Characterization of arsenic resistant bacteria and their potential role in bioremediation of industrial waste water

Abstract

Arsenic is toxic metalloid, which is found everywhere on earth. Pakistan has large number of industries which release their waste including arsenic and other heavy metals in our environment and pollute it. So there is a need of arsenic detoxification. In the present study, arsenite resistant bacteria were isolated from the waste water of three different districts in Pakistan, and their arsenite oxidizing potential and mechanism of arsenic resistance were determined. All the isolated bacterial strains were screened on the basis of resistance against arsenite as well as their ability to oxidize it into arsenate which is 100 times less toxic than arsenite. The bacterial isolates were dubbed as 1S1, CS2, AS2, SH2, AS1, AS2C, AS3, AS4, AS5, AS6, AS7, AS8, and AS9. Identification of bacterial strains were carried out on the basis of morphological characteristics, biochemical testing, and 16S rRNA. They showed similarity with Microbacterium sp. strain 1S1, Micrococcus luteus strain AS2, Brevibacterium sp. strain CS2, Dietzia sp. strain SH2, Microbacterium sp. strain AS1, Micrococcus sp. strain AS2C, Microbacterium sp. strain AS3, Microbacterium sp. strain AS4, Staphylococcus sp. strain AS5, Staphylococcus sp. strain AS6, Microccus sp. strain AS7, Staphylococcus sp. strain AS8, and Microbacterium sp. strain AS9. Minimum inhibitory concentration (MIC) against arsenic was determined for all isolated bacterial strains. All the strains exhibited high resistance against arsenite ranging 20-75 mM and against arsenate the MIC range was 110-520 mM. Cross metal resistance against Pb+2, Cd+2 , Cr+6, Hg+2, Se+3, Co+2, and Ni+2 was determined and all bacterial strains showed fair resistance against these metals. All the bacterial strains showed optimum growth at 37 and pH 7 except the AS4 and AS6. Both AS4 and AS6 showed optimum growth at 30 . The screening of arsenite oxidizing bacteria was carried out through AgNO3 method. The bacterial strains 1S1, xi CS2, AS2, AS1, AS2C, AS8, and AS9 showed arsenite oxidizing ability while strains SH2, AS3, AS4, AS5, AS6, and AS7 were not able to oxidize arsenite into arsenate. The arsenite oxidizing ability of Microbacterium sp.1S1 was 40, 65, 85, and 98% after 24, 48, 72, and 96 hours while the arsenite oxidizing ability of AS2 and CS2 was 92 and 48 % after 96 hours of incubation. All the three bacterial strains (1S1, AS2, and CS2) showed optimum arsenite oxidizing ability at 37 and pH 7. Pilot scale study for the arsenite bioremediation trial of our bacterial strains revealed that 1S1 has ability to remove 72 % arsenite from industrial waste water and 95 % from distilled water after 8 days while strains AS2 and CS2 can remove 68% and 32%, respectively. Thus, this microbially purified water up to 95 % could possibly be used for irrigation of agricultural crops. The bioremediation efficiency (E) of strain 1S1 was 99 % after 8 hours while for strain AS2 and CS2, the bioremediation efficiency (E) was 99 and 96 % after 10 hours. Illumine and nanopore sequencing revealed that there is a genes fragment aioB gene, responsible for arsenite oxidition in all arsenite oxidizing bacteria. The arsenite oxidizing fragment of size 9.6 kb was cloned in puC19 vector and electporated in E. coli. Apart from aioB gene, there are some other genes in all the isolated bacteria such as arsC1, arsR1, ACR3, and trxB found after illumine and naopore sequenceing. Transcription analysis was performed to confirm the expression of aioBgene in the presence and absence of arsenite (20mM) after certain interval of time in log phase. It was seen that aioB gene was equally expressed in the absence and presence of arsenitein Microbacterium sp. strain 1S1 and Micrococcus luteus strain AS2. xii Bisorption and subsequent arsenite accumulation inside the cell were confirmed through FTIR, and SEM-EDX analysis. Infrared spectra of bacterial strains, when grown normally, showed characteristics absorption peaks of amino, hydroxyl, and carboxyl groups ascertained their existence on cell surface. However, when bacterial strains were challenged with 15 mM arsenite, alternation was detected in the region of 3278 cm-1 and 1741 to 1038 cm-1 . All the bacterial strains behave differently under arsenite stress condition. The ratio of GSH and GSSG was increased up to 40 % under 15 mMarsenite in strain 1S1 while non-protein thiols were increased up to 78. 57 % in the same strain. The concentration of different antioxidant enzymes i.e., SOD, CAT, POX, and APX was varied under arsenite stress. The SOD was significantlydecreased while CAT was significantly increased under arsenite stress (15 mM). SDS-PAGE analysis revealed that proteins of molecular weights 50, 62, 37, 30, 25, and 20 kDa were overexpreesed in Microbacterium sp. strain 1S1 under 15 mM arsenite stress. The 2D-gel analysis of Micrococcus luteus revealed that SOD was down-regulated and thioredoxin reductase was up-regulated under arsenite stress. Total proteins in Microbacterium sp. strain 1S1 were identified through MS in the absence and presence of arsenite (15 mM). It was found that 24 proteins in Microbacterium sp. strain 1S1 were expressed or induced due to arsenite stress while it was found that 42 proteins were suppressed or block due to arsenite stress. Multiple metal tolerance and high arsenite oxidizing potential make these bacterial strains an ideal candidates to be utilized for bioremediation of heavy metal contaminated sites to reclaim the environment. Thus these microbes are laying an impending foundation for green chemistry to exterminate the toxic levels of environmental arsenite.