Microbially Mediated Arsenic-Sulfur Transformation And Microbial Survival Strategies In High Arsenic Hot Springs In Northeastern Tibet

Arsenic（As）is a toxic metalloid element which is ubiquitous in hot springs.Arsenic from geothermal fluid is continuously released into the surface water and groundwater with the discharge of geothermal fluids,which threatened environmental safety and biological health.It is important to understand As geochemistry in hot springs.The mobility and biological effectiveness of As in hot springs are mostly effluenced by its forms.The form of As is largely influenced by sulfur（S）element,and the As and S cycles in hot springs are closely linked.Microorganisms are the most critical members of hot spring ecosystems.The composition,diversity and interactions of microorganisms in hot springs are largely influenced by the geochemical processes of As and S.Microorganisms have developed a series of survival strategies to adapt to high As or S hot springs.Meanwhile,microbial metabolism also greatly effected on As and S transformation in the hot springs which have not been fully understood thus far.In this study,the biogeochemistry of As and S in hot springs in northeastern Tibet was studied with integrated methods including molecular biology,bioinformatics,and traditional microbial pure culture techniques.The main results are summarized as follows:（1）Geochemical characteristics of high As or S hot springs and their microbial community structure in northeastern TibetThis study examined the geochemical characteristics,microbial community structures,microbial diversities and key environmental factors of hot springs in northeastern Tibet.The results showed that the hot springs in the region are mainly neutral-alkaline,with HCO₃-Na being the main water type.The As concentration in the hot springs was generally high,with a maximum of 14.0 mg/L and an average of 7 mg/L.The main As form was AsO₃^3-（62.0%of the average total As value）.The hydrochemical characteristics of Cl^-,SO₄^2-,alkalinity and sulfide influenced the concentration of As in the hot springs.The pe+p H affected the As form in hot springs.The AsO₃^3-was dominant in hot spring with low pe+p H,and AsO₄^3-was dominant in hot spring with high pe+p H.High As and low As geothermal waters have distinctly different geochemical characteristics.At the phylum level,the microbial communities were mainly composed of Proteobacteria and Aquificae in northeastern Tibetan hot springs,and those with high As concentrations are dominated by Hydrogenobacter,Thermus,Sulfurihydrogenibium,Thiothrix,and Roseiflexus.Sulfide,AsO₃^3-,AsO₄^3-,and SO₄^2-were the key environmental factors shaping the microbial community structures in northeastern Tibetan hot springs.Arsenic concentration and its form also influence the microbial diversity of hot springs.Hot springs with higher As concentrations have higher microbial community diversity in a certain range of As concentrations.The microbial interaction network relationships were dense and the positive correlation between microorganisms was much greater than the negative correlation.（2）Metabolic potential and strategies of microbial communities in high As or S hot springsBy comparing in key metabolic potentials including As,S,C,and N in four hot springs with different As and S concentrations in northeastern Tibet,the coupling relationship between microbial As or S oxidization and N fixation was further verified through the results of cultivation and metagenomic assembled genomes（MAGs）.The metabolic strategies of microorganisms adapted to high As and S environments of hot springs and their possible metabolic coupling relationships were revealed.The results showed that there were significant differences in metabolic potentials among hot springs with different geochemical characteristics.The ars B,ars C,and acr3 were the most common As detoxification genes,while the abundance of aio B and ars M gradually increased with the increase of As concentration in the hot springs.The abundance of ars C1（Grx-ars C）was higher in high As hot springs,while the abundance of ars C2（Trx-ars C）was higher in low As hot springs.Microorganisms in hot springs with high S and As concentrations had higher nitrogen fixation rates,while those in low As and S concentration hot springs had higher carbon fixation rates.Many MAGs in hot springs have S and As oxidation-reduction related metabolic genes,indicating that As and S might be important electron donors or acceptors for microorganisms in hot springs.The geochemical characteristics of hot springs shape the microbial functional groups,which were proved in the four hot springs.Daba hot spring with the highest As concentration has the most MAGs with AsO₃^3-oxidation and As methylation metabolic potential,while Dazuo hot spring with the highest S concentration has the most MAGs with S oxidation metabolic potential.The genome analysis also provide evidence for the coupling between microbial nitrogen fixation and AsO₃^3-oxidation and S oxidation in hot spring environments.The results of microbial mat cultivation further confirmed that S oxidation and AsO₃^3-oxidation of hot spring microorganisms could promote nitrogen fixation.However,when the concentration of added AsO₃^3-was too high（>1 m M）,it may inhibit microbial nitrogen fixation to some extent in the early stage,but the inhibition could be recovered later.The S oxidation of microorganisms had a positive effect on nitrogen fixation than AsO₃^3-oxidation.