Nitrate Transformations Catalyzed By The Arsenic Redox Microorganisms And Their Environmental Influences

Arsenic redox microorganisms play key roles in the As-biogeochemical cycle.Envrionmental factor compounds often change the valence and solubility of arsenic in the environment by affecting or regulating the metabolic activities and physiological functions of arsenic-metabolizing microorganisms.In these processes,the environmental factor compounds themselves also transform into other forms.However,little is known about how the interaction between environmental factors and anaerobic arsenic-metabolizing microorganisms.Nitrate is a typical nitrogen contaminant in the groundwater environment and a terminal electron receptor in the respiratory electron transport chain.Nitrate is also one of the most ubiquitous environmental factors in the arsenic-contaminated environment.Existing studies have shown that some arsenic-reducing organisms can respire As（Ⅴ）as well as nitrate.However,it remains to be elucidated for how nitrate affects the microorganisms-catalyzed dissolution and reduction of arsenic.On the other hand,nitrate can be used as electron acceptor to participate in the As（Ⅲ）oxidation catalyzed by anaerobic arsenite-oxidizing bacteria.However,little is known about the fate of nitrate denitrification during this process.In order to further explore the nitrate transformations catalyzed by the arsenic redox microorganisms and their environmental influences,three different samples of arsenic-contaminated soils or sediments were collected from different environments,including the deep underground of Jianghan Plain,the realgar mining area in Shimen and the hot springs in southern Tibet.The interaction between Arsenic redox microorganism and nitrate and its effect on environment were systematically analyzed by the microbiology and molecular biology.The main conclusions of this paper are as follows:（1）Inhibitory effect of nitrate/nitrite on the microbial reductive dissolution of arsenic.In order to know how nitrate affects the microorganisms-catalyzed dissolution and reduction of arsenic,we collected soil samples containing high-contents of arsenic from the Shimen Realgar Mine area.Microcosm assay indicated that addition of nitrate/nitrite significantly inhibited the dissolution,reduction and release of As caused by the biological catalysis of microbial communities in the soils.After 5 days,As（Ⅲ）decreased by 26.9-33.6%.Meanwhile nitrate/nitrite was reduced into N₂O,N₂or NO.Quantitative PCR analyses of arsenate-resipring reductase（Arr）genes indicated that the presence of nitrate/nitrite siginificantly decreased the Arr A gene abaundances by 59.85-93.86%in the sediment microcosms.To further investigate the molecular mechanism of this finding,we isolated a representative dissimilatory arsenate-respiring strain Shewanella sp.GL90 from the soils to perform the arsenic release assay.GL90 is able to efficