The Isolation and Identification of Novel Arsenic-Resistant Bacteria from an Arsenic-Contaminated Region—A Study to Understand the Efficiency of Bacteria for Arsenic Removal from Aqueous Media

Abstract

Drinking water sources with groundwater arsenic (As) contamination face multifaceted challenges in the removal and supply of fresh drinking water resources. To eradicate this problem, bioremediation has evolved to become more effective than other chemical and physical removal processes in its cost-effectiveness, high removal efficiency, and lesser production of secondary by-products or waste. Thus, this study aimed to treat As from aqueous media and to detoxify highly toxic forms of As by the isolated bacteria from As-affected areas. We isolated two new Gram-positive bacteria, which are reported here (Bacillus sp. and Bacillus cereus), with As⁵⁺ minimum inhibitory concentrations (MICs) of 4500 mg/L for the Bacillus sp. and 1000 mg/L for Bacillus cereus; meanwhile, for As³⁺, the MICs are 600 mg/L for both isolates. Bacillus sp. and Bacillus cereus can also effectively convert the highly toxic and easily mobile As³⁺ to As⁵⁺ in aqueous media. This study also demonstrates that these bacteria can remove a significant proportion of As³⁺ and As⁵⁺ (averaging 50% for both) from aqueous media. These As-resistant bacteria from the As-affected area can be used and upscaled for the treatment of As for a safer drinking water supply.

1. Introduction

To meet the growing needs of this overpopulated planet, increased industrialization, urbanization, and enhanced agricultural activities are consequently causing pollution in almost all components of the biosphere. During the past few decades, heavy metal concentrations in soil and water have increased tremendously from such anthropogenic activities as well as geogenic sources [1]. Arsenic is one of these heavy metals (metalloid), which has both anthropogenic and geogenic sources in the natural environment [2], and it has been reported as one of the major contaminants in the environment in almost 70 countries [3,4]. The severity of the harmful effects of arsenic contamination has been realized through new arsenic-affected areas being reported almost every day [5].

Although soil and water arsenic contamination has been reported all over the world, the problem is more prominent around Asia, specifically Southeast Asia. In India, groundwater arsenic contamination was first reported in Chandigarh [6], and in 1980, a second case was reported in West Bengal’s Lower Gangetic Plains [7]; gradually, several reports of groundwater arsenic contamination started being shared from almost 14 states of India [8,9,10]. Although very small amounts of arsenic come to groundwater systems from anthropogenic activities like the excessive abstraction of water from shallow as well as deep aquifers, pumping for irrigational uses, the use of arsenic-containing fertilizers, etc. [11], the majority of arsenic comes from geogenic sources. It can be noticed in the map of arsenic contamination in the Bengal Basin that most arsenic contaminations occur in and around the catchment area of the Ganges–Bramhaputra–Meghna river system, which comes from complex sources including arsenic-containing parent rocks situated in the Himalayan region and its foreland areas [12], sediments of the Bengal Basin and Bihar mica belt, and arsenic-enriched fluvial sediments widely transported from the Himalayas [13].

During the early 1970s, due to the huge pollution load on surface water, people started using groundwater as a clean and perennial source of drinking water, mainly in the Bengal Basin, which in turn resulted in some negative impacts, like the extensive withdrawal of groundwater leading to exposure to arsenic of a significant proportion of the local population through their drinking water [13]. In the past decade, groundwater arsenic contamination has become one of the biggest public health concerns all over the world [14]. Maximum amounts of arsenic enter the human body through drinking water, which may cause lethal effects like cancer in the bladder, digestive system, lungs, and skin after the long-term consumption of water containing more than 50 µg/L of arsenic in drinking water [15]. Even if the concentration is less than 50 µg/L, health effects like severe nausea, vomiting, irregular heartbeat, unusual bleeding, exhaustion, and nervous system injury may be experienced. It also has an impact on regular blood circulation by breaking red blood cells as well as inhibiting the production of blood cells. The non-cancer effects include brain damage, liver enlargement, darkening of the skin, and damage to the immune system, cardio-vascular system, and reproductive system [16].

