Identification and Evaluation of Arsenic Tolerant Bacteria for Arsenic Mitigation in Contaminated Soil

Research Article

J Bacteriol Mycol. 2020; 7(8): 1156.

Identification and Evaluation of Arsenic Tolerant Bacteria for Arsenic Mitigation in Contaminated Soil

Prosun TA1, Md. Younus Ali2, Most. Monira Yesmin3, Brun MS4, Md. Badiuzzaman Khan1, M. Harun-or Rashid4*

1Department of Environmental Science, Bangladesh Agricultural University. Mymensingh- 2202, Bangladesh

2Fibre Quality Improvement Division, Bangladesh Jute Research Institute, Dhaka, Bangladesh

3Department of Agronomy, Bangladesh Agricultural University. Mymensingh- 2202, Bangladesh

4Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Germany

*Corresponding author: Harun-or Rashid, Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Germany

Received: October 14, 2020; Accepted: November 03, 2020 Published: November 10, 2020


A total of seventy two bacterial strains were isolated from different areas of Bangladesh to find and evaluate Arsenic (As) tolerant bacteria for mitigation of arsenic contamination and other biotechnological application. Strain colonies were circular, groove and flat in shapes and size ranged from 0.3 mm to 5.8 mm with white, off white, orange, yellow color. Among the strains, the strain TAN-8 was able to grow in high concentrations (28 mM) of arsenic. The highest arsenic tolerant strain TAN-8 showed maximum growth at 37°C and at pH-7 after 34 h of inoculation. The strain TAN-8 was arsenic metabolizing bacteria since it produced violet color in silver nitrate test and suggesting that this strain uses arsenic for its own growth and development. The ERIC-PCR fingerprinting of arsenic tolerant TAN-8 strain showed seven different DNA bands with 400 bp to > 1500bp long. Sequencing and phylogenetic analysis of 16S rRNA gene confirmed that the strain TAN-8 was Klebsiella pneumoniae (100%). The strain TAN-8 was capable of effective metabolism of arsenic and survives in high arsenic condition along with high temperature and pH. Thus, the strain TAN-8 could be used for mitigation of arsenic contaminated environment and to reduce arsenic uptake by crops grown in Arsenic contaminated soil.

Keywords: Arsenic Tolerant Bacteria; Mitigation of Arsenic, Arsenic Uptake by Crops


As: Arsenic; BINA: Bangladesh Institute of Nuclear Agriculture; ATSDR: Agency for Toxic Substances and Disease Registry; LB: Luria Bertani; AgNO3: Silver Nitrate; cm: Centimeter; ML: Maximum Likelihood; K2P: Kimura Two-Parameter; ERIC: Enterobacterial Repetitive Intergenic Consensus; bp: Base Pairs


Heavy or toxic metals are trace metals with a density at least five times higher than water and are detrimental to human health [1]. Ecosystems are exposed to heavy metals heavily due to industrial and mining activities, and they also occur during fuel production from waste materials. These result in unnatural and harmful presence of heavy metals in environment because they form stable molecules and do not breakdown organically [2]. The ATSDR [3] (agency for toxic substances and disease registry) reported that arsenic is one the most harmful element for the ecosystem since it deposits heavily in lipid tissues of organism, which in turn accumulate in even heavier proportion in the upper level of the trophic chain. Sodium arsenite exposure can occur by inhalation or skin absorption which causes skin irritation, burns, itching, stomach pain, nausea, vomiting, diarrhea, convulsions, carcinogenic and teratogenic effects etc. [4]. Significant level of Arsenic exposure may affect nervous system leading to a number of conditions ranging from weakness, poor coordination, or “pins and needles” sensations to the extent of paralysis and even death [5,6]. Arsenic toxicity in crop plants caused by transfer of arsenic from contaminated soil is of great concern because of its potential health hazards [7] because about 30% of the total arsenic ingestion is caused by arsenic polluted rice and other food sources [8]. Breakdown of heavy metals by microorganisms is known as bioremediation (Retrieved from Microorganisms can interact with metals via many mechanisms such as biosorption, biotransformation, bioaccumulation, biomineralisation, microbiallyenhanced chemisorption of metals, biodegradation of chelating agents and bioleaching. Some of which may be used as the basis of potential bioremediation strategies [9]. Bacteria could be used to mitigate arsenic from soil and plants. For example, Mallick et al. [10] used two arsenic resistant bacteria (Kocuria flava and Bacillus vietnamensis) for reducing arsenic accumulation in rice. By 84.8% and 82.2%, respectively. Wang et al. [11] showed that Populus deltoides LH05-17 (Poplar plant) is an efficient arsenic accumulator but high concentration of arsenic reduces its growth. Agrobacterium radiobacter D14 was used with Populus deltoides LH05-17 and it helped to tolerate even 300 mg /kg arsenic in soil and showed 54% arsenic removal. Therefore, bioremediation with microbes could have a remarkable impact in solution that issue. Sequencing of the 16S rRNA gene along with REP-PCR and ERIC-PCR are powerful techniques in bacterial taxonomy and their identification [12]. Isolation and identification of As resistant bacteria could be an avenue for microbial remediation of this heavy metal. Thus, the objective of the present study was to isolate and identify arsenic tolerant bacteria and to evaluate their arsenic tolerance level, their morphology and physiology for their further application in mitigation of arsenic contaminated environment and to reduce arsenic uptake by rice.

