of specific aberrations in 100 dividing cells (metaphase, anaphase, and telophase) per onion bulb. The percentage of mitotic index (MI), chromosomal abnormality index (CAI), nuclear abnormality index (NAI), and micronucleus index (MN) were calculated by following formulae (Maity et al. 2020).

Statistical analysis
For all the analysis, three replicates were carried out in triplicate and results were represented as mean ± standard deviation (SD) to validate the accuracy of the experiment. For the confirmation of statistically significant differences, data were evaluated using one-way analysis of variance (ANOVA) followed by the use of post hoc tests for Tukey's test (p ≤ 0.05), using Origin Pro 8.5 software.

Biochemical and molecular identification of potent chromate resistant bacteria
A total of three morphologically different Cr(VI) tolerant bacterial strains were screened from CMQW on an LB agar plate supplied with 100 mg/L of Cr(VI) and considered chromate-resistant bacteria (CRB). Among these strains, CR02 has shown the highest tolerance ability at 520 mg/L of Cr(VI) stress. The optimum condition with pH (7.5) and temperature (35 °C) was found to be the ideal environment for the growth of CR02 strain. Strain CR02 was characterized by a mesophilic, white-colored, irregular, undulate margin, raised elevation, rod-shaped, and Gram-negative bacterium. The strain has shown negative toward oxidase, amylase, H 2 S production, Voges-Proskauer (VP), phosphate solubilization, starch hydrolysis, gelatinase activity, and citrate utilization as carbon source; whereas shows positive results toward methyl red, urease, catalase, indole, nitrate reductase, cellulase, chitinase, protease, and fermentation of dextrose. Identified characteristic features of the strain CR02 were compared with Bergey's manual of determinative bacteriology, and this bacterium is contingently identified to be a member of genera Rhizobium.
The MTL of strain-CR02 toward different metals was found to be (mg/L): As (260), Cd (100), Co (220), Cr (520), Hg (30), Mo (650), Ni (180), Pb (380), and Zn (1250), respectively. It was evident that the isolated strain has the lowest tolerance for HgCl 2 and maximum for ZnSO 4 . Isolated strain-CR02 might have developed the resistance and detoxification mechanism of Cr(VI) through adsorption, uptake, and biotransformation mechanisms to withstand the toxic environment (Das et al. 2021). However, strain CR02 was found to be sensitive toward Hg (30 mg/L) and Cd (100 mg/L). Industrial effluents and mine drainage contain varying types of contaminants, including heavy metals (Ontanon et al. 2018;Sahoo et al. 2020;Sahoo et al. 2021). In our previous report, a multi-metal tolerant strain Enterobacter sp. AMD01, isolated from an iron-ore mining site, could efficiently able to remove about 95% of toxic metals from acid mine drainage (Sahoo et al. 2020). Thus, for proper biological treatment and management of wastewater, there must be a search for tolerant microorganisms and their resistance to toxic environments. It could also be evident that strain-CR02 might maintain the physiological adaptation mechanisms such as biosorption, accumulation, and transformation to overcome the metal toxicity along with its reduction. Ontanon et al. (2018) have reported multi-metal resistant strain Bacillus sp. SFC500-1E form tannery sediments showed bioremediation efficiency in removing Cr(VI). Tan et al. (2020) have also reported chromium-reducing bacteria Bacillus sp. CRB-B1 isolated from sewage treatment plant with 420 mg/L of chromium MIC value. Furthermore, isolated strain CR02 has shown resistance to antibiotics clindamycin and trimethoprim. It was observed that strain-CR02 has 8. 04, 7.07, 3.80, 11.34, 5.31, 3.46, 7.54, 4.15, 18.09, and 4.15 mm 2 zones of inhibition against penicillin-G, chloramphenicol, streptomycin, tetracycline, amikacin, vancomycin, azithromycin, erythromycin, ciprofloxacin, and gentamycin, respectively. Previous studies have also documented that certain chromate-resistant bacterial stains have resistance capacity against most of the tested antibiotics Khusro et al. 2014). Application and improper management of antibiotics cause the development of antibiotic resistance genes (ARG). Antibiotics pose a range of risks and have significant effects on human and animal health. As a result of this, antibiotic-resistant and multi-drug-resistant bacteria are developed in the environment. Furthermore, the application of antibiotic-resistance bacterial strain in bioremediation work could not be suitable for the treatment of heavy metals.
