Arsenic, a heavy metal with an atomic number of 33 and an atomic mass of 74.92, is found in Group V of the periodic table (Szpunar et al., 1992). It has a 10-hour half-life and has no flavor or scent (Szpunar et al., 1992). Arsenic can be classified as a heavy metal because of its high atomic weight and density. Metals and metalloids that can pollute the environment as well as have harmful & fatal effects on biota are basically referred to as "heavy metals" in environmental literature (Ali and Khan, 2018). According to Ali and Khan, (2018) It can also be defined as naturally appearing metals with an atomic number greater than twenty (20) and an elemental density greater than five (5) gcm-3 (Ahmed et al., 2022).

Groundwater reservoir poisoning by naturally occurring arsenic is a worldwide problem. Drinking groundwater tainted with arsenic has impacted over 170 million people (Shakoor et al., 2016). Most people in Bangladesh, India, Pakistan, Taiwan, Canada, Brazil, Indonesia, Hungary, Mexico, the United States, Vietnam and China are badly affected by the chronic concentration of arsenic in groundwater which is close to 4000 μg/l (Shakoor et al., 2016). However, in Southeast Asia, there is a substantial danger of arsenic poisoning for around 100 million people (Shakoor et al., 2016; Rahman et al., 2011). Because it is difficult to remediate arsenic below that level, 50μg/L is still the frequently accepted standard in many developing nations, including Bangladesh (WHO, 2008). The nation with the highest rate of arsenic poisoning in drinking water is Bangladesh. The WHOs recommended level for arsenic in water is five times lower than the 50 μg/L limit set by the Bangladeshi government (Abedin et al., 2002; Uddin et al., 2025).

There are basically over 8.6 million tube wells in Bangladesh, according to estimates from UNICEF in 2008 (Uddin et al., 2011). Among all of these, 4.75 million tube wells (55%) had their arsenic levels tested; 3.3 million (39%) had green marks indicating the ground water is totally safe while 1.4 million (16%) had red marks meaning the high levels of arsenic is dangerous to use as drinking water sources (Uddin et al., 2011). Compared to other heavy metals, arsenic is more hazardous (Sher et al., 2019). The toxicity of a chemical mainly relies on its bioavailability, concentration, speciation and lack or presence of agonists as well as antagonists (Sher et al., 2019; Ali and Khan, 2017). There are two forms of it in the environment: arsenate (As5+) and arsenite (As3+) where arsenite is relatively more toxic compared to arsenate (Sher et al., 2019). Since As3+ is more soluble than As5+, its toxicity threshold is 100 times greater compared to arsenate (Sher et al., 2019). Arsenic toxicity can be lowered by employing several microorganisms which carry the ability to oxidize As3+ into As5+ with the help of a beneficial arsenite oxidizing enzyme (Rai et al., 2023).

“Bioremediation mainly refers to the process of using specific microorganisms to remove, contain and transform toxic or hazardous compounds into less poisonous and dangerous forms in the environment” (Rai et al., 2023). The interaction of different types of microorganisms with various hazardous metal ions led to the development of the concept regarding bioremediation of toxic metals (Rahman et al., 2020). Active transport is the basis for some metal detoxification processes. Although metal ions cannot be destroyed, their toxicity is reduced by the interactions of microbes. The following is how microorganisms reduce the toxicity of metals: 

1. Through oxidation and reduction processes, microorganisms can alter the oxidation states of metals. For example, in arsenic, oxidation occurs when As3+ becomes As5+ and reduction occurs when As5+ becomes As3+ (Sher et al., 2019).
2. Toxic metals can be eliminated from the contaminated site by using microorganisms to make the metal more soluble (Sher et al., 2019).
3. By precipitating the metal ions out of the soil solution, microorganisms can render them immobile (Sher et al., 2019).

Numerous bacterial species that are crucial to the detoxification and elimination of various metal ions, including arsenic, have been identified and isolated (Sher et al., 2019; Rai et al., 2023; Khan et al., 2015). “Green in 1918 identified the first bacteria which holds the ability to oxidize arsenite known as Bacillus arsenooxydans” (Sher et al., 2019). In the meanwhile, numerous arsenic-oxidizing bacteria have been identified and isolated (Sher et al., 2019) like “Alcaligenes faecalis, Pseudomonas arsenitoxidans, Microbacterium lacticum, Agrobacterium tumefaciens, Microbacterium oxydans, Pseudomonas stutzeri, Aeromonas sp., Agrobacterium sp., Comamonas sp., Enterobacter sp., Pseudomonas sp. and Pseudomonas lubricans” (Sher et al., 2019; Mou et al., 2020).

Three genes that are simultaneously transcript make up the arsenic-resistance operon or Ars operon (Rai et al., 2023). “It is composed of three genes: arsR which codes for the repressor gene; arsB which codes for the arsenic efflux pump and arsC that basically codes for the intracellular arsenate reductase enzymes” (Rai et al., 2023). This operon has two more genes, ArsA and ArsD, in addition to these three. Consequently, arsRDABC is the whole-gene sequence in this main cluster (Rai et al., 2023). Seven of the 61 ORF involved in metal resistance processes were identified from the whole genome sequence of Pseudomonas putida (Cánovas et al., 2003). The objective of this study is to isolate and characterize arsenic resistant bacteria from arsenic-contaminated groundwater samples and observe their oxidation activity to use them for bioremediation. 

Area of the Study  

The area was selected based upon the availability of arsenic contaminated area of Moharajpur union, Chapainawabganj, Bangladesh. A total of 15 samples were taken from villages named Mintowla, Namo Miapara, Miapara, Tamlipara, Hindupara and Pukurtuli (Fig. 1).  

Fig. 1: Site map of the study area Moharajpur union, Chapainawabganj, Bangladesh.

Sample Collection  

Sample was taken from a number of residential areas in Chapainawabganj, Moharajpur Union, where the water source was still in use. The sampling took place in 2021 between November 28 and December 3. From the water source (dug wells, pumps, and tube wells), 500 milliliters of each sample were taken and placed in a new water bottle. Samples were appropriately labeled, and a record book was used to record the sources information (such as name, address, and kind of water source). Without compromising the waters physiological qualities, samples were transported securely to the Faculty of Biotechnology and Genetic Engineering lab, Sylhet Agricultural University. 

Physico-Chemical Analysis of Water  

The HACH EZ Arsenic Test Kit was used to measure the samples arsenic levels in parts per billion on the same day they were collected. Volunteers from DASCOH assisted in measuring the samples pH and temperature using their equipment. 

Isolation of Bacteria from Water Sample  

The water sample was diluted for several folds (up to 10-5) and 50 µl sample was taken in an agar plate. The plates were then incubated at 37℃ for a whole day (24 hours). The different morphological isolates were selected and subcultured in nutrient agar medium for obtaining a pure culture for further arsenic resistance test.  

Isolation of Arsenic Resistance Bacteria

Several pure colonies of microorganisms were obtained and cultured for the entire night in a nutrient broth medium. Agar plates containing 1.33 mM sodium arsenite (NaAsO2) were mainly used to hold 100 ml of the culture that had developed overnight. For 72 hours, the plates were incubated at 30°C. Following effective purification by repeated growth on agar plates, the colonies exhibiting resistance to As3+ were chosen and kept at 4°C. Because their colonies developed more on the agar plates treated with sodium arsenite than other cultures, CM-11, CM-12, CM-31, CM-61, CM-101, CM-102, and CM112 were chosen.

Minimum Inhibitory Concentration (MIC) Determination  

The minimum concentration of a drug required to fully halt the development of a microbe is defined as the minimum inhibitory concentration (Dey et al., 2024). The agar dilution method was genuinely used to establish the minimum inhibitory concentration (MIC) of arsenite at which colony growth will not occur (Aridi et al., 2024; Pandey et al., 2015). A culture of bacterial isolates in the exponential growth phase in nutrient broth was aseptically added to agar plates which were supplemented with varying concentrations of arsenite (10, 20, 40, 100, 200... up to 2000 mM/L) (Ghodsi et al., 2011). At 35°C, the plates were incubated for 48 hours (www.biosciencejournals.com). The morphology of the colonies following incubation was used to firmly determine the minimum concentration of arsenite. Positive tolerance was demonstrated by the minimum concentration of arsenite that permitted the isolates to develop (Hassen et al., 1998; Shahen et al., 2019).  

Morphological Identification and Biochemical Tests  

Gram staining was used to identify morphology, and the results were examined under a microscope. Using the Microbiology Laboratory Manual as a guide, several biochemical assays were conducted to primarily identify target bacterium (Shakoori et al., 2010). To establish a pure culture, the examined bacteria were grown on nutrient agar plates at 37ºC in an incubator. Tests for indole, citrate utilization, methyl red (MR), catalase, oxidase, triple sugar iron (TSI) and Vogues Proskeur (VP) were conducted, and the results were documented (Shakoori et al., 2010). 

Antimicrobial Activity  

Chloramphenicol, Ampicillin, Ceftazidime, Cefepime, Erythromycin, Cefotaxime, Gentamicin, Ceftriaxone, Colistin and Amoxicillin discs were used to analyze the antimicrobial resistance on the isolates. Antimicrobial activity was carried out using the disc diffusion method also known as Kirby-Bauer method (Yousufi et al., 2012; Bauer et al., 1966). Initially, an agar plate was used to prepare the inoculum. Each colony was then touched with a loop and placed into 5-6 milliliters of nutritional broth. To achieve the desired turbidity, the broth culture was next incubated for 24 hours at 37°C (Bou et al., 2007). After that, 40 milliliters of Muller-Hinton agar media were added to each plate (Bou et al., 2007). 

Then, using a micropipette, 80 μl of the bacterial culture was transferred to the plate (www.hindawi. com). The dried Muller-Hinton agar plate surface was spread using a sterile cotton bud. Using sterile forceps, five discs were pressed into the center and four corners of an agar plate with the least amount of space possible while the plate was aseptic. The plates were inverted carefully and finally incubated at 37°C for 15 minutes after the discs were applied. Each plate was examined after 16 to 18 hours of incubation. There was a constant, circular zone of inhibition on the surface. The total zone of inhibition (as seen with the naked eye) and the discs diameters were measured. Zones were measured to the closest whole millimeter (mm) by using a ruler (Hire et al., 2014). 

Physico-Chemical Characteristics of Water  

On the same day that the samples were collected and the physico-chemical based characteristics of the water were also measured. Although the temperature was typical, there were instances where the tubewell was placed in a chilly or shaded area with lower temperatures than others. Additionally, the water from deep wells and excavated wells was somewhat colder than the water from other sources. The pH range ranged between normal and slightly alkaline, with little variation. On the same day, the HACH EZ arsenic test kit was used to measure the arsenic level (Table 1). The following are the overall findings:

Table 1: Physico-chemical properties including temperature, pH and arsenic level (ppb) of fifteen water samples. 

Morphological Characteristics of Isolates  

Isolates were observed under microscope in 100x lens. Gram staining was performed while the violet color firmly indicates gram positive and pink color viewed through microscope indicates gram negative bacteria (Fig. 2). The shape and appearance were recorded too. Summary of these characteristics are given on Table 2.  

Fig. 2: Gram staining of two isolates: CM-11 and CM-112; where CM-11 was gram negative (pink color) and CM-112 was gram positive (violet color).

Table 2: Morphological Characteristics (Color, Shape and Appearance) of Seven Isolated Bacterial Species.  

Biochemical Characteristics  

Several biochemical tests like catalase, MR-VP, citrate utilization, TSI, indole etc. were performed following the appropriate protocol (Table 3). Summary of these results are given as follows: 

Table 3: Summary of Biochemical Analysis of Isolated Bacterial Species; where CM-11, CM-12, CM-31, CM-61, CM-101, CM-102 and CM-112 were assumed as Acinetobacter sp., Serratia sp., Corynebacterium sp., Corynebacterium sp., Klebsiella sp., Staphylococcus sp. and Corynebacterium sp. respectively.

Legend: + = Positive, - = Negative, A= Acidic, K= Alkaline, MR= Methyl Red, VP= Voges- Proskaur, NC: No change.  

 

Minimum Inhibitory Concentration  

After repeatedly growing on different concentrations of sodium arsenite, minimum concentration was measured (Table 4).   

Table 4: Minimum Inhibitory Concentration of Seven Isolates in Sodium Arsenite.

Some of the incubation periods were extended to observe the growth on the same concentration (Fig. 4). A microbial community must evolve a detoxification mechanism to get past the growth restriction when it is subjected to a selective stressor, such as a high concentration of arsenic, for an extended length of time (Huang et al., 2010). So, the highest concentration of arsenite tested was 2000mM, in which CM-31 and CM-112 showed only a few colonies after 10 days of incubation, but they lost the capability of the arsenite oxidation [Fig. 4(d)].  In summary, the MIC is as follows:

Fig 4: MIC of arsenite determination for isolate CM-112; where (a), (b), (c) named plate 3, 8 and 9 contained 800 mM, 1500mM and 1600mM arsenite respectively in which growth of CM-112 decreased with the increase of concentration of arsenite; (d) Plate 10 Showed the growth of CM-112 on 2000mM of arsenite with a prolonged incubation period (>12 days).

