Isolation and Identification of Unique Arsenotolerant Exiguobacterium indicum DSAM62 from Arsenic Rich Environment

Arsenic infiltration via bio-geological and anthropogenic mean's has been a global concern in the developing countries due to the severe toxic effects of the compound which leaches to the ecological niche. Herein, we report one such arsenotolerant bacteria isolated from a contaminated soil collected from a district in West Bengal (India), where an arsenic concentration of the soil sample was found to be 0.51 mg/kg. In the present study, several bacteria were isolated from the soil sample, out of which LD50 value of arsenic for the novel tolerant bacteria was found to be 500 mg./l which was also determined by INT assay. The efficacy of arsenate uptake by this bacterium was 52.20% from its respective LD50 concentration. After 16S rDNA sequencing, the bacteria showed 99% similarity with Exiguobacterium indicum based on nucleotide homology and meta-phylogenetic analysis. LCMS FAME revealed that the unique fatty acids were monosaturated C:10:03 OH (0.53%), C12:1 (0.16%), C11:03 OH (0.38%), terminally branched Anteiso C14:0 (0.74%), monosaturatedC15:1w5C, monosaturatedC16:0 N Alcohol, Anteiso 16:0 (0.36%), Iso- C16:0 3OH (0.33%), Iso- C18:1 H (0.25%), C18:3w6C (6,9,12) (0.53%), C18:1 2OH (0.52%), which lead us to conclude that the bacteria was a new and unique bacterial strain. So, on the basis of FAME analysis, the unique bacteria were named as Exiguobacterium indicum Strain DSAM62, which can be used as a potential bio accumulator in agricultural fields for bioremediation of arsenic. The fully annotated sequence of the genome and the plasmid of the novel strain revealing key regulatory genes were also investigated and results can be correlated with this tolerant behavior. The annotated sequence was submitted to NCBI genome database which currently having an accession number of PRJNA555453.


Introduction
Arsenic is a toxic element of the environment and it is a member of group V of the periodic table. So, it is classified as a heavy metal (Wackett, 2004). It mainly exists in the nature in two forms, the predominant pentavalent arsenic As [V] and trivalent arsenic As [III] (Cullen and Reimer, 1989) because arsenate As [V] is immobilized in the solid phase and arsenite As [III] primarily exists as uncharged H 3 As 0 3 with the pKa value of 9.2. Less toxic methylated form of arsenic dimethylarsinic (DMA) and monomethylarsonic (MMA) are generated by microorganisms under oxidizing conditions (Wang et al., 2007). Groundwater contamination with bioavailable arsenic has been found in West-Bengal, India and Bangladesh (Smith et al., 2002) as a direct result of irrigation in crop fields by contaminated water. Rice grains are also affected by bioaccumulation of arsenic (Meharg and Rahman, 2003) which exhibits the most common route of arsenic poisoning through food chain (Chowdhury, 2004) and causes skin cancer, liver and lung cancers (Wang et al., 2001). The most common way for heavy metal removal techniques from industrial effluents are chemical precipitation, chemical oxidation or reduction, ion exchange, filtration, electrochemical treatment, reverse osmosis, membrane technologies and evaporation recovery (Ahluwalia and Goyal, 2007). These methods are expensive and have some drawbacks in the higher concentration of heavy metal removal (Nourbakhsh et al., 1994). Therefore, innovative, low-cost and eco-friendly microbes involving bioremediation techniques for heavy metal mitigation from the industrial sludge are developed. Bacteria can absorb, adsorb, chelate and biochemically modify arsenic species into much less toxic forms of reduced arsenic with the help of proteins helping in metal binding and modification, these activities are regulated by the inducible gene expression upon arsenic stress. The present study involves the isolation, characterization, arsenic tolerance studies, uptake ability and identification of a novel bacteria isolated from the arsenic contaminated soil from a rural area of West-Bengal.

Arsenic Content Analysis of Soil Sample by ICP-OES
Arsenic concentration of the collected soil sample from Shyamnagar was measured by ICP-OES after preparing the soil sample by wet digestion method (USEPA, 1996). 2 gm. of soil sample was mixed with 5 ml. of 69% HNO 3 in 100 ml Borosil glass conical flask and it was incubated for overnight. After the incubation the sample was kept in block tubes and the block tubes were kept in the Block Digester (AIM 600 Digestion System, Australia). Arsenic content of the soil sample was analyzed by Thermo Scientific, Model No. ICAP 6000 at 193.759 nm wavelength in UV region.

Characterization of the Collected Soil and Water Samples
The soil sample was collected from paddy rhizospheric soil from Shyamnagar. Then, the deionized water was added into the soil in 1:10 ratio. Then the mixtures were filtered and pH, EC content and specific gravity of all the collected samples were measured.

Bacterial Isolation by Serial Dilution Method
The colloidal soil sample was measured up to 1 ml and dissolved in 9 ml of sterile, deionized water to prepare a dilution of 10 -1 . From this, 10 -2 and 10 -4 dilutions were prepared in sterile, de-ionized water. The 10 -2 and 10 -4 dilutions were used for pour-plating with fresh, sterile Nutrient Agar (HiMedia) media to obtain isolated bacterial colonies. For this, 0.2 ml of sample aliquots were placed in sterile petri-plates and to it, the nutrient agar (HiMedia) was added and swirled gently. After solidification, the plates were incubated for about 24 hours, at 37˚C. Numerous colonies were obtained in each plate. Few colonies were selected on the basis of their frequency at random for further studies, i.e., morphological and biochemical characterization.

Biochemical Characterization of the Isolated Bacteria
Since bacterial organisms are similar in morphological and cultural characteristics, four biochemical tests are used to differentiate between these bacteria. InVic and Sugar Utilization test were performed for the identification of the bacteria at preliminary stage.
For indole test, the bacterial strains were inoculated in tryptophan broth and they were incubated at 37˚C for 24 hours. After 24 hours of incubation, 0.5ml of Kovac's reagent was added to it, without shaking. Development of reddish ring was taken as a positive result.
For methyl red test, 48 hours old bacterial cultures in dextrose phosphate broth were taken and about 0.5 ml methyl red indicator was added to it, followed by shaking. Development of red coloration was taken as a positive result.
The bacterial strains were inoculated in dextrose phosphate broth for Voges-Proskauer test and they were incubated at 37˚C in BOD Incubator Shaker for 48 hours old cultures in dextrose phosphate broth were taken and to it about 0.6ml Barritt's reagent was added with vigorous shaking. Development of copperish red color was considered as a positive result.
For citrate utilization test, the bacterial strains were inoculated in Simmon's Citrate Agar (HiMedia) media and they were incubated at 37˚C for 24 hours. After 24 hours of incubation, the change in color of media from green to blue was taken as a positive result.
For chrome agar test, 24 hrs old cultures in Hi chrome agar (HiMedia) media were taken and the characteristic color of the respective cultures was observed.
For sugar utilization Test, each tube containing TSI agar (HiMedia) was taken. Small amount of the experimental bacteria from fresh culture was inoculated by using of the sterile technique into the tubes by means of slant and stab inoculation method with an inoculating loop. Then, the tubes were then incubated at 37˚C for 24-48 hours in BOD Incubator. After 24-48 hours the color of both the stab and slant of agar slant cultures were observed.

Arsenic Tolerance Study
The bacterial cultures were inoculated into sterilized 30 ml. of nutrient broth supplemented with increasing concentrations of arsenic [As(v)] like 10 mg./l, 30 mg./l, 50 mg./l, 70 mg./l, 80 mg./l, 100 mg./l, 200 mg./l, 300 mg./l, 500 mg./l. 800 mg./l and 1000 mg./l and they were incubated at 37˚C for 24 hours and 48 hours. After the incubation, OD 580 values were taken for each bacterial sample to find the LD 50 value and the maximum arsenic tolerance level of each bacterial strain.