It was evident that microbes producing biofilm are favorable for bioremediation work. Because the biofilm formation process enhanced the tolerance toward contaminants and environmental stress, and facilitates to degrade of a wide variety of pollutants through various catabolic pathways. Microbes are trapped in a self-synthesized matrix in biofilm which protects against stress and pollutants. Bacterial biofilms play a key role in metal biosorption from wastewater (Gupta et al. 2015;Schmid and Sieber 2015). Bio-film producing efficiency of strain-CR02 was found to be moderate under the control condition without Cr(VI), while strong biofilm formation activity was observed under 100 mg/L Cr(VI) condition. The study suggested that strain-CR02 has the potential for the development of biofilm in response to Cr(VI) stress. Pan et al. (2014) have reported on the bioreduction of Cr(VI) to Cr(III) which was subsequently immobilized by extracellular polymeric substances within the biofilm of Bacillus subtilis. Kang et al. (2005) have also reported that EPS of biofilm has greater absorption of Cd 2+ and Pb 2+ in a water environment, facilitating the efficient microbial remediation processes.
Microbes produce different types of polysaccharides, which play an important role in the development of biofilm and help for biosorption of metals, flocculation, adherence to the surface, and mediator for the cell-to-cell interaction in the toxic environment (Gupta et al. 2015;Schmid and Sieber 2015). The polysaccharides secreted by bacteria within a biofilm are known to sequester heavy metals. Intensive studies for bacterial biofilm have also been reported for its wide-scale application in biotechnology, wastewater treatment, human infections, etc. (Jachlewski et al. 2015). Hence, the current study focused on the validation of the biofilm formation activity of Rhizobium pusense CR02 and its efficiency in removal of chromium.
Based on sequence alignment of quarry 16S rDNA gene sequence with available sequences in NCBI databank, phylogenetic tree analysis was constructed. The isolated strain-CR02 was confirmed as Rhizobium pusense (Agrobacterium pusense) with 98% of sequence similarity (Fig. 2). Sequenced 16S rDNA gene has been submitted to the NCBI-GenBank database and detailed information is available in the GenBank with accession number MZ617264. Very little information on the bioremediation of Cr(VI) by Rhizobium sp. is available in the database. Both biochemical and physiological findings confirm the survivability of bacterium in a toxic environment with a high concentration of different heavy metals. This study might ascertain our knowledge of the ecological adaptations of the strain-CR02 in a toxic chromite mine water environment.

Growth inhibition and DNA stability profiling
Survivability of the selected strain was noticed to be decreased with increasing concentration of Cr(VI) as evident from the CFU/mL value (data not shown), that with an increase in Cr(VI) concentration, the survivability of the bacterium is getting decreased. The IC50 value for the bacterium CR02 toward Cr(VI) was found to be very high (358.4 mg/L) (Fig. S1a). Furthermore, the genotoxicity of varying concentrations of Cr(VI) on bacterial DNA profiles was evaluated against the control. Comparative investigation of the genomic content (both concentration and band intensity) of bacteria was found to be reduced with increasing Cr(VI) concentration (Fig. S1b). Current findings were found to be similar to the previous reports where a high concentration of Cr(VI) leads to damage of DNA by oxidation of guanine residue (Kasprzak 2002), chromosomal aberrations, mutation, the constraint of polymerase activity, and other genotoxic effects (Tsou et al. 1997;Das et al. 2021).

Removal of Cr(VI) and detoxification potency in batch culture
The reduction of TCr, Cr(VI), and other toxic metals in CMQW after 24 h was calculated by taking about one gram of cultured bacterial pellet under the defined assay conditions. Removal efficiency for toxic metals in CMQW by strain Rhizobium pusense CR02 has been shown in Table 1. The concentration of removed Zn, TCr, and Cr(VI) was evaluated as 6.55, 2.34, and 1.36 mg/L, respectively, after 24 h of incubation with Rhizobium pusense CR02 biomass (1.0 g). It was evident that Rhizobium pusense CR02 had high adsorption efficiency toward different metal ions. Rhizobium pusense CR02 has shown the highest Cr(VI) removal efficiency (46.32 ± 1.69 h mg/L) from the aqueous solution with 50 mg/L of Cr(VI), 2% (w/v) sucrose after 72 h of incubation (Fig. 3, Table 2) in comparison to LB without sucrose having 100 mg/L of Cr(VI) (26.91 ± 1.30 e mg/L). Tan et al. (2020) have reported Bacillus sp. CRB-B1 with maximum Cr(VI) reduction efficiency of 97.04% at 150 mg/L of Cr(VI) at optimum assay conditions (35 °C, 30 h, and 150 rpm). The present investigation suggested that the Cr(VI) reduction efficiency of Rhizobium pusense CR02 has increased Cr(VI) reduction efficiency in LB sucrose (2%) medium.
The kinetics of Cr(VI) reduction by Rhizobium pusense CR02 in the aqueous solution at different initial Cr(VI) concentrations against time was studied by exponential decay equation [Eq. (1)] (Camargo et al. 2003;Das et al. 2014) and it was fitted well (Fig. 3b) with the linearized form of the exponential rate of Eq. (3).

Incubation (h)
Here LB broth media have different concentration of Cr(VI) (10, 20, 50, and 100 mg/L) without sucrose, represented as LB10, LB20, LB50, and LB100, respectively. Similarly, LB medium having different concentration of Cr(VI) (10, 20, 50, and 100 mg/L) with (2%) sucrose represented as LBS10, LBS20, LBS50, and LBS100, respectively.  Table 2 Cr(VI) removal efficiency of Rhizobium pusense CR02 under the assay condition Cr(VI) reduction data at different concentrations were represented as mean ± SD (n = 9), followed by Tukey's test. Different letters as superscripts of respective incubation period denote significant difference between the means of treatments and the same superscripts denotes means difference is not significant at p < 0.05  Das et al. (2014) have also reported the rate constant values for Cr(VI) reduction at 10-100 mg/L with Bacillus amyloliquefaciens CSB-9 isolated from chromite mine soil of Sukinda, India, have been reported to be the highest (2.8 × 10 −2 /h) at 10 mg/L of Cr(VI) and lowest (0.44 × 10 −3 /h) at 100 mg/L. A similar finding was also reported on a moderately halophilic bacterium Halomonas smyrnensis KS802, which shows the highest Cr(VI) reduction rate at low initial Cr(VI) concentration and lowest rate constant at higher initial Cr(VI) concentration (Biswas et al. 2018).

EPS formation and Cr(VI) removal activity
Bioflocculants are considered biopolymers, basically produced by bacteria during the growth period, and these biodegradable flocs are very efficacious in the removal of metals and suspended particles from solution (Aljuboori et al. 2013). It was evident that bioflocculant production is greatly influenced by different nutrient parameters of media; Ayangbenro et al. (2019) have reported two bacterial isolates (Pantoea sp. and Pseudomonas koreensis) from mining sites with 71.3 and 56% of flocculating activity as carbon sources for glucose and sucrose, respectively. In this present study, it was observed that Rhizobium pusense CR02 has the least flocculating activity when lactose is used as a carbon source and maximum with sucrose (data not shown). Optimization result of different carbon sources has confirmed that sucrose acts as an efficient carbon source for EPS and flocculant production in Rhizobium pusense CR02. However, EPS and biofilm formation activity were found to be increased under Cr(VI) treated condition. The produced EPS might have contributed to the flocculation and adsorption of chromium ions from the solution (Gupta et al. 2021). Eckbo et al. (2022) have reported that Cr(VI) reduction increases with increasing soil total organic carbon (TOC) contents. Different workers have reported sucrose as the commendatory carbon source for bioflocculant production in bacteria (Subudhi et al. 2016;Ayangbenro et al. 2019), which is very much similar to Rhizobium pusense CR02 in this study. Furthermore, the flocculating activity of Rhizobium pusense CR02 under control, and 100 mg/L of Cr(VI) condition was found to be 78.29 ± 2.78 and 97.06 ± 2.20, respectively. The current report on Rhizobium pusense CR02, has shown the highest flocculent activity under Cr(VI) condition and confirms sinew toward metal removal. A previous study has revealed the role of bioflocculant from strain Pantoea sp. having a removal efficiency of 80.5%, 52.5%, and 51.2% for Pb, Cr, and Cd from wastewater, respectively (Ayangbenro et al. 2019). Furthermore, extracted EPS from Rhizobium pusense CR02 under Cr(VI) condition was found to be maximum (8.906 ± 0.28 g/L) as compared to the control condition (2.370 ± 0.09 g/L). Batool et al. (2015) have also reported a high concentration of total exopolysaccharides (mg/g of fresh weight) production under chromium conditions in Ochrobactrum intermedium Rb-2, similar to our findings in the present study. Extracted EPS from Rhizobium pusense CR02 comprised 22.62% (w/w), 46.32% (w/w), and 2.16% (w/w) of protein, carbohydrate, and lipids (dry weight) under control conditions, respectively. However, EPS extracted from Cr(VI) treated bacterial culture was noticed to be with higher content of protein (38.74% w/w), carbohydrate (66.12% w/w), and lipids (5.80% w/w) of dry weight. The amount of secreted carbohydrates under the Cr(VI) treated condition has also been found to be maximum in comparison to the control condition (Mungasavalli et al. 2007). Cr(VI) removal (mg/L) efficiency of extracted EPS was found to be 2.53 ± 0.09, 3.35 ± 0.05, 4.55 ± 0.05, and 8.0 ± 0.1 after 1 h, 2 h, 4 h, and 6 h of incubation, respectively (Fig. 4). The previous study has reported that indigenous Enterobacter cloacae SUKCr1D from the Sukinda chromite mines have shown the potential for reduction of Cr(VI) into Cr(III) through EPS secretion with 31.7% of Cr(VI) reduction at 10 mg/L (Harish et al. 2012). Though Cr(III) has not been measured in the present investigation, it is expected that Rhizobium pusense CR02 isolated from the same habitat, i.e., Sukinda chromite mines, might have reduced Cr(VI) into Cr(III) through EPS-based reduction. The structure and chemical composition of EPS plays an important role in the removal of metals and flocculation activity for wide-scale application (Gupta et al. 2021). Sheng et al. (2005) have reported EPS with a high number of amino groups might have enhanced binding and degradation efficiencies for different pollutants. Thus, from the above findings, this could be concluded that increased content of major cell wall constituents such as protein and carbohydrate are the attributes of interaction of chromate ions onto the bacteria surface facilitating detoxification of Cr(VI).

Localization of chromate reductase enzymes and Cr(VI) reduction mechanism
The present investigation revealed the presence of about 268-bp-long putative chromate reductase gene R (ChR) in Rhizobium pusense CR02 and the absence of chromate reductase gene T (ChrT). A phylogenetic tree showed that the gene ChR of strain-CR02 has a close relationship with chromate reductase of Rhizobium sp. BK251. Identified ChR sequence was submitted to NCBI (GenBank) with the accession number (OL616125). Presence of ChrR gene in Pseudomonas aeruginosa (Aguilar-Barajas et al. 2008), Staphylococcus simulans (Kalsoom et al. 2021), and Serratia sp. GP01 (Sahoo et al. 2021) has been reported to have a significant role in chromate resistance and reduction. The presence of chromate reductase gene (ChR) in Rhizobium pusense CR02 might have a paramount contribution to its adaptation to extremely toxic environments and the reduction of Cr(VI) as well.