Oxidation of Arsenic  

Arsenite was converted to arsenate when the isolates were incubated with arsenite agar plates for five days. The existence of arsenite oxidase is confirmed by the appearance of a brown to dark brown color while the enzyme is present and arsenite is converted to arsenate (Fig. 5). 

 

Table 5: Oxidation of arsenic; this table demonstrates the presence of arsenite oxidase in all of the seven isolates. 

Legend: + = Positive, - =Negative  

Fig. 5: Oxidation of arsenic assay with silver nitrate; where brown color indicates the presence of silver arsenate, which means oxidation occurred due to the presence of arsenite oxidase indicating positive test results.

Antibiotic Resistance  

Antibiotic resistance was observed after disc inoculation and incubation, where zone of inhibition was measured with a normal scale in mm (Table 6) summary of these data are as follows:  

 

Table 6: Antibiotic resistance profiling of isolated bacteria against 10 different antibiotics using the disc diffusion method.

Data are the means of three replicates (n=3) ± Standard deviations.  

According to Fig. 6, Susceptibility rate: Chloram-phenicol and Gentamicin: 100%, Cefotaxime and Ceftriaxone: 85.71%, Colistin: 71.42%, Erythromycin 57.14%, Cefepime: 42.85% and Ampicillin 14.28%; Intermediate resistance rate: Cefepime: 57.14%, Ampicillin: 42.85%, Amoxicillin: 28.57% and Colistin, Cefotaxime & Ceftriaxone: 14.28%; Resistance rate: Ceftazidime: 100%, Amoxicillin 71.42%, Ampicillin & Erythromycin: 42.85% and Colistin: 14.28%.

Fig. 6: Antibiotic resistance: zone of inhibition where clear area around the discs indicates the zone of inhibition.  

The present study emphasized isolation as well as characterization of bacteria showing arsenic resistance. From the data collected, it can be observed that there are huge number of variations in the level of arsenic concentration in the water, ranging from 25 ppb to 250 ppb. In the same study area, Chapainawabganj, Ohno et al. (2005) found 14 ppb to 2630 ppb and Islam et al. (2017) found 2.5 ppb to 150.6 ppb arsenic concentration in the contaminated groundwater. All the water samples were kind of neutral to alkaline (pH 7.02-7.98) in nature which is genuinely suitable for most bacterial growth (Dey et al., 2015).  

The isolated species from the present samples were assumed to be Acinetobacter, Serratia, Corynebacterium, Klebsiella and Staphylococcus species. Several similar bacterial species have been isolated for arsenic resistance and oxidation-reduction. Shakoori et al. (2010) have reported Klebsiella oxytoca, Corynebacterium freundii, Corynebacterium freundii; Satyapal et al. (2016) reported several species along with Acinetobacter; Ryan & Colleran, (2002) reported Serratia marcescens and Serratia meliloti and Ji & Silver, (1992) reported Staphylococcus aureus for arsenic resistance.

This research proves the potential of isolated arsenic-resistant bacteria to serve as economical green alternatives for replacing existing metal decontamina-tion methods. The isolated bacteria showed high tolerance to arsenite through their demonstrated minimum inhibitory concentration (MIC) values in addition to their ability to produce arsenite oxidase which aids in detoxifying arsenite [As(III)] into the safer arsenate [As(V)]. The bacterial strains demonstrate potential for bioremediation because their enzymatic abilities make them suitable agents to treat arsenic in contaminated sites including water sources and soil as well as industrial waste. The bacterias distinctive metabolic traits together with their robust nature create conditions where they can potentially be utilized to develop commercial enzymes suitable for industrial production. Studies must investigate whether these isolated bacteria contain antimicrobial resistance genes to guarantee their appropriate use in open environments and analyze their influence on the environment. Further research should be done with inspections of resistance genes at the molecular level regarding enzyme production enhancement and the creation of large-scale bioremediation systems using these isolates. To determine the performance of these microbial agents under different environmental settings, both in situ trials and pilot-scale applications needed to conduct for confirmation.

The investigation, conceptualization, writing reviews, supervision was conducted by M.A.M.; writing-original draft, data collection, and laboratory experiments were done by A.P.; and M.R.A.; writing-critical reviews, editing, and data analysis were prepared by S.K.S. All authors have read and agreed to the published version of the manuscript.

The study did not involve the use of animal models or human participants. This study was conducted in compliance with the faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University ethical standards and guidelines. All experimental procedures were carried out in accordance with institutional and international ethical standards for laboratory-based research.

We thank the faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University, Sylhet for the laboratory support. 

This article is original work and the authors declare that there are no conflicts of interest relevant to this article.