In spite of arsenic being toxic to most of the life forms on earth, including bacteria, in various times from past decades to recent years, different research groups have reported quite a few types of bacteria to be highly or moderately arsenic-resistant because of their potential to cope with or to remove harmful arsenic from their surrounding environment through various detoxification mechanisms [17,18,19]. Reductions in arsenic uptake by methylation, the oxidation of arsenite to less toxic arsenate [20,21], the adsorption of negative arsenic ions onto the bacterial cell wall by positive amino groups present there, chelation, compartmentalization, the sequestration of arsenic by cysteine-rich peptides, exclusion, immobilization, and dissimilatory arsenate respiration are some of the common detoxification mechanisms [19]. Because of the above-mentioned potential of microbes, scientists in recent years have been more interested in bioremediation methods over chemical methods—which have high costs and cause irreparable damage by producing toxic by-products—in the cleaning of contaminated ecosystems [22,23]. The advantages of bioremediation are that it is relatively inexpensive, environmentally benign, and is a green technology; hence, it should be more acceptable to the public. In addition, the use of microbes in the bioremediation of arsenic-contaminated soil also improves soil quality by promoting nitrogen fixation and reducing the bioavailability of other inorganic and organic contaminants; in addition, they can control the speciation and cycling of arsenic in the ecosystem [24,25]. Quite a few types of bacteria from genera Acidithiobacillus, Aneurinibacillus, Bacillus, Deinococcus, Desulfitobacterium, Pseudomonas, etc., have already been reported to be resistant to high concentrations of arsenic [21,26,27,28], but very few of these reports have tried to use these resistant bacteria for the removal of arsenic from contaminated water.

Purbasthali II block is located in the Burdwan district of West Bengal, India, and it is known for having high levels of arsenic in the groundwater, which poses a serious health risk to the local population [29]. According to this study, the groundwater in Purbasthali II and other nearby areas in the Burdwan district have arsenic concentrations that exceed the permissible limit of 10 µg/L as recommended by (WHO). The study found that more than 50% of the wells in the area had arsenic concentrations above the WHO guideline value, putting the health of the local population at risk, which requires urgent action to address the problem.

Thus, from the above backdrop, the major objectives of this study were (1) to isolate and identify microorganisms resistant to arsenic from this particular region affected by arsenic (Purbasthali II block of Burdwan district, West Bengal), (2) to determine the minimum inhibitory concentration of arsenate and arsenite for the isolated bacteria, and (3) to examine their efficiency for the transformation of arsenic to a less toxic form and for the removal of arsenic from contaminated water.

2. Materials and Methods

2.1. Study Area

As Purbasthali II block in Burdwan district, West Bengal, India, was previously reported for arsenic contamination [21,30], the collection of soil samples was conducted from this particular area that is located between 23°29′28.3″ N and 23°31′54″ N latitude, and 88°17′4.3″ E to 88°21′56.1″ E longitude.

The area is climatically tropical dry and subhumid throughout the year with an average annual temperature of 25 °C; its highest temperatures (40 °C) are during the summer months and lowest during winter (15 °C), respectively. The region receives adequate rainfall during the monsoon season, typically between June and September, which is crucial for agriculture in the area. The present study area is geologically located on the alluvial Ganges delta and is composed of sedimentary rock formations, including sandstones, shales, and clays. The presence of arsenic in the sedimentary rock formations in the region is a major factor contributing to the high levels of arsenic contamination in the groundwater [29].

A total of fourteen sites from twelve villages (Figure 1) in this block were selected for the collection of soil samples, namely Kalyanpur (A1, A2, and A3), Misbahpur (B), Paschim Atpara (C), Purba Atpara (D), Kamalnagar (E), Laxmipur (F), Natun Laxmipur (G), Dhamas (H), Sinhari (I), Tamaghata (J), Majida (K), and Rukuspur (L).

2.2. Collection of Soil Samples

The areas close to the arsenic-affected tube wells (3–5 m around the wells) were selected for the collection of soil samples. After removing 1 cm of the topsoil, approximately 100 g of soil samples was collected, thoroughly mixed, transferred into sterilized polyethylene zipper packets with proper levels, and stored in thermal boxes as soon as possible following retrieval. In total, 5 samples were collected from each village during the months of May–June, i.e., in the pre-monsoon or summer season, and stored at 4 °C for subsequent analysis. Microbial analysis was carried out within 24 h of soil sample collection.

2.3. Chemicals and Reagents

All the chemicals used in the present study were AR-grade chemicals that were procured from M/S. Merck India Ltd. Double-distilled water was used to prepare all of the reagents and standards. For the preparation of standard As(V) and As(III) solutions, sodium arsenate hydrate (Na₂HAsO₄·7H₂O) and sodium arsenite (NaAsO₂) were used, respectively. Media used for microbial analysis were procured from HiMedia Laboratories Pvt. Ltd., India. Before being used for any experiment, each piece of glassware was carefully cleaned by soaking it in 15% HNO₃ and then rinsing it again with double-distilled water.

2.4. Habitat Characterization

Soil samples were air-dried, ground to a fine powder, and sieved for further analysis at controlled laboratory conditions. A 1:10 (weight/volume) soil–water suspension was prepared, and pH and conductivity were measured using a digital pH and conductivity meter (Model 304-Systronics) [30]. The standard spectrophotometric methods of Walkley–Black (1934) [31], Kjeldahl [32], and Olsen’s method [33] were used for the estimation of soil organic carbon, nitrogen, and available phosphorus, respectively. The exchangeable water-soluble potassium was determined by the ammonium acetate extraction method developed by Sarkar and Halder in 2010 [34]. Exchangeable sodium in the soil solution was measured with the help of a flame photometer (Systronics Flame Photometer 130) by adjusting the blank and standard solutions [34]. For the measurement of soil arsenic content, first, 0.5 g of dry soil powder was added to an acid mixture (9:1 sulfuric and nitric acid) and digested until it was evaporated to dryness. The residue was diluted up to 100 mL with double-distilled water and the quantitative determination of arsenic in this solution was carried out spectrophotometrically following the standard silver diethyl dithiocarbamate (SDDC) method of APHA [35] (2005) [21,28,36,37] with a minimum detectable limit of 1 µg [38].

2.5. Isolation of Bacteria

The bacteria that are capable of surviving in arsenic-contaminated environments by evolving mechanisms to detoxify arsenic or to use it as an energy source are known as arsenic-resistant bacteria. The serial dilution of the soil sample was performed by mixing 1 g of soil with autoclaved distilled water and 1 mL from the 10⁻⁵ dilution was inoculated onto a nutrient agar plate and incubated at 37° C (±1) for 24 h. The colony’s morphology was studied under a microscope. To purify different colonies, morphologically distinct colonies from the agar plates were randomly selected and streaked onto a nutrient agar slant and placed in the incubator for another 24 h. Nutrient broth media was prepared with different concentrations of arsenite and arsenate solutions, and the isolates were cultured in these media. The growth of the bacterial culture was measured after 24 h in terms of optical density at the wavelength 660 nm. The same media without any bacterial culture were used as blanks to observe the growth of the isolates under arsenic stress [21,28,39].

2.6. Viable Cell Count

A viable cell count experiment was executed for the verification of the arsenic resistance of isolated strains [21,40]. All isolated strains were first inoculated in nutrient broth prepared with 100 mg/L of arsenate or arsenite and incubated for up to 72 h. Then, every 24 h, cultures were transferred onto nutrient agar plates prepared with and without arsenic solution, and the colony-forming units (CFUs) were counted after 24 h of incubation.

2.7. Establishment of Minimum Inhibitory Concentration

A minimum inhibitory concentration (MIC) is the lowest concentration of a chemical that is needed to inhibit the growth of a particular microorganism, typically a bacterium or fungus [41]. MIC testing is an important tool for determining the effectiveness of a chemical against a specific microorganism. The test involves exposing the microorganism to various concentrations of a particular chemical and then observing the growth of the microorganism in each concentration.

In our study, the MIC of arsenic for the isolated strains was determined following the method developed by Brown (2007) [42]. All the selected isolates were inoculated in nutrient broth media amended with different concentrations of either sodium arsenate (100 mg/L to 1000 mg/L with a 100 mg/L intermediate difference and then up to 5000 mg/L with a 500 mg/L intermediate difference) or sodium arsenite (100 mg/L to 1000 mg/L with a 100 mg/L intermediate difference) and then incubated in a shaker incubator at 37 °C (±1) for 24 h. Two sets of control (one for sodium arsenate and the other for sodium arsenite) were also prepared without bacterial inoculation. The growth of the isolates under various arsenic stresses was measured in terms of the optical density of the culture medium at 660 nm.

2.8. Morphological and Biochemical Characterization and Scanning Electron Microscopy of the Isolated Arsenic-Resistant Bacteria

Gram staining, which is a common morphological characterization technique, was carried out in order to differentiate the bacteria on the basis of the chemical and physical properties of the cell walls by detecting the presence of peptidoglycan on the cell wall of Gram-positive bacteria [43] (Holt et al., 1994). Some selected biochemical tests, namely the enzyme activity test (catalase, indole, oxidase, and urease), methyl red test, nitrate reductase, Voges–Proskauer, ability to produce H₂S, salt (sodium chloride) tolerance test, utilization of citrate, carbohydrates, starch and lipid, and antibiotic sensitivity test were carried out according to the standard method of Pelczar et al. [44] (1957), Lacey [45] (1997), and Brown [42] (2007). For studying the surface morphology of the isolated arsenic-resistant bacteria, they were observed under a 15 kV scanning electron microscope (HITACHI, S-530, SEM, and ELKO Engineering) after proper treatment and preparation [21].

2.9. Determination of Optimum pH and Temperature

Nutrient broth was prepared in different test tubes and the pH was adjusted using either a mild HCl solution or mild NaOH solution to attain pH 2, 4, 6, 8, and 10. Isolated bacteria were then inoculated into these media and incubated at 37 °C in a continuous shaking condition. After 24 h, bacterial growth was measured in a spectrophotometer at 660 nm against a nutrient broth solution of the same pH without bacteria.

In another set of experiments, each strain (in nutrient broth) was transferred into four test tubes and then placed into four different temperatures: 30 °C, 37 °C, 45 °C, and 60 °C. Optical density was measured after 24 h against the blank nutrient broth solution [46].

2.10. Arsenic Bioremediation Test by the Isolated Bacteria

Isolated bacteria were cultured in nutrient broth media in two sets. In the first set, the broth was prepared in 100 mg/L of sodium arsenite solution, and the second was prepared in sodium arsenate of the same concentration. All the sets were placed in the shaker incubator at 37 °C ± 1 with continuous shaking. Control nutrient broth solutions were also prepared for both arsenite and arsenate without bacteria. In every 24 h (up to 72 h), 10 mL of culture solution was taken and centrifuged at 10,000 rpm for 10 min in order to separate the bacterial cells from the solution. Then, the arsenic concentration of the supernatant was measured following the SDDC method according to APHA [21,30].

2.11. Determination of Arsenic Oxidation and Reduction Potentiality of the Isolated Bacteria

Silver nitrate solution was used for the determination of the reduction of arsenate or the oxidation of arsenite by the isolated novel bacteria [21,28,46]. Bacteria were cultured on the nutrient agar plate amended with either sodium arsenate or sodium arsenite for 24 h. Control plates were also prepared for both cases without bacteria. After the addition of silver nitrate solution to the plates, the formation of brown precipitation confirmed the presence of silver arsenate, and the yellow precipitation confirmed the presence of silver arsenite.

2.12. Genomic DNA Sequencing

A chromous genomic DNA isolation kit was used to isolate the genomic DNA from pure culture of each strain, and the ~1.5 kb fragment of the genomic DNA was amplified by high-fidelity PCR polymerase. The bidirectional sequencing of the PCR product was performed in a Genetic Analyzer (ABI 3130 Genetic Analyzer), where universal Bacillus-specific forward and reverse primers were used. The sequence data were analyzed in the NCBI BLAST program and aligned with other similar sequences collected from the GenBank database using the ‘ClustalW Submission Form’ (http://www.ebi.ac.uk/clustalw/), which were analyzed by ClustalW24. The evolutionary distances between closely related sequences with the isolated bacteria were calculated to prepare the phylogenetic tree by following the ‘Neighbor Joining’ method developed by Saitou and Nei [47].

3. Results

3.1. Soil Physicochemical Properties

The results of the physicochemical characterization of the collected soil samples are presented in Table S1 (Supplementary Information). From each sampling site, five samples were collected, and the results presented in Table S1 are the mean values of these five samples. The soil in the entire area was characterized as alkaline, as the average pH value of all soil samples was 7.88. On average, the total organic carbon and total nitrogen content of the soil samples in this region were 1.55% and 0.05%, respectively, in the dry season. The phosphorus, sodium, and potassium concentrations were recorded at 182 kg/ha, 63.9 kg/ha, and 52 kg/ha, respectively. Although the average soil arsenic concentration of this area was 1.17 kg/ha, the highest contamination was recorded from three samples (A1, A2, and A3) collected from the same village, Kalyanpur.