Materials and Methods

Collection of Samples

Tannery effluents and municipal solid wastes samples were collected from Hazaribagh tannery industrial area, Dhaka and bypass area, Mymensing, Bangladesh. A total of 22 samples were collected from those locations. Among them, 14 were soil and 8 were water samples. Water and soil samples were collected in sterilized plastic bottles and bags and then transported to Bangladesh Institute of Nuclear Agriculture (BINA) and preserved at 4°C in fridge until bacterial isolation process was initiated [13].

Isolation of heavy metal resistant bacteria

For the isolation of heavy metal resistant bacteria, at first Luria Bertani (LB) agar plates (Peptone 10.00 g/L, yeast extract, 5.00 g/L, NaCl 5.00 g/L, and agar 18-20.00 g/L: pH 7.00) were prepared with different concentration of arsenic. 10 g of soil and 10 mL of water from each sample were added to 90 mL sterile water and a dilution series up to 10-5 was prepared according to Azad et al. [14] to isolate desired bacteria. 10 μL aliquots from each dilution (10-2 to 10-5) were poured on Luria Bertani agar plates that contained 4 mM of arsenic. Control plates were also prepared without including arsenic into the LB media. Sterilized spreader was used for spreading the diluted aliquot by rotating the plate. Inoculated plates were incubated at 37°C for 2-3 days. Meanwhile bacterial colonies appeared. Individual colonies with distinct morphologies were picked and streaked on Luria Bertani agar medium containing 6 mM of arsenic to get pure single colonies [13]. Distinct single colonies were grown in liquid LB medium. All the cultures were stored in 50% glycerol at -80°C for further study.

Evaluation of Arsenic tolerance

72 isolates were tested for their resistance to arsenic. LB-agar plates were prepared with different concentrations of arsenic. The starting concentration of arsenic in this test was 7 mM which was gradually increased to 42 mM. Freshly cultured bacteria were streaked on arsenic containing LB plate at 37°C for 4 days and the growth response of bacteria was observed in different concentrations of arsenic. Arsenic tolerance was assessed according to standard protocol of European Food Safety Authority [15].

Determination of growth curves

For the determination of growth curves, maximum arsenic tolerant strain TAN-8 was grown with and without arsenic stress in 100 mL Luria Bertani broth prepared in conical flask. Experiments were performed in triplicates. Medium was inoculated with 10 μL bacterial cultures and incubated for 44 h at 37°C in shaking incubator. One (1) mL of bacterial sample was drawn in a cuvette with the help of micropipette in laminar air flow every two hours. Optical density of TAN-8 broth was determined at 600 nm using spectrophotometer (Model: Eppendorf ----). A growth curve was plotted by taking optical density on Y-axis and incubation time on X-axis [16].

Determination of optimum pH and temperature

About 20 mL of sterilized Luria Bertani broth was taken in 50 mL flasks. The pH range of medium was adjusted from 4.0 to 10.0. Each pH was taken in triplicates with and without arsenic stress (20 μg/L). They were then inoculated with 10 μL of fresh culture of each bacterial isolates and incubated at 37°C in shaking incubator. After 24 h optical density was noted in spectrophotometer at 600 nm. A graph was plotted between optical density along Y-axis and pH along X-axis. The optimum pH of the strain was determined by graph. For the temperature measurement, sterilized 50 mL Luria Bertani broth was prepared in 100 mL conical flask. After inoculation with 10 μL culture, bacteria were grown in flask with and without arsenic stress (20 μg/L) that were kept at different temperatures ranging from 25°C to 40°C for 24 h. Experiments were conducted in triplicate. Optical density of each strain was noted and graphs were plotted taking optimum density along Y- axis and temperature along X- axis [16].

