Kumari Tripti* and Shardendu TO UNRAVEL THE SURVIVAL STRATEGIES OF Bacillus licheniformis UNDER DIFFERENT ARSENIC STRESS BY ASSESSMENT OF PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS Kumari Tripti* and Shardendu LABORATORY OF ENVIRONMENT AND BIOTECHNOLOGY PATNA SCIENCE COLLEGE, PATNA UNIVERSITY PATNA – 800005, INDIA

Introduction Two arsenic tolerant bacteria were isolated from rhizosphere of Amaranthus viridis, identified as Bacillus licheniformis DAS-1 and Bacillus licheniformis DAS-2 Isolated from arsenic contaminated region located at 85º 32’ E longitude and 25º 11’N latitude (Bihar) on the Earth. The survival strategies study was done in the form of growth, uptake/ removal, transformation of arsenic species, biochemical characterization ( enzyme assay) of bacterial cell under different concentration of arsenic stress.

Scientific Background Arsenic (Z=33), a metalloid is considered as most significant potential threat to human health even in the rural areas due to its ubiquity and toxicity. The most available forms are As(V)[arsenate] and As(III)[Arsenite], their abundance in soil environment is influenced by microbial transformation. Arsenic is taken by crops and to next trophic level resulting health hazards. Removal of arsenic from environment and to trace out the occurrence, distribution and potential of native flora for arsenic tolerance is of great importance for human welfare.

Bacillus licheniformis DAS-1 and Bacillus licheniformis DAS-2 Experimental Plan Bacillus licheniformis DAS-1 and Bacillus licheniformis DAS-2 Survival strategies of bacteria in presence of arsenic [ As(V) and As(III)] Growth profile uptake/removal transformation/reduction Biochemical characterization Bacteria were isolated and identified by 16SrDNA sequencing. Survival strategies of bacteria was determined by growing in TYEG medium enriched with different levels of As(V) and As(III) along with variation in pH. Growth profile was measured by turbidometry. uptake/removal and transformation/reduction was measured by estimating the amount and forms of arsenic in residual media and in bacterial cell biomass after digestion and determination by spectrophotometer (Azure B). Enzyme assay (arsenate reductase) was done as a biochemical characterization of bacterial cell in response to arsenic stress.

Growth profile Bacillus licheniformis DAS-1 Growth pattern of Bacillus licheniformis in arsenic containing TYEG broth, over the range of arsenic concentrations. Fig.(A) As(V) and Fig.(B) As(III] enrichment Bacillus licheniformis DAS-1 MIC for As(V) is 10mM and for As(III) is 7mM. Bacillus licheniformis DAS-2 MIC forAs(V) is 8mM and for As(III) is 6mM.

Uptake /removal & transformation/reduction of As(V). On Y-axis concentration of As(V) left in residual media at the same time concentration of As(III) formed in media [uptaken As(V) reduced to As(III)] and concentration of total arsenic (As) accumulated in bacteria [As in biomass], at different time point (x-axis) of growth phase 100% removal 42% reduction 99% removal 80% reduction 75% removal 76% reduction 68% removal 64% reduction DAS-1 DAS-2

Uptake /removal of As(III). On Y-axis concentration of As(III) left in residual media(after uptake/removal) and concentration of total arsenic (As) accumulated in bacteria (As in biomass)at different time point (X-axis) of growth phase 99% removal 40% accumulation 99% removal 80% accumulation 40% removal 30% accumulation 56% removal 32% accumulation DAS-1 DAS-2

pH dependent uptake/Removal of As(V) and As(III) On Y-axis, the concentration of As(V) and As(III) left in residual media at the different time point (x-axis) of growth phase. DAS-1 [Change in pH significantly (p < 0.05) reduced the uptake/removal of As(V) [p = 0.021 with acidic pH and p = 0.049 with basic pH] and As(III) [p=.03 with acidic pH and p=0.04 with basic pH ] DAS-2 [Change in pH significantly (p <0.05) reduced the uptake/removal of As (V) [p = 0.017 with acidic pH and p = 0.021 with basic pH] and As(III) [p=0.016 with acidic pH and p=0.013 with basic pH ]

Biochemical Assay Arsenate reductase assay,enzyme activity was detected by measuring NADPH absorption at 340 nm over the time range of 0 to 10mins DAS-1 Absorption decreasing at the rate of 0.0246 ABS/min. Specific activity of enzyme 0.088-0.31 µmole/min/mg. Concentration of protein in the reaction was 0.152µg/mL] DAS-2 Absorption decreasing at the rate of 0.030 ABS/min. Specific activity of enzyme 0.24-0.55 µmole/min/mg. Concentration of protein in the reaction was 0.181µg/mL

Conclusion Bacillus licheniformis DAS-1 and DAS-2 are able to tolerate both As(V) as well as As(III) at variable level. Both bacteria are capable of removing about 100% of As(V) and As(III) from media with lower concentrations enrichment. Removal of arsenic is pH dependent and neutral pH is most favorable for maximum uptake/removal . Reduction of uptaken As(V) into As(III) is one of the survival strategy of both the bacteria for tolerating As(V) toxicity. Arsenate reductase assay provided the evidence for the existence of system for reducing As(V).

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