3.2. Isolation and MIC of Arsenic Resistant Bacteria

From all the soil samples of Purbasthali II block, thirteen morphologically distinct colonies were picked up for the examination of arsenic resistance potentiality. Seven out of these thirteen isolated colonies showed an arsenic resistivity of varying levels among which particularly two isolates showed the highest capability. These two isolates, namely SS6 and SS10, were selected for further experimentation. The growth of the isolate SS6 first increased with increasing arsenate concentration in the media up to 2000 mg/L, and then started to decrease gradually and finally stopped growing at a concentration of 4500 mg/L of arsenate (Figure 2a). But, the growth of the isolate SS10 was completely inhibited by a 1000 mg/L (Figure 2b) arsenate concentration. In the case of arsenite, both the isolates first showed increased growth up to a 300 mg/L concentration, and there was absolutely no growth at 600 mg/L (Figure 3a,b) but very little growth was observed at a 550 mg/L arsenite concentration. Hence, the MICs of arsenate were 4500 mg/L and 1000 mg/L for SS6 and SS10, respectively, and for arsenite, they were 600 mg/L for both isolates.

3.3. Presence of Viable Cells

This particular experiment was executed to determine whether the isolated bacteria were truly arsenic-resistant and to examine the influence of arsenic on their growth. Both bacteria in the plates without arsenic showed increased growth up to 48 h and stopped growing at 72 h. But, in the case of the plates supplemented with arsenate and arsenite, both bacteria continued their growth increment in terms of CFU/ml for up to 72 h (Figure 4a,b). In both cases, the bacterial growth was slightly better in terms of CFUs in the presence of arsenic than in its absence.

3.4. Morphology and Biochemistry of the Isolates

The colony characteristics of isolate SS6 were round, white, smooth, opaque, flat, and multilayer concentric, whereas isolate SS10 had round and white-colored colonies with a smooth surface, they were flat and concentric, and the appearance of the periphery of the colony was opaque with a transparent center. Both isolated bacteria were Gram-positive, and from the scanning electron micrograph, it was observed that individual bacterial cells were rod-shaped (Figure 5a,b) and spore-forming in nature. The two bacterial strains were positive for catalase enzyme production and neither of them could produce indole or the urease enzyme. In the case of the oxidase enzyme, isolate SS6 was negative and SS10 showed a positive result. Isolate SS6 and SS10 were both acid-producing bacteria, as they showed a positive result in the methyl red test. Both isolates were negative in the V-P test, nitrate reductase test, and citrate utilization test. The bacteria could not produce H₂S gas as there was no black patch when they were cultured in TSI media. The results of the TSI test are summarized in

Table 1. Growth results of the isolated bacteria on Triple Sugar Iron (TSI) agar medium.

Isolate No.	Fermenter	CO2 or Other Gas Production	Butt Color	Slant Color	H2S Production
SS6	Glucose fermentation only	+ve	Yellow	Pink	−ve
SS10	Glucose, lactose, and sucrose non-fermenter	−ve	Pink	Pink	−ve

. Neither of these two bacteria was able to hydrolyze starch and gelatin, but they were able to hydrolyze lipids (fats) to glycerol and fatty acids, as they have the lipolytic enzyme lipase. Various carbon source utilization and acid/gas production patterns were different for these two bacteria, which are presented in

Table 2. Acid (+/−) and gas production (gas) by bacterial isolates against various carbon sources.

Carbon Source	SS6	SS10
Glucose	+/gas	+
Sucrose	+	+
Lactose	+/gas	−
Mannitol	+/gas	−
Arabinose	+	+
Fructose	+	+
Maltose	+	+
Salicin	+	+
Inositol	+	+
Mannose	+	+
Aesculin	−	+

. Sodium chloride is not mandatory for all bacteria, but sometimes, the addition of NaCl to growth media has a positive influence on the growth of certain bacteria, which are known as halotolerant bacteria [48,49]. Isolate SS6 and SS10 were mildly halotolerant in nature and SS6 could tolerate up to 6% while isolate SS10 was able to tolerate up to 4% of salt (Figure 6a,b). Two of the most significant environmental variables that might affect the growth of bacteria are pH and temperature. The metal accumulation property is also greatly affected by the pH of the solution [46]. The pH range of 6 to 7 was ideal for the growth of these two bacteria, although they were able to survive in a wide range of pH from 4 to 10 (Figure 7a,b). Compared to other organisms, bacteria are more resilient to environmental stressors. Every species, however, has unique traits and a preferred temperature range for growth and reproduction. The maximum growth of the isolated bacteria occurred at 37 °C, and like pH, they could survive in a wide range of temperatures from 30 °C to 60 °C (Figure 8a,b). So, based on the optimum temperature of the growth of the bacteria, they were identified as mesophilic in nature.