Arsenic Analysis by ICP-OES
The bacterial cultures were centrifuged (Remi Cold 300 Isolation and Identification of Unique Arsenotolerant Exiguobacterium indicum DSAM62 from Arsenic Rich Environment Centrifuge Machine, Model No.C-24) and the supernatants were taken for acid digestion. Then the samples were prepared with 69% w/v HNO 3 , using de-ionized water. Acidification to pH less than 2 was done 16 hours before the ICP-OES analysis. Necessary dilutions were made if the arsenic content exceeded the calibration standards. The nebulizer flow and plasma power were optimized (i.e., the plasma power adjusted between 1300-1400 W) using a 5 mg./l arsenic solution. Next, the nebulizer flow was incrementally adjusted between 0.90 and 0.60 L/min for radial and axial plasmas, in that order. After observing change in intensity, the best flow was established and used for the analyses. The most sensitive lines for arsenic lie in the UV region (193.759 nm.) and an appropriate spectrophotometer (Thermo Scientific, Model No. ICAP 6000 Duo) were used. For correlating the results, the arsenic standard is diluted with 1% ultrapure HNO 3 and analyzed on the instrument. After the run, the peaks and baselines are reset, the blanks are identified as global blanks, any interfering elements are identified for subsequent subtraction.

Cell viability Test Using INT Assay
The bacteria was grown in tryptic soy broth (HiMedia) with 500 mg./l, 300 mg./l arsenic As (V) and without arsenic As (V) at 37˚C for 24 hours at BOD Incubator Shaker. After the incubation, 100µl. of the inoculums were inoculated in 3 ml. of 30% TSB containing 0.5 mg./ml of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazoli um chloride (INT) (SRL). Then it was incubated for 30 minutes at 37˚C in BOD Incubator Shaker at 40 rpm shaking speed. After the incubation the supernatant was separated and removed by centrifugation and 1ml. of Dimethyl Sulfoxide (DMSO) (Merck) was added to each tube. The cell viability was quantified by measuring the absorbance at 490 nm wavelength in UV-Visible Spectrophotometer (Optizen) using DMSO as a blank (Steifel, et al., 2016).

SEM-EDX
To study the effect of arsenic on cellular morphology of S6C2 bacteria, 1% inoculum was added into LB broth containing LD 50 concentration of arsenic [500 mg./l of As(V)] and without arsenic (control) and incubated at 37°C in BOD Incubator Shaker incubator (100 rpm). Samples were harvested at mid-log phase and the samples were prepared for SEM and EDX according to De et al. (2008) and Daware and Gade, (2015). Following incubation, both control and arsenic stressed bacterial cells were harvested by centrifugation at 6,000g for 10 min, 4°C.The pelleted cells were washed twice with 0.1M potassium phosphate buffer solution (PBS; pH 7.2) and fixed overnight in 2.5 % glutaraldehyde in 0.1M PBS at 8°C. The cells were washed twice again with PBS. The cells were dehydrated through a series of different concentrations (from 30% to absolute) 1 ml. of ethanol at 10,000 rpm for 2 minutes. Finally, the pellet was dissolved in 200 µl of 100% ethanol concentration for half an hour. Finally, 2 μl of each sample was placed on 1mm clean and grease free mini round glass cover slips. The glass cover slips were coated with platinum and examined under SEM with the acceleration voltage of 20 kV (Zeiss Evo 18 Special Edition). Energy dispersive x-ray spectroscopy (EDS) (Zeiss Evo 18 Special Edition) was performed to detect the presence of arsenic over cell surface.

Identification of Bacteria by 16 S rDNA Sequencing
According to Paul et al., 2015, DNA was isolated from S6C2 bacteria. Its quality was evaluated on 1.0% Agarose Gel, a single band of high-molecular weight DNA has been observed. Fragment of 16S rDNA gene was amplified by 27F and 1492R primers. A single discrete PCR amplicon band of 1500 bp was observed when resolved using Agarose gel. The PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forward primer and reverse primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 16S rDNA gene was generated from forward and reverse sequence data using aligner software. The 16S rDNA gene sequence was used to carry out BLAST with the database of NCBI genbank database. Based on maximum identity score first ten sequences were selected and aligned using multiple alignment software program MEGA 7 followed by construction of the distance matrix and construction of the phylogenetic tree.

Identification of Strain Specific Bacteria by Fatty
Acid Methyl Ester (FAME) Analysis According to Kunitsky et al., 2005, the fatty acids were extracted from the growing bacterial culture by a procedure which consists of saponification in dilute sodium hydroxide/methanol solution (15g sodium hydroxide,50ml methanol, and 50ml distilled water) followed by derivatization with dilute hydrochloric acid/methanol solution (40.62ml certified 6.0N hydrochloric acid and 34.37ml methyl alcohol pH value below 1.5) to give the respective methyl esters (FAMEs). The FAMEs were then extracted from the aqueous phase by the use of an organic solvent (50ml hexane and 50ml methyl tert-butyl ether) and the resulting extract was analyzed by GC using Agilent HP-ULTRA 2 column which was of 25m length, 0.200mm diameter, 0.33µm film and its temperature limit is 60°C to 325°C.In that gas chromatography, Hydrogen was used as mobile phase, Flame ionization detector as detector and its flow rate was 1.3ml/min. The software automated all analytical operations and used a sophisticated pattern recognition algorithm to match the unknown FAME profile to the stored library entries for identification by RTBA6 method.

Whole Genome and Plasmid Sequencing
The DNA extraction from the bacteria S6C2 was performed using standard kit based protocols (Oyedara et al., 2018). The DNA sample quality were checked using Nanodrop and quantitated using Qubit ds DNA BR assay (Thermo Scientific, USA). The genomic DNA was fragmented using Covaris to generate 300-400bp fragments. 100 ng of fragmented DNA was used to generate sequencing library using NEBNext Ultra II DNA Library Prep Kit for Illumina (#E7645S). In brief, the fragmented DNA was subjected to end repair followed by A-tailing and adapter ligation. Ampure bead based size selection was performed to obtain the library of desired size. The size selected DNA were enriched by PCR amplification using Illumina index adapter primers. The amplified product was purified using ampure beads to remove unused primers. The library was quantitated using Qubit DNA High Sensitivity quantitation assay and library quality was checked on 2100 using Agilent 7500 DNA Kit. The library was quantitated using Qubit DNA HS quantitation assay (Thermo Scientific) which specifically quantitates dsDNA assay. For sequencing, the libraries were diluted to 3Nm Using RSB and 5 uLs of the diluted library was mixed with an equal volume of freshly prepared 70.1 N NaOH to allow the library to get denatured. The pH of the reaction was neutralized by adding 5 Ul of freshly prepared 200Mm Tris-HCl and were taken on to Cbot for cluster generation on a HiSeqX high output flow cell. Cluster generation and sequencing was performed on the Illumina 2000 system. The following pipelines were used FastQC -To run and provide a QC report to check the quality of the sequences Galaxy (Unicycler + MOB) -To assemble the draft genome and to identify plasmid presence. RAST -To annotate the nuclear genome &amp; Plasmid genome CONTIGuator -To finish the Genome assembly RNAmmer -To identify the 5s/8s, 16s/18s, and 23s/28s ribosomal RNA in full genome sequences. Genomic DNA. The circular plasmid map of the bacteria was created by CGVIEW Server. After the whole genome annotation, the existence of unique ClpB protein was identified and the bacterial whole genome sequences were submitted at NCBI under the Accession Number PRJNA555453.

