Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Study of an Arsenic Metabolizing Bacteria from Arsenic Contaminated Soil of Chandpur District, Bangladesh

Roksana Khanam
Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh

Ripa Moni
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Md. Zahidul Islam
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Md. Morsaline Billah
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh

Umme Salma Zohora
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Farah Sabrin
Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh

Mohammad Shahedur Rahman*
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Abstract/ Executive Summary:

Arsenic is a toxic metal found as inorganic oxyanion arsenate As(V) and arsenite As (III) species. The disposal of toxic heavy metals such as arsenic poses high risk to environment. The present study was undertaken to isolate arsenic-metabolizing bacteria from arsenic contaminated soil of Chandpur district, which is one of the most arsenic contaminated area in Bangladesh and later these bacteria were screened for their ability to metabolize arsenate. Out of ninety eight isolates, ten were found to be capable of metabolizing arsenic in Yeast Extract Mannitol (YEM) medium containing 2 mM arsenate at 37ºC. One of the bacterial isolates designated as I-25 was found to produce an extracellular enzyme which can reduce As(V) into As(III) and able to grow in presence of up to 500 mM arsenate. Subsequent molecular identification of this enzyme producing bacterial isolate using 16s rRNA sequence analysis was correlated with previously identified isolate as Bacillus aryabhatti. Further characterization of the enzyme showed that optimum pH of the extracellular enzyme by the bacterial species was 7 and optimum temperature for the enzyme activity was 60ºC. The bacterial isolates can be exploited for the study of possible bioremediation of arsenic contamination.

Keywords: Arsenic metabolizing bacteria, Bacillus aryabhattai, Bangladesh

Location: Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Arsenic

Objectives:

The present study also investigates optimization temperature, pH and optimum arsenate concentration for maximum arsenate reduction for the extracellular enzyme produced by this bacterium.

Methodology:

Sample collection: Soil sample was collected from Saharasti upazilla of Chandpur district, Bangladesh. Sample from subsurface soil (0-15 cm in depth) were collected, placed in plastic bag and kept on ice or at 4ºC until further analysis. Chemicals and stock solutions: All buffers and solutions were prepared with distilled water and sterilized by autoclaving at 121ºC for 30 min. As(III) stock solutions (10 mM) were prepared from stock sodium arsenite (NaAsO₂) and As(V) solutions (10 mM) from sodium arsenate heptahydrate (Na₂HAsO₄.7H₂O). Stock solutions were stored at 4ºC in the dark. A starch-iodine complex (mixture of aqueous starch and Lugol’s iodine solution) was prepared freshly before use. Bacterial culture preparation and identification of isolate: For the bacterial culture preparation, 1 g of each soil sample was taken in 100 mL of 0.1 M phosphate buffer solution and mixed well by vortexing for 3 min. Each sample was serially diluted with sterile saline water and plated on Yeast Extract Mannitol (YEM) agar medium. After 16 h of incubation, stocks were prepared by dissolving the culture in equal volume with 40% glycerol. After single colony isolation, the morphological, biochemical and molecular characteristics of the isolated bacterial strains were also evaluated as previously described by Rahman et al. (2018). Screening of arsenic metabolizing Bacteria: Precultures were prepared from each single colony in 5 mL YEM (without arsenate) broth and incubated at 37ºC for 24 h at 120 rpm. Then preculture was centrifuged, washed twice with sterile buffer solution and resuspended in 2 mL sterile water. Twenty μL preculture was inoculated into YEM broth medium containing 2 mM arsenate and incubated under the above condition. Then cultures were centrifuged at 10,000 × g for 5 min and supernatant was taken for further analysis. Next, 1mL of supernatant was added into test tube followed by addition of 30 μL of starch-iodine complex (Mandal et al., 2007). The test tubes were incubated in dark for 10 min. Then optical density (OD) was measured by using spectrophotometer (Mecasys Optizen Pop UV/Vis Spectrophotometer, Korea) and highly active bacteria were identified from these bacterial isolates. Identification of localization of the active enzyme: Firstly, 24 hours cultures were grown in YEM medium which contained arsenate. Then they were subjected to centrifugation at 10000 × g for 5 min and supernatant was removed. Pellet was washed twice with autoclaved phosphate buffer. Then the pellet was resuspended into 1 mL of autoclaved YEM medium (without arsenate). 100 μL of resuspended aliquots were inoculated into autoclaved 5 mL YEM medium (without arsenate) for 24 h shaking at 37ºC, 120 rpm. After 24 h of incubation, the tubes were centrifuged and supernatant (4 mL) was taken into another autoclaved test tubes. Then 1 mL arsenate (10 mM) was added into 4 mL of supernatant. After 2 h of incubation in shaking water bath (37ºC, 120 rpm), 2 mL supernatant was withdrawn and 60 μL starch-iodine complex was added into it and incubated in dark. If color change occurred, the enzyme responsible for arsenic metabolism was considered as extracellular one. In the event of no color change, sonication was used for cell disruption. In this case, pellets were washed twice with autoclaved buffer solution by centrifugation at 10000 × g for 2 min. Then supernatant was aspirated and the pellet was dissolved into 2 mL autoclaved YEM medium. Then the cells were disrupted by using ultrasonicator at 37 ºC for 3 h. After sonication, Lugol’s iodine was added and incubated in dark. Color change indicated that the enzyme responsible for arsenic metabolism was intracellular one. Determination of optimum metabolizing condition: For determining the optimum growth of the bacterial strains, different arsenate concentrations (0 mM, 2 mM, 16 mM, 64 mM, 128 mM, 250 mM and 500 mM) in YEM medium were considered in different test tubes and autoclaved at 121⁰C for 30 min. Then freshly prepared bacterial culture inoculated into these concentrations and incubated for 24 h. Finally, the absorbance was measured at 600 nm. For optimization of the temperature requirement on enzyme activity, large amount of supernatant were used. For this, 100 mL 2 mM arsenate containing YEM medium were prepared in a conical flask with 50 mL medium. Then the flask was autoclaved at 121ºC for 30 min and 500 μL of freshly prepared bacterial culture was inoculated into each flask. After 24 h incubation, 10 mL of culture was taken into 15 mL falcon tube and centrifuged at 10,000 × g for 20 min. Then supernatant was collected into test tubes (5 mL each) and stored at -20ºC. Test tubes were kept in different temperatures (30ºC, 40ºC, 50ºC, 60ºC, 70ºC, 80ºC and 90ºC) for 10 min and then cooled at room temperature. Finally starch iodine complex was added and after an incubation period, optical density was measured at 580 nm using spectrophotometer.

Source of Information: Jahangirnagar University J. Biol. Sci. 8(1): 57-65, 2019 (June)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This study provides insight into identification, levels of arsenic resistance and reduction of arsenate by novel bacterial isolates which could play an important role in arsenic cycling in the arsenic-contaminated soils in Bangladesh. Isolated bacteria from arsenic contaminated soil were capable of metabolizing arsenic from the culture media. Highly As (V) resistant isolates could be a excellent candidate for the study of arsenic bioremediation. Further research is necessary to explore the possibility to use these bacteria in the other culture conditions to remove arsenic from the contaminated soil.

Knowledge Management: Journal