3.5. Bioremediation of Arsenic

In our study, the bacteria, isolated from arsenic-affected soil and unaffected by elevated levels of arsenate and arsenite, were also examined for their potentiality to remove arsenic from a chemically defined culture media supplemented with particular concentrations of either arsenate or arsenite. According to the results, the bacteria were able to remove significant amounts of arsenate and arsenite from the nutrient broth medium. The removal of arsenic increased gradually with time, but after 72 h, the percent removal did not increase further and reached an equilibrium condition. The patterns of arsenic removal by the isolates are presented in the following bar graphs for better realization (Figure 9a–d).

3.6. Microbial Oxidation of Arsenic

In the present study, following the inclusion of silver nitrate to the overnight grown culture plate, the solution turned brown (Figure 10) in all cases, indicating arsenate’s presence in the media. In the arsenate-amended plates, arsenate was already present because the bacteria lacked the ability to reduce arsenate whereas in the arsenite-amended media, arsenate was created by the bacteria-oxidizing arsenite.

3.7. Identification of the Isolates

The two isolated highly arsenic-resistant bacteria were identified on the basis of biochemical analysis and similarity with other closely related bacteria whose gene sequences were obtained from the GenBank database, which were then used to prepare the phylogenetic trees for both isolates in order to determine the relation between other bacteria. The isolate SS6 was identified as Bacillus sp. (Figure 11) and SS10 as Bacillus cereus BS14 (Figure 12). The 16S rRNA sequence data of these two unique bacterial species were submitted to GenBank and the bacteria were given specific accession numbers, which are KT462573 for SS6 and KT462576 for SS10.

4. Discussion

Wide variations were observed in the physicochemical properties of the soil samples collected from this arsenic-contaminated area. Among all the physicochemical properties, soil organic carbon showed a positive correlation with the microbial diversity in low concentrations. Other properties like nitrogen, phosphorus, potassium, and sodium did not imply any direct effect on the microbial diversity of soil, but these have effects on the growth of the vegetation, which indirectly impacts soil organic carbon content [28].

The microbial communities in highly arsenic-rich environments eventually evolved themselves to be highly resistant to arsenic in order to maintain their natural life cycle without being affected by the toxic effect of arsenic. In our present study, we are reporting two novel bacterium species isolated from arsenic-contaminated soil samples, which can tolerate up to 4500 mg/L and 1000 mg/L of arsenate and 600 mg/L of arsenite concentrations. The tolerance towards high concentrations of arsenic of the isolated bacteria is due to the ecological fitness of the bacteria in the stress condition as well as due to the prolonged exposure to arsenic-contaminated environments [4,50]. There are previous reports of arsenic-tolerant bacteria isolated from different environmental media, which are able to withstand a varying range of arsenic concentrations [4,19,21,28,51,52,53,54,55]. But, these two bacteria were able to tolerate more extreme concentrations of arsenic than those previously reported. It is already well documented that the bacteria that reside in highly heavy metal-contaminated environments must develop some mechanisms of detoxification for their survival under stress conditions [28,40,53]. The metal-resistant bacteria can withstand high-stress conditions because they are able to chemically transform into less toxic forms through some processes like methylation–demethylation, oxidation–reduction, precipitation, etc., which are parts of their normal metabolic process [28,56,57]. This detoxification mechanism often includes biofilm formation around the cell, which restricts the entrance of arsenic to the interior of the cell and prevents the damage of cell organelles [58] or the extrusion of arsenic from the bacterial cells with the help of specific ars genes. This gene comprises a single transcriptional array containing either arsRBC or arsRDABC. Among all these genes, arsB is a kind of membrane protein that mainly takes part in the pumping out of arsenic from the cell in order to detoxify the inside environment [28]. The thicker cell walls of the Gram-positive bacteria also restrict the entrance of arsenic into the cells, thereby rendering the resistance mechanism [59]. But, in most of the cases reported, the arsenic resistance of the bacteria is due to the presence of specific genes [40,60,61,62,63]. From the viable cell count test, it was observed that the growth of the isolates was better in the media amended with arsenic than in the normal media because the bacteria were able to utilize arsenic as a supplementary energy source [28,40]. One repressor protein, arsR, is also responsible for the resistance of the bacteria against arsenite, as the protein has a very specific binding site for arsenite and can discriminate it effectively [19].

