Diversity And Functional Characterization Of The Genes And Microorganisms Involved In Arsenite Oxidation From The Extreme Environments And Their Influence On Arsenic Release

Extreme environment includes the deep underground,acid mine drainage,hot springs,and hypersaline environment.Extreme microorganisms are an important component of the earth's biosphere.However,the diversity and functions of the genes and microorganisms involved in arsenite oxidation and their biogeochemical processes remains to be characterized.To address this issue,we chose the high arsenic sediment from the typical underground water,aicd mine drainage and hotspring.We systematically analyse and compare the diversity and functions of the genes and microorganisms involved in arsenite oxidation in the extreme environments and further reveal the critical roles of microbial community in the transformation and release of arsenic from the those high-arsenic sediments under aerobic condition.We also domanstrate the key role of arsenite-oxidazing organism in the arsenic geobiochemical cycle and their influence on the formation of high-arsenic groundwater.Our work gain insights into the molecular mechanism by which microorganisms drive arsenic oxidation in the high-arsenic sediments,and provide foundations for the environmental-friendly remediation of high-arsenic groundwater.Geochemical features of the sediments from deep underground shows that they containrelatively same contents of total arsenic?6.74-27.90 mg/kg?and TOC?0.27-8.37 g/kg?,but contain relatively high contents of Fe?12.13-62.50 g/kg?,SO₄^2-?14.53-863.87 mg/kg?,NH₄⁺?3.12-52.77 g/kg?and Cl^-?3.88-511.76mg/kg?and relatively low contents of TOC?0.27-8.37g/kg?,TON?0.21-1.26g/kg?,NO₃^-?0.23-3.03 mg/kg?and Mn?0.28-2.23 g/kg?.Microbial communities of deep underground contained thirty-one different phyla.Among them,the most abundant phyla was Firmicutes?63.20%of the total microorganisms?,followed by Proteobacteria?24.77%?,Actinobacteria?6.88%?.CCA showed that microbial communities structure were significantly shaped by several key environmental factors Cu?R²=0.9564,P=0.075?,Sb?R²=0.9088,P=0.018?,Al?R²=0.8969,P=0.033?,TON?R²=0.7358,P=0.017?,K?R²=0.7355,P=0.023?,Fe?R²=0.6314,P=0.078??NO₃^-,NH₄⁺,TOC and total As also had an impact on the community structures.The triplot analysis also indicated that TOC was positively associated with TON,K,Sb;Fe was positively associated with Al,Cu,Sb,K,TON.TOC has significant impact on 114 m,223m,1 m,200 m,but less impact on 65 m,30 m,95 m,175 m,145m.The arsenite oxidation of the microbial communities indicated that the microbial community from deep underground were able to efficiently oxidize As?III?using yeast extract or sodium bicarbonate as the sole carbon source.When 0.2%yeast extract or 10 mM NaHCO₃ was added to the cultures from deep underground,As?III?was completely oxidized into As?V?in 48-95,72-288 hours,respectively.Bioinformatics analysis of the aioA genes from the total genomic DNA of the samples from deep underground indicated that we identified one hundred and fourty-nine different arsenite oxidase genes,including three novel families and six novel subfamilies of AioAs family.Ninety-eight different arsenite oxidase genes were identified from deep underground,including a unique group of AioAs that represent an evolutionary intermediate between the AioAs of?-Proteobacteria and?-Proteobacteria,and three novel subfamilies of AioAs family affiliated with?-Proteobacteria and?-Proteobacteria and other seventy six new members of the AioA family affiliated with?-Proteobacteria,?-Proteobacteria,?-Proteobacteria,?-Proteobacteria,respectively.AioA proteins from the deep underground share 34 to 99%sequence identities with other known AioAs from bacteria and archaea.The arsenic release assay of the microbial communities from the sediments of deep underground indicated that the concentration of the soluble arsenic of the samples 114 m,65 m,95 m,200 m,135 m,175 m,223 m,30 m,1 m after incubation for 28 days in the slurry reached the maximum 10.78?M,10.78?M,4.69?M,3.48?M,3.19?M,2.89?M,2.01?M,1.60?M,1.41?M,0.52?M,respectively.This suggests that significant aerobic oxidization and re-absorption or re-immobilization of arsenic occured in the microcosm.Geochemical features of the sediments from acid mine drainage shows that theycontain relatively same contents of total arsenic?973.54-13387 mg/kg?,but contain relatively low contents of TOC?0.27-8.37 g/kg?and relatively high contents of SO₄^2-?1240-60680 mg/kg?.The microbial communities of acid mine drainage contained twenty-four different phyla.Among them,the most abundant phyla was Firmicutes?86.29%of the total microorganisms?,followed by Proteobacteria?11.89%?.The arsenite oxidation of the microbial communities indicated that the microbial community from acid mine drainage were able to efficiently oxidize As?III?using yeast extract or sodium bicarbonate as the sole carbon source.When 0.2%yeast extract or 10 mM NaHCO3 was added to the cultures from acid mine drainage,As?III?was completely oxidized into As?V?in 48-72,48 hours,respectively.Bioinformatics analysis of the aioA genes from the total genomic DNA of the samples from acid mine drainage indicated that we identified Thirty-one different arsenite oxidase genes from acid mine drainage,including a unique group of AioAs that represent an evolutionary intermediate between the AioAs of?-Proteobacteria and Archeae,and two novel subfamilies of AioAs family affiliated with?-Proteobacteria and?-Proteobacteria and other twenty-nine new members of the AioA family affiliated with?-Proteobacteria,?