In this study, the chromate reductase activity of the crude extracts of CFECs, ICs, and MCs was recorded as 3.06 ± 0.012, 3.60 ± 0.13, and 1.89 ± 0.01 U/mL of protein, respectively (Fig. 5c). Strain Rhizobium pusense CR02 revealed efficient Cr(VI) reduction activity for both extracellular and cytosolic soluble reductase enzymes; furthermore, it reflects the involvement of membrane proteins in Cr(VI) reduction process. Most of the bacterial chromate reductase was also reported from both cytosolic and membrane-bounded fractions of cells, however, few reports also described extracellular chromate reductase activity (Mishra et al. 2021). Li et al. (2016) have reported high Cr(VI) reduction enzyme activity of 6.60 and 6.78 U/mL in cell-free extract (CFE) and periplasmic content of Pseudoalteromonas sp., respectively. Results of Cr(VI) reduction efficiency (%) for cellfree extracted crudes (CFECs), intracellular contents (ICs), and membrane contents (MCs) of Rhizobium pusense CR02 were found to be 74.8 ± 0.89%, 84.4 ± 0.56%, and 49.6 ± 0.62%, respectively, after 60 min of incubation under the defined assay conditions (Fig. 5b). Though, all the three localized components played important role in the Cr (VI) reduction, ICs have shown the highest reduction activity and lowest with MCs. From the above findings and floc formation activities as discussed earlier, the CFECs might be involved in both the adsorption and Cr(VI) reduction process. This could be concluded from the above findings that chromate reductase activity of strain Rhizobium pusense CR02 is associated with intracellular, extracellular, and membrane components.

Antioxidant assay
The total protein content of the bacterial crude extract was estimated as 6.8 mg/mL. Increased activity of SOD (47.14 U/mL), catalase (32.15 U/mL), and peroxidase (58.49 U/ mL) in Rhizobium pusense CR02 were noticed in response to 100 mg/L of Cr(VI). Ilias et al. (2011) have reported that metal-induced oxidative stress enhanced the production and accumulation of free radicals, leading to increased detoxifying enzyme concentration in the living system. The expression of protective enzymes (SOD, catalase, and peroxidase) under normal conditions was found to be low and thought to be increased due to stress conditions to counter the reactive oxygen species (Sahoo et al. 2021). Though Lenartova et al. (1998) have reported induction of SOD activity as the first line of defense against the generation of ROS, peroxidase-mediated reaction of oxidative polymerization helps in strengthening cell wall formation in repose to metal-induced stress (Cosgrove 1997).

Morphological assessment and electrokinetics of cell surface
The result of the FESEM-EDX micrograph, under control conditions (without treatment), revealed the uniform distribution of rod-shaped, elongated cells with smooth and regular surface characteristics and the absence of a Cr peak [Fig. 6a(i)]. However, bacterial cells under both Cr(VI) and CMQW conditions have shown distinct changes in morphology and distribution. Compactly arranged bacterial cells having more porous structures, roughness in surface texture, and disordered cell size with globular appearance were found in response to CMQW [ Fig. 6a(ii)]. Similarly, Cr(VI) treated bacteria cells showed the morphological changes, cell surface characteristics, and distribution pattern of strain-CR02 were observed. EDX-analysis confirms the disintegration and bio-adsorption of Cr(VI) onto the bacterial cell surface [Fig. 6a(iii)] in Cr(VI) aqueous condition. Changes in cell surface morphology (size, shape, roughness, porous structure, and swelling) of different bacteria like Bacillus subtilis, Bacillus cereus IST105, Escherichia coli, and Serratia sp. GP01 with response to Cr(VI) toxicity has been reported by different groups of authors. Bacteria might have developed the adaptation system to arrange themselves in groups and secretion EPS to reduce the exposure and controlled intake of toxicants (Schembri et al. 2003;Naik et al. 2012;Samuel et al. 2013;Sahoo et al. 2021). Das et al. (2014) have reported the changes in cell morphology with the coagulated, porous structure of cell surface and roughness and immobilization of Cr(III) species on the Bacillus amyloliquefaciens cell surface in Cr(VI) treated conditions as analyzed with SEM-EDX. Both FESEM-EDX and reductase assay clearly suggested the high removal potency of Rhizobium pusense CR02.