Very little action has been taken to employ the numerous arsenic-resistant bacteria that have been identified over time from all over the world as a viable method of removing arsenic from the environment. As this method produces less waste and is less expensive than many other removal methods, microbial bioremediation has always been superior. In our study, both bacteria have the ability to reduce arsenic concentration in arsenic-amended media. The isolate SS6 could remove almost 60% of arsenate and 50% of arsenite, and for isolate SS10, the removal efficiencies were 53% and 51%, respectively, from nutrient broth media amended with arsenate or arsenite in 72 h. The decrease in the concentration of arsenic in the media is due to the biotransformation and precipitation of arsenic by some specific genes as suggested by Salam et al. [55].

When treating water that has been contaminated with arsenic, the microbial oxidation of arsenate is always more advantageous than the conventional chemical oxidation process, as it does not need the addition of oxidizing chemicals that might create additional problems in the water quality. These two bacteria were also able to oxidize arsenite to less toxic arsenate, which works as a part of the metabolism of arsenic in many bacterial cells. As previously reported, the optimum temperature for the growth of the arsenic-oxidizing bacteria is 40–50 °C [46], our studied bacteria could also grow in a wide range of temperatures, with 37 °C being the optimum. The aoxR, aoxB, and aoxC genes present in the genomic DNA are mainly responsible for the arsenic oxidation potentiality of bacteria [53,55]. The presence of these specific genes responsible for arsenite oxidation was reported previously by Chang and Kim [60] in the arsenic-resistant bacteria isolated from the Damyang and Woopo wetlands in China and in the indigenous arsenic-resistant Pseudomonus bacteria isolated from the middle Gangetic plain of Bihar, India [53]. In some arsenite-oxidizing bacteria, a specific enzyme—called periplasmic arsenic oxidase—is responsible for the oxidation of arsenite. Two protein subunits, namely Fe-S—which is the small part—and Mo-pterin—which is the large unit—joined together to form this specific enzyme, and the gene that acts behind the production of this enzyme is identified as aioA [64]. Recently, research has been ongoing to commercialize the purified form of this enzyme to be used in the laboratory for the bioremediation of arsenic on a large scale and in the development of arsenic biosensors [46]. Most of the heterotrophic arsenite-oxidizing bacteria use the oxidation process as a detoxification mechanism while some bacteria utilize arsenite as an alternative energy source; these are mainly autotrophic in nature [65].

Our study reports—for the first time—the natural presence of arsenic-resistant bacteria in the arsenic-affected soil of Purbasthali II block, Purba Bardhaman district, West Bengal, which also exhibits the unique properties of removing and transforming arsenic into something less toxic as a mechanism of general metabolism as well as detoxification (Figure 13). These novel strains could be exploited in the bioremediation of arsenic from contaminated drinking water as this process is emerging as superior to conventional chemical and physical methods, although the efficiency can differ in the actual polluted field than in the laboratory condition.

5. Conclusions

Metals and metalloids can easily accumulate in the biological system, which is directly responsible for several instances of metabolic and physiological damage. Parallel to the Industrial Revolution, recent advancements in science and technologies have created new vistas for overcoming this problem through the use of natural resources including microorganisms rather than conventional chemical methods. This is absolutely a new pathway for the abatement of toxic substances from the environment; it is also cost-effective and relatively safe. In this study, we have reported two highly arsenic-resistant bacteria isolated from arsenic-affected soil of Purbasthali II block, Purba Bardhaman district, West Bengal. These two Gram-positive, rod-shaped, and spore-forming bacteria were able to withstand 4500 mg/L and 1000 mg/L of arsenate, respectively, and 600 mg/L of arsenite. Almost 50% of the arsenic could be removed by culturing these bacteria in the arsenic-amended media. They were also able to oxidize arsenite into its less toxic form arsenate, but neither of the isolates was able to reduce arsenate to arsenite. The oxidation of arsenite by bacteria is considered to be a detoxification mechanism as the end product arsenate is far less toxic than arsenite. On the basis of 16S rDNA sequence analysis, the isolates were identified as Bacillus sp. (KT462573) and Bacillus cereus strain BS14 (KT462576). Moreover, if these two unique bacteria can be exploited as novel agents for the treatment of arsenic-contaminated water, it will be beneficial for the human population as well as another step towards achieving sustainable development goals.