（3）Microbially mediated in the geochemistry of As coupled with S in alkaline hot springsTwo alkaline hot springs（designated Gulu and Daba）with different total As concentrations（0.88 mg/L and 12.42 mg/L,respectively）and different sulfide/As ratios（3.97 and 0.008,respectively）were selected to compare the changes in geochemical parameters and the differences in the processes of microbial community succession during the outflow of hot spring water.The relationship between microbial functional group succession and As-S geochemical processes in the hot spring environment was revealed.The results showed that the concentrations of As and S compounds in the Gulu and Daba hot springs differed,but the As-S trasformation processes were similar.Thioarsenates was formed at the hot spring vent and is rapidly converted to AsO₃^3-and SO₄^2-along the drainage channel,followed by the oxidation of AsO₃^3-to AsO₄^3-.The sulfide/As ratio affects microbial communities by influencing microbial diversity,community composition and microbial interaction networks.Sulfate,total As and monothioarsenate were the key environmental factors shaping microbial communities.The potential interactions between microorganisms with low sulfide/As ratios were stronger than those of high sulfide/As ratios in co-occurrence network,which implied that microorganisms respond to high As stress by collaborating with each other.Differences in the forms and concentrations of S and As affected the succession of microbial functional groups.There were multiple microbial populations working in synergy at each step of As-S compound transformation.The relative abundances of sulfur reduction genes（dsr AB）and arsenate reduction genes（ars C）were higher upstream in GL outflow explaining high thiolation.Arsenite oxidation genes（aio AB）were relatively abundant downstream in GL outflow and at the vent of DB explaining low thiolation.Sulfur oxidation genes（sox ABXYZ）were abundant in GL and DB outflows.Putative sulfate-reducing bacteria（SRB）,such as Desulfuromusa and Clostridium,might be involved in forming thioarsenates by producing reduced S for chemical reactions with AsO₃^3-.Sulfur-oxidizing bacteria（SOB）,such as Elioraea,Pseudoxanthomonas and Pseudomonas,and arsenite-oxidizing bacteria（AsOB）such as Thermus,Sulfurihydrogenibium and Hydrogenophaga,might be responsible for the oxidation of As-bound S,thereby desulfurizing thioarsenates,forming AsO₃^3-and,further abiotic or biotic oxidizing to AsO₄³.（4）Arsenic detoxification strategy of microbial mats in high As hot springsMicrobial mats from the high As hot spring（Daba）in northeastern Tibet was selected to investigate arsenic detoxification strategy of microbial mats in hot spring.The role of extracellular polymers in microbial resistance to As stress was investigated.The existence of metabolic interactions between cyanobacteria and their epiphytic bacteria was confirmed by mixed culture experiments.And a metabolic interaction model between the microbial taxa within the microbial mat of high As hot springs were constructed through genomic and metagenomic analyses.The results showed that the hot spring microbial mat（HSMM）could oxidize 100μM AsO₃^3-within 6 days under the anaerobic light condition without any additional organic carbon and electronic acceptors.Whereas the AsO₃^3-oxidization was stopped under anaerobic dark conditions but restarted when additional nitrate or oxygen was added.Batch experiments indicated that no AsO₃^3-was oxidized by the dominant cyanobacteria Thermoleptolyngbya sichuanensis XZ-Cy5 in the microbial mat in the organic carbon-free medium.Heterotrophic bacteria enrichment in the microbial mat can oxidize AsO₃^3-in the organic carbon-contain medium.Without the epiphytic bacteria,the photosynthetic system of the dominant cyanobacterium XZ-Cy5 in HSMM was destroyed with 5 m M AsO₃^3-,whereas epiphytic AsOBs decreased the damage by converting it to arsenate.The metagenome results indicated that HSMM had multiple carbon fixation pathways such as the Calvin cycle,reductive acetyl Co A（WL）pathway and tricarboxylic acid cycle（r TCA）.It also contains nitrogen cycle genes,such as nir K,nor BC,nos Z and nif H,as well as aio AB,ars C,ars M and SOX sulfur oxidation system.These genes are distributed in different taxa with frequent metabolic interactions between them.Cyanobacteria provided oxygen,organic carbon,nitrogen sources for other bacteria.Entangled filamentous cyanobacteria and their extracellular polymeric substances can serve as substrates for the colonization of other microorganisms.Other bacteria can reduce As toxicity in the microenvironment by oxidizing AsO₃^3-and provide growth factors such as vitamins.These results suggest that cooperation and cross-feeding drive microbial adaptation to high As hot spring environments.The innovation of this thesis are:（1）Analyzing the geochemical process of As coupled with S driven by the microbial community succession along the drainage channels of hot springs.（2）Revealing the survival strategy of cyanobacteria and heterotrophic bacteria in hot spring microbial mats,which adapt to high As and oligotrophic hot spring environment through metabolic interactions.