The electrokinetics study of bacterial cell surface under control and 100 mg/L of Cr(VI) treated condition was assessed through zeta potential analysis at pH 7.4. The zeta potential values indicated that the surface charge of Rhizobium pusense CR02 differs both in control (−17.0 ± 0.3 mV) and Cr(VI) treated conditions (−9.5 ± 1.9 mV) (Fig. 6b). It was observed that zeta potential was found to be less negative with subsequent interaction with Cr(VI) ion as compared to the control [Fig. 7b(i),b(ii)]. The less negative value of bacterial surface charge under Cr(VI) treated condition after 48 h of incubation facilitated adsorption followed by reduction of Cr(VI) to Cr(III). Binding of positively charged Cr(III) ions onto the bacterial surface leads to less negative surface charge in comparison to control. Previous studies have also reported on shifting of bacterial surface charge toward less negative in Corynebacterium paurometabolum, Sphingopyxis macrogoltabida SUK2c with increasing Cr(VI) concentration (Prabhakaran and Subramanian 2017;Prabhakaran et al. 2019). Li et al. (2020) have also reported the bioreduction of Cr(VI) by Bacillus subtilis and adsorption of positively charged Cr(III) ions, shifting zeta potential values toward less negative in comparison to control.
FTIR analyses of untreated bacteria, Cr(VI) treated bacteria, extracted EPS from control, and Cr(VI) treated bacteria have revealed the pattern of functional groups involved for Cr(VI) binding mechanistic in Rhizobium pusense CR02 (Fig. 6c). The distribution of reduced Cr(III) ions mainly depends upon the media composition and type of strain used for the reduction of Cr(VI) study Das et al. 2021). There is the possibility of the presence of organic acids and amino acids in the growth medium, which might compete with bacteria for coordination with Cr(III). Thus, IR spectra of the bacterial cell and extracted EPS from both control and Cr(VI) conditions could confirm the involvement of different functional groups in the binding of reduced Cr(III) Sahoo et al. 2020;Das et al. 2021). It was observed that the stretch of the hydroxyl group shifted from the range of 3710.42 (bacterial cells under control condition) to 3724.26 cm −1 (bacterial cells under Cr treated condition). Furthermore, some of the common banding patterns have been observed in the range of 3841-3723 cm −1 of both Cr-treated, EPS and bacterial cells, indicating the interaction of Cr ions to both EPS and bacterial cells as well. Band stretching at 3300-3000 cm −1 corresponds to the -OH and -NH functional groups for glucose, hydroxyl, and proteins (Mary Mangaiyarkarasi et al. 2011;Das et al. 2021). The characteristic band stretching at 3249 and 3293 cm −1 for EPS and control bacterial growth conditions indicates the presence of hydroxyl and proteins functional groups, whereas the shift in the banding toward 3239 and 3282 cm −1 under Cr(VI) treated conditions for both extracted EPS and bacterial biomass confirm the interaction of Cr-ions with protein and hydroxyl groups. The presence of a visible peak at 1713-1715 cm −1 is the -CO bond of the lipid group of EPS and the shift might be due to the Cr ions' interaction to EPS. Further -NH bending of primary and secondary amide at 1600-1550 cm −1 range confirms the involvement of peptide bond of protein, and the minor shift among these peak ranges and intensity compared to control is due to the interaction of Cr ions (Das et al. 2021). Spectra range between 1070 and 1080 cm −1 corresponds to stretching vibration of -CO bond of polysaccharide, which is found only in bacterial cells. However, there was a shift of spectral range 1081.31 cm −1 of bacterial cells (control condition) to 1076.15 cm −1 under Cr treated bacterial cells. Appearance