Identification Of Arsenate Reducing Bacteria And Their Potential Metabolic Traits In Hydrothermal Sediments In Tibet By The DNA-SIP Coupled To Metagenomic Analysis

Tibet is a typical high-arsenic geothermal ecosystem in China.In recent years,the serious arsenic pollution problem in the region has endangered the health of local residents.Therefore,resolving the mechanism of arsenic contamination is urgently needed.Studies have reported that arsenic-reducing microorganisms drive the release and transport of arsenic,which can reduce stable,low-toxicity pentavalent arsenic to mobile and highly toxic trivalent arsenic.However,research on microbial-driven arsenic reduction mechanisms in Tibetan hot spring systems is limited.A systematic study of microorganisms and their arsenic reduction mechanisms in Tibetan hot spring ecosystems will reveal the mechanisms of arsenic release in the region and provide new insights for arsenic pollution prevention and control.This study constructed an anaerobic microcosm culture system to simulate the anaerobic environment at the sediment near Riduo hot spring in Tibet.Acetic acid was used as an electron donor and carbon source and As(Ⅴ)as an electron acceptor to verify whether microorganisms are involved in the process of arsenic reduction.The stable isotope tracing technique(DNASIP)was used to identify functional strains of As(Ⅴ)-reducing microorganisms by labeling them with 13C-acetate.The functional microorganisms mediating anaerobic arsenic reduction processes and their metabolic mechanisms in arsenic-contaminated environments under thermal spring systems were explored in combination with metagenomic high-throughput sequencing.In this study,we found that microcosm culture experiments at a single arseniccontaminated oligo carbon nutrient-rich hot spring in Riduo,Tibet,showed that microorganisms in the system could use acetic acid as a single carbon source and electron donor to drive the As(Ⅴ)reduction process.DNA-SIP experiments showed that microorganisms could successfully label As(Ⅴ)-reducing functional strains by assimilating 13C-acetic acid and incorporating isotopically labeled carbon into their genomic DNA.16S rRNA amplicons sequencing showed that the dominant functional microorganisms driving the anaerobic arsenic reduction process in this contaminated sediment were two genera of bacteria,Pseudomonas and Thermincola,respectively.The metagenome sequencing results revealed that Pseudomonas and Thermincola genera contain both genes related to the arsenic respiration reduction pathway and the arsenic efflux pathway.Their diverse arsenic metabolism pathways make them the main microorganisms responsible for arsenic reduction in Tibetan hot spring sediments.The identification of carbon fixation-related genes,nitrogen transformation,sulfur transformationrelated genes,and other metal resistance genes in these two genera suggests that they may respond to this oligotrophic hot spring environment through other alternative non-arsenic transformation pathways,contributing to their environmental dominance in the genus.Further pure bacterial isolation and culture of Pseudomonas and Thermincola can be followed and applied to arsenic-contaminated sites for implementation into practical soil microbial remediation projects.Therefore,this project traces the core microorganisms involved in the arsenic reduction process by a cutting-edge method combining high-throughput sequencing technology and DNA-SIP.The association between indigenous microorganisms and arsenic transport and transformation in Tibetan hot spring sediments was investigated.The metabolic information of arsenic-reducing microorganisms in sediments under anaerobic conditions was also explored.The results of this study are expected to provide theoretical support for microbial remediation of arsenic in hot spring environments,which is of great environmental remediation significance for mitigating arsenic pollution problems.