Microbial Isolation and Characterization of Arsenic Degrading Microbes from Soil and its RAPD Analysis for Bioremediation
Authors: Shanmugam Kaushika , Kumar Kalaivani , Abhimanyu Srinisha , Selvaraju Sri Sowmiya , Narayana Sri Lakshmi B , Sharanprasath R S , Kumar Naveen T , Manikandan R , bala Hari S 
Published: March 2023

The aim of this work is to isolate the microbes possessing arsenic degrading property from contaminated soil, collected from Cauvery River at Pallipalayam, Erode District. Six microbial strains were grown well in 40Mm sodium arsenate as a sole carbon source amended M9 minimal media. Based on the zone of clearance, three microbial strains were found to be potent arsenic degrading microbes and they are identified as Bacillus spp., Staphylococcus spp., and Pseudomonas spp. They may potentially be used in the bioremediation of arsenic and other contaminants. It infers that the presence of arsenate reductase (ArcC) gene in three of the microbial strain and they were taken for further studies. Genomic DNA isolation protocol was standardized and DNA isolation was performed. ArcC gene-specific primers were designed using Primer3 bioinformatics tool. Genetic diversity among the strains was studied by RAPD analysis using four different primers. Dendrogram was constructed using Unweighted Pair Group using Arithmetic Averages and NJ tools. The presence of genetic diversity was observed from the analysis. Polymerase chain reaction amplification and sequencing of amplified gene products are to be done in future.

Background:
The aim of this work is to isolate the microbes possessing arsenic degrading property from contaminated soil, collected from Cauvery River at Pallipalayam, Erode District. Six microbial strains were grown well in 40Mm sodium arsenate as a sole carbon source amended M9 minimal media. Based on the zone of clearance, three microbial strains were found to be potent arsenic degrading microbes and they are identified as Bacillus spp., Staphylococcus spp., and Pseudomonas spp. They may potentially be used in the bioremediation of arsenic and other contaminants. It infers that the presence of arsenate reductase (ArcC) gene in three of the microbial strain and they were taken for further studies. Genomic DNA isolation protocol was standardized and DNA isolation was performed. ArcC gene-specific primers were designed using Primer3 bioinformatics tool. Genetic diversity among the strains was studied by RAPD analysis using four different primers. Dendrogram was constructed using Unweighted Pair Group using Arithmetic Averages and NJ tools. The presence of genetic diversity was observed from the analysis. Polymerase chain reaction amplification and sequencing of amplified gene products are to be done in future.

Methods:
The soil sample was collected from Cauvery River, Pallipalayam. Arsenate, arsenic bioremediation, arsenic reducing gene, RAPD, and genetic diversity were used.

Results:
With the dilution concentrations, 10−5 and 10−6 microbial population was obtained in M9 minimal media. From the pure colonies of isolates, TA1, TA2, TA4, and TA5 genomic DNA was extracted using the protocol mentioned above. The culture was inoculated in LB broth and kept in incubation at 37°C for overnight. From overnight culture, genomic DNA was extracted. RAPD analysis for the isolates was performed using four different random primers namely RBA-1, RBA-4, RBA-5, and RBA-6.

Conclusion:
Three of the isolates designated as TA2, TA4, and TA5 were found to be potent arsenic degarding microbes. They are able to degrade sodium arsenate of about 40mM. It infers that they can be potentially used in bioremediation of arsenic. Isolation of ArcC gene from the isolates is in progress. Sequencing will reveal the nature of amplified products. If the amplified genes are cloned and mass production of ArcC gene could be obtained.