of some low-intensity peak in Cr(VI) treated bacterial cells and EPS between 750-600 cm −1 represents the involvement of Cr=O vibration of reduced Cr-species as shown in Fig. 6c Biswas et al. 2018;Das et al. 2021). The broadening of the absorption peak at 618 cm −1 in all the groups, indicating the presence of polysaccharides on the cell surface and EPS, which might be involved for Cr adsorption on Rhizobium pusense CR02 (Gupta et al. 2015). The presence of typical band stretching of 1116, 1643, 1396, and around 2926 cm −1 indicates the existence of phosphoric acid (P=O or P-O), amide (-CO-NH-), carboxyl (-COOH) and hydroxyl (-OH) of extracted EPS (Zhu et al. 2019) and the minor spectral shift, decrease in intensity among these vibrations of Cr(VI) treated EPS shows the interaction of reduced Cr(III) ions with EPS. The phenomenon of adsorption of Cr ions onto the bacterial cell surface and EPS might be occurring due to modification of functional groups in response to chromium. Results of the IR study support the processes like chromium reduction, enzymatic and EPSbased Cr(VI) reduction, FESEM-EDX, zeta potential characteristics of Rhizobium pusense CR02. But the significantly reduced Cr(III) end product might have the chance of getting reoxidized to Cr(VI) (Zhu et al. 2019) hence, further investigation was focused on the characterization of bacterial reduced products.

Characterization of bacterial reduced products
The XRD diffractograms of both Cr(VI) treated and untreated bacterial cells have shown (Fig. 7a) different peak values at 2θ (degree). Cr(VI) treated bacterial biomass have shown peak values at 2θ (degree) of 7. 04, 27.45, 31.77, 45.50, 56.57, 63.34, and 75.33; with a high-intensity peak at 31.77. Mishra et al. (2021) have also reported some similar findings at 2θ (degree) values of 25.77, 27.22, 31.65, 45.27, and 38.20 in Microbacterium paraoxydans. The present investigation suggests the presence of possible Cr(III) reduced products in the form of different chromium oxides on the bacterial cell surface .
The XPS spectra of Cr(VI)-treated bacterial cells revealed a convoluted peak in the chromium zones (Fig. 7b). The binding energy at 577.52 and 587.25 eV corresponding to Cr2p 3/2 and Cr2p 1/2 orbitals were assigned for Cr(III) forms that is Cr(OH) 3 and organic complexes (Cr 2 O 3 ), respectively (Park et al. 2008;Jin et al. 2016;Tan et al. 2020). Similarly, the optimum fitting peak at a binding energy of 581.82 eV is attributed to the Cr2p 3/2 orbital. However, the higher binding energy of 580-580.5 and 589.0-590.0 eV was characterized as CrO 3 since Cr(VI) draws electrons very strongly than Cr(III) (Park et al. 2008). Chen et al. (2018) have reported the binding energy peak at 588.9 and 579.9 eV for the orbitals of Cr2p 1/2 and Cr2p 3/2 , respectively. Observed binding energy in the current findings probably is attributed to Croxides and Cr-hydroxides, indicating the strong evidence for the reduction of Cr(VI) to a stable product of Cr(III) by Rhizobium pusense CR02. Many workers have reported the assigned eV values for Cr2p 3/2 orbitals at 577.2-576.5 eV as Cr(III) compounds (Cr 2 O 3 ), whereas Cr(VI) forms are characterized by higher eV values at 578.1 or 579.2 for K 2 Cr 2 O 7 (Liu et al. 2015;Jin et al. 2016;Mishra et al. 2021). FESEM-EDX, zeta potential, FTIR, and other enzymatic studies confirmed the binding of Cr(VI) ions onto the cell surface possibly through EPS production facilitating the sequestration of Cr(VI). Furthermore, the XPS study confirmed the mechanistic of adsorption in conjugation with the Cr(VI) reduction by Rhizobium pusense CR02. A detailed graphical representation of Cr(VI) resistance and reduction mechanism in Rhizobium pusense CR02 was depicted in Fig. S3.