-Proteobacteria,respectively.AioA proteins from the acid mine drainage share 42%to 95%sequence identities with other known AioAs from bacteria and archaea.The arsenic release assay of the microbial communities from the sediments of deep underground indicated that the concentration of the soluble arsenic of the samples T1A?T1B?T4 after incubation for 28 days in the slurry reached the maximum 152.22?M,118.20?M,158.08?M,respectively.Geochemical features of the sediments from hot spring shows that it contains relatively same contents of total arsenic?8.64 mg/kg?,but contain relatively high contents of TOC?34 g/kg?.We also analyzed the microbial communities using 16S rRNA clone library technique.The results showed that the microbial communities of hot spring contained seven different phyla.Among them,the most abundant phyla was Proteobacteria?54.46%of the total microorganisms?,followed by Firmicutes?31.68%?,Bacteroidetes?4.46%?,Chloroflexi?3.96%?.The arsenite oxidation of the microbial communities indicated that the microbial community from hot spring were able to efficiently oxidize As?III?using yeast extract or sodium bicarbonate as the sole carbon source.When 0.2%yeast extract or 10 mM NaHCO₃ was added to the cultures from hot spring,As?III?was completely oxidized into As?V?in 216,264 hours,respectively.When 10.0 mM glucose,10.0mM lactic acid,were each added to the cultures in hot spring,As?III?was also completely oxidized into As?V?.When 10.0mM ethanoic acid was added to the cultures,the oxidation efficiency was similar to that of the microcosm without inoculation of bacterial cells;this suggests that the arsenite-oxidizing microorganisms from the sample cannot use ethanoic acid as the sole carbon source.Bioinformatics analysis of the aioA genes from the total genomic DNA of the samples from hot spring indicated that we identified twenty different arsenite oxidase genes from hot spring,including a unique group of AioAs that represent an evolutionary intermediate between the AioAs of?-Proteobacteria and Archeae,and one novel subfamilies of AioAs family affiliated with?-Proteobacteria and other eightheen new members of the AioA family affiliated with?-Proteobacteria,?-Proteobacteria,respectively.AioA proteins from the hot spring share 44%to 93%sequence identities with other known AioAs from bacteria and archaea.One thermophilic bacterial was isolated from the sediment of hot spring.When the cultures of GHS311from hot spring were incubated at 30°C,the strain GHS311 was not able to grow;when the incubation temperature increased into 45°C,GHS311 got optimal growth,suggesting that the optimal temperature for the growth of GHS311 was 45°C.A protein homology search against the GenBank database indicated that the amino acid sequence of AioA311shares 84.7%-57.6%identities to those of cultured bacterias;this suggests that the primary structure of the thermotolerant arsenite oxidase from GHS311 is similar to those of the bacterial non-thermotolerant arsenite oxidases.The arsenic release assay of the microbial communities from the sediments of deep underground indicated that when 10mM NaHCO₃ was added to the cultures,the concentration of the soluble arsenic of sediment after incubation for 28 days in the slurry reached the maximum 5.79?M and3.09?M,respectively.Twenty-six arsenite-oxidizing bacterial strains were isolated from the deep underground using an enrichment strategy.Their 16S rRNA genes were cloned and sequenced.Phylogenetic analysis indicated that those strains are affiliated with?-Proteobacteria,?-Proteobacteria,?-Proteobacteria,Actinobacteria,Bacteroidete,respectively.Heterotrophic and chemolithoautotrophic arsenite oxidizers from Jianghan Plain are able to completely oxidize As?III?into As?V?in 34-60,14-60 hours,respectively.Seven arsenite oxidase genes were identified from these cultivable strains,which are affiliated with the arsenite oxidase families of?-Proteobacteria,?-Proteobacteria or?-Proteobacteria.Horizontal gene transfer occured in two strains,of which are Pseudorhodoferax sp.N105-12 and Shinella sp.N114-3.The interation between arsenite-oxidazing bacteria and arsenopyrite under aerobic conditions shows that the arsenite-oxidazing bacteria can promote or inhibit the arsenic release from arsenopyrite.The arsenic inhibite concentrations of N105-5 and N105-12 after incubation for 28 days are13.8?M and 12.4?M,respectively;the arsenic promete concentrations of N105-16,N114-3,N125-8,N125-10 and N105-5 after incubation for 28 days are 9.3?M,5.9?M,4.1?M and 6.5?M,respectively.We finally compare the diversity and functions of the genes and microorganisms involved in arsenite oxidation in the extreme environments.The data indicated that total arsenic of samples from the acid mine drainage contain relatively high contents?973.54-13387 mg/kg?than deep underground?6.74-27.90 mg/kg?,which is higher than hot spring?8.64 mg/kg?;the TOC of samples from the hot spring contain relatively high contents?34 g/kg?than deep underground?0.27-8.37 g/kg?,which is equal to acid mine drainage?4.3-5.1 g/kg?.Illumina Miseq high-throughput sequencing results showed that the diversity of microbial communities at phylum level in deep underground is higher than acid mine drainage and hot spring.The most abundant phyla in these three extreme environments were totally different.The arsenite oxidation of the microbial communities in the three extreme environments indicated that the oxidation efficiency of microbial community in acid mine drainage is higher than deep underground and hot spring.The oxidase diversity of microbial community in deep underground is higher than acid mine drainage and hot spring.The concentration of arsenic release of the microbial communities in acid mine drainage is higher than deep underground and hot spring.