Verification of arsenic transforming ability of arsenic tolerant strain

Silver nitrate (AgNO3) method was used to verify the transforming ability of bacterial strain. Arsenic tolerant bacterial strains were streaked on Luria Bertani agar plate containing 10 mg/L of arsenic. Plates were filled with 0.1 M AgNO3 solution and then incubated at 37°C for 48 h [17].

Seedling emergence test

The bacterial strain (TAN-8) was grown in LB broth. Exponentially growing cells’ broth cultures were used for inoculation. Rice seeds (advanced line CC-2 and advanced line -1) were surface sterilized using 70% ethanol in Erlenmeyer flask for 1 min and were treated with 3% sodium hypochlorite for 3 min followed by six to seven times washing with sterile water. After that, the seeds were soaked in TAN-8 bacterial broth for one hour. Seeds soaked in sterile LB broth (Without TAN-8) were treated as control. After soaking, the air-dried seeds were used for germination in sterilized Petri dishes containing agar 1% and kept at 28°C for 10 days [18].

Germination trait parameters

Germination rate was calculated according to the method by Krishnaswamy and Seshu [19]. Measurement of root and shoot length was taken after 10 days of seed setting. Measurement of root and shoot length was carried out as follows- five seedlings were randomly selected from each Petri dish and measured with a measuring scale and expressed in centimeters (cm) [20].

DNA isolation, ERIC-PCR and sequencing of 16S rRNA gene

Bacterial culture was grown at 37°C overnight in LB medium and the DNA was isolated following the protocol described by Chen and Kuo [21]. Extracted DNA was dissolved in TE buffer and the concentration was measured by UV-spectrophotometry. The PCR amplifications were performed with about 100 ng of template DNA. ERIC-PCR primers (forward: ATGTAAGCTCCTGGGGAT and reverse AAGTAAGTGACTGGGGGTGAGC) described by De Bruijn [22] were used. The PCR product was visualized on 3% (w/v) agarose gel. The conditions for ERIC–PCR were at 95°C for 5 min for Initial denaturation. Thirty PCR cycles containing denaturation at 94°C for 30 s, annealing at 52°C for 1 min, and elongation at 65°C for 8 min were used. The final elongation was at 65°C for 16 min.

For species identification, the 16S rRNA gene was amplified by using the primers described by Weisburg et al. [23]. The forward (AGAGTTTGATCCTGGCTCAG) and reverse (AAGGAGGTGATTCCAGCC) primers were used to amplify 16S rRNA gene. DNA amplification was performed in a bio-red gradient thermal cycler, PTC-200. The PCR product was visualized on 1.5% (w/v) agarose gel. The PCR conditions for the amplification of 16S rRNA gene were: initial denaturation at 95°C for 5 min. Thirty PCR cycles containing denaturation at 95°C for 1 min, annealing at 57°C for 1 min, and elongation at 72°C for 1.5 min were used. The final elongation was at 72°C for 15 min. Amplified PCR product was purified using PCR clean up system from Promega, USA. Sequencing was performed using an ABI 3730 automated capillary sequencer (Applied Biosystems) with the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 by 1st BASE (Singapore). To confirm the observed sequences quality, both strands were sequenced from arsenic tolerant strain.

Phylogenetic Analyses

The sequences were aligned with Bio Edit [24]. Phylogenetic trees were reconstructed using the Neighbor-Joining (NJ) algorithm [25] and Maximum Likelihood Methods (ML) in MEGA version-7 [26] using the Kimura two-parameter (K2P) model [27] and GTR model [28]. Bootstrap support for each node was evaluated with 1000 replicates.


During the period of study, a total of 72 bacterial strains were isolated from 22 soil and water samples from selected locations of Bangladesh. All strains had different types of colonies. Bacterial colony shapes were circular, some of them were groove and the remaining was flat. Colony size of the isolates was ranged from 0.3 to 5.8 mm. Most of the colony colors were white and off white, whereas the rest of the colonies were orange, yellow and transparent (Table 1).