Sub-chronic assay
The toxicity of CMQW and detoxified CMQW by Rhizobium pusense CR02 on the onion root was assessed through chromosomal aberration. Root morphology and genotoxicity results supported the findings of bioremediation of CMQW by Rhizobium pusense CR02 after 72 h (Figs. 8 and S4). Although morphological assessment of onion root length under detoxified CMQW (3.6 ± 0.19 b cm) was not similar to the result of control (7.6 ± 0.31 a cm), it was found to be very much significant in comparison to CMQW (1.4 ± 0.12 c cm) alone. Genotoxicity assessment revealed a significant reduction in the mitotic index of CMQW, unlike control and detoxified CMQW. Mitotic index (%) of control, detoxified CMQW, and CMQW was estimated to be 41.45 ± 1.71 c , 30.25 ± 3.33 a , and 16.27 ± 1.83 b , respectively. Inhibition of mitotic index after exposure to metal ions might be due to the disturbed cell cycle (blocking of G1 stage and inhibition/suppression of DNA synthesis) or dysfunction of chromatin (Gupta et al. 2018). Present findings showed an increase in significant chromosomal abnormalities due to toxic CMQW on the onion root cells (Table 3), leading to both physiological (stickiness, c-mitosis, lagging, and vagrant) and clastogenic aberrations (fragments and bridges) (Gupta et al. 2018). Many workers have extensively employed micronucleus and chromosomal aberration assay in onion root meristematic cells to validate the toxicological efficacy of environmental pollutants (Silveira et al. 2018;Gupta et al. 2018). However, the most common chromosomal aberration was observed due to the dysfunction of chromatin causing bridge formation, stickiness, and failure of the spindle (c-mitosis and laggard chromosome), which leads to a high chance of aneuploidy (Leme and Marin-Morales 2009). It was evident from genotoxic assessment (Fig. 8) that elevated metalloids present in CMQW reduced the mitotic index as well as enhanced chromosomal aberrations in the root meristem, unlike control and detoxified CMQW.

Conclusion
In the present study, isolated strain Rhizobium pusense CR02 has shown the remediation capacity against high concentration of Cr(VI). The remediation capacity might be attributed due to high EPS production and its strong chromate reductase activity. However, the involvement of three localized chromate reductase enhanced the enzymatic reduction. Further, elevated antioxidant level reflected resistance capacity of the strain against Cr(VI) toxicity. Changes in functional groups and negative surface charge supported the interaction and bioaccumulation of Cr(VI), hence assist the bioconversion of Cr(VI) into Cr(III). Genotoxicity study confirmed substantial detoxification of CMQW by indigenous isolate. The study provided new insights on mechanisms of Cr(VI) reduction by Rhizobium pusense CR02, which may have great potential for ex-situ and in-situ bioremediation of Cr(VI) contaminated wastewater.

Acknowledgements
We would also acknowledge University Grants Commission (UGC), Govt. of India for providing NFOBC Fellowship. We  Chromosomal aberration data of different treatments were represented as mean ± SD (n = 9), followed by Tukey's test. Different letters as superscripts of respective rows denote significant difference between the means of treatments and the same superscripts denotes means difference is not significant at p < 0.05 would also like to acknowledge CIF, OUAT, and Bhubaneswar for ICP-