Isolation of Cellular Protein and SDS-PAGE Analysis
The sample preparation was done according to Mathabe, 2014. The bacteria (S6C2) was grown in control (without arsenic) and 500 mg./l of As(V) treated condition. After the incubation the bacterial cells were centrifuged 15,000rpm for 15 minutes (Remi C-24). The pellet was obtained and the cellular proteins were extracted with 100 µl of 0.4% Triton-X. Then, the samples were centrifuged at 15,000 rpm for 15 minutes. The resulting pellet was resuspended in 4X loading buffer (consisting of 10Mm Tris-HCl pH 6, 277 mM SDS and 40% Glycerol) and it was sonicated (Hielscher UP 200S, Hielscher Ultrasonics GmbH, Germany) to recover the cellular proteins. After that, it was centrifuged and the supernatant was retained. SDS-PAGE analysis (Biorad, Richmond, CA) of the crude extract of the cellular proteins of the control and 500 mg./l As(V) treated samples were done.

Arsenic Concentration of Soil Sample
Arsenic concentration of the collected soil sample from Shyamnagar was 0.51 mg./l ( Table-1).

Physical Characterization of Soil Sample
Some physical characterization of the soil sample was done. The temperature of the soil sample was 25˚C. It was clay type because its pH value was 7.34. EC value and Specific Gravity value were 0.00247 dS/m and 0.997 respectively (Table 1).

Isolation of Bacteria
The bacterial colonies were isolated from 10 -2 and 10 -4 dilution plates. Numerous colonies were obtained in each plate. The four colonies were selected at random for further studies, i.e., morphological and biochemical characterization [ Table 2 and Figure

Biochemical Characterization of the Isolated Bacteria
The results are tabulated in Table 3 and Table 4.

S6C1
Red slant/Yellow Stab Glucose fermentation with acid production. Proteins catabolized aerobically (in the slant) with alkaline products.

S6C2
Red slant/Yellow Stab Glucose fermentation with acid production. Proteins catabolized aerobically (in the slant) with alkaline products.

S6C3
Red slant/Yellow Stab Glucose fermentation with acid production. Proteins catabolized aerobically (in the slant) with alkaline products.

S6C4
Red slant/Yellow Stab Glucose fermentation with acid production. Proteins catabolized aerobically (in the slant) with alkaline products.

Arsenic Tolerance Study
The isolated bacterial strains were inoculated in the nutrient broth (HiMedia) supplemented with increasing concentration of arsenic [As (V)] from 5 mg./l to 1000 mg./l because the bacterial strains have arsenate reduction ability. The LD 50 value of S6C1, S6C2, S6C3 and S6C4 were 30 mg./l, 500 mg./l, 300 mg./l and 50 mg./l respectively [ Figure

SEM-EDX
The scanning electron micrographs of S6C2 bacterial cells without arsenic (control) and with exposure to arsenic [500 mg./l of As(V)] were studied in mid-log phase. Cellular morphology of S6C2 bacteria in the presence of As(V) and was changed in mid-log phase and the cells became elongated [ Fig. 3.(a) and (b)]. The energy dispersive X-ray spectroscopy (EDX) analysis showed a distinct EDX signal corresponding to arsenic peak was observed in presence of As(V) loaded cells [

Bacterial Viability Test by INT Assay
Based on the higher arsenic As (V) tolerant ability, S6C2 and S6C3 bacterial cells were tested for viability assay with the help of INT. The decreasing amount of cell viability of S6C2 and S6C3 bacteria were 41.34% and 43.35% respectively [Supplementary Table 1, Figure. Table 6).  The number of base substitutions per site from between sequences are shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Kimura 2-parameter model. The analysis involved 11 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 1494 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

Cellular Protein Isolation and SDS-PAGE Analysis
Two distinct bands were found in the As(V) treated sample at about 96 kDa and 76 kDa. Among these two bands only 96 kDa molecular weight band was found in case of 500 mg./l of As(V) treated sample, which may correspond to be ClpB protein as the gene for that protein is present in the genome assembly and previous reports (Tsuchiya et al., 2019) do predict the role of the protein in response to arsenic stress ( Figure. 6). This needs to be tested further to confirm the role of the protein under conditions of arsenic uptake in this bacterium.

Discussion
According to Castro-Severyn et al., 2017 andAlbarracin et al., 2016, psychrophilic bacteria Exiguobacterium was normally found in the extreme environmental condition of Chilean Altiplano, Atacama desert, Antarctic ice (Carneiro et al., 2012), Himalayan glaciers (Chaturvedi and Shivaji, 2006), soda lakes (Borsodi et al., 2005), hyperthermophilic hot springs (Vishnivetskaya et al., 2009), and deep-sea hydrothermal vents (Vishnivetskaya et al., 2009 andCrapart, 2007). They had been reported to persist in effluent of paper mills and in rhizospheres of Lemna minor (Kashamaet al., 2009 andTang et al., 2013). The genetic differentiation of Exiguobacterium were found due to the ecology of diverse habitats (e.g., CuatroCiénagas: Rebollar et al., 2012). According to Castro-Severynet al., 2017 Exiguobacterium sp. Strain SH31 was arsenic resistant and it was able to survive in the media supplemented with 100 mM of Arsenic; i.e. As(V). Pandey and Bhatt, 2015 described in their study that Exiguobacterium sp. (As-09) was tolerant to arsenic upto 700 mM, which was isolated from an arsenic contaminated soil from Rajnandgaon district, Chattisgarh, India. Sacheti et al, 2013, isolated Exiguobacterium sp. PS from the arsenic contaminated site in India. According to the study of Belifore, et al., 2013, that arsenic resistant Exiguobacterium sp. S17 was isolated from a high-altitude Andean lake stromatolite.
In the present study, the four bacteria were isolated from the soil sample of Shyamnagar, 24 Parganas (North). The LD 50 value of the isolated S6C1, S6C2, S6C3 and S6C4 bacteria were 30 mg./l, 500 mg./l, 300 mg./l and 50 mg./l of arsenic As (V) respectively. The uptake ability of S6C1, S6C2, S6C3 and S6C4 bacteria were 11.46%, 52.2%, 11.56% and 10.84% respectively. Depending on their higher arsenic tolerance ability the cell viability test through INT assay were done in case of two bacteria viz. S6C2 and S6C3 bacteria among these four bacteria. In this assay, the decreasing cell viability value of S6C2 and S6C3 bacteria were found to be 41.34% and 43.35% in their respective LD 50 value i.e 500 mg./l and 300 mg./l of arsenic As(V) respectively and the amount was the near about 50%. From these results, it was concluded the fact that the bacteria S6C2 isolated shows an extreme tolerance towards this heavy metal and this indicates that further molecular studies must be performed in order to understand the molecular basis of this tolerance in order to shed light on the fact that how they can be used in contaminated fields as a potential bioremediator of this heavy metal. Metal tolerant bacteria usually have the ability to internalize and metabolize heavy metals to more non toxic gaseous forms (Chen et al .2014) such that a part of the arsenic in the biomass can be encompassed within the atmosphere essentially reducing the gross quantitated level of arsenic from the soil.

Conclusions
From the present study it is concluded that isolated unique Exiguobacterium indicum Strain DSAM62 is highly arsenic tolerant and its efficacy of arsenic uptake is 52.2% from its respective LD 50 dose of arsenic, which will be utilized in arsenic mitigation strategy of agricultural and industrial sludge treatment.16S rDNA sequence of Exiguobacterium indicum Strain DSAM62 was deposited at NCBI GenBank under Accession Number MH819520.Whole genome sequence including plasmid sequence of Exiguobacterium indicum Strain DSAM62 was deposited at NCBI under Accession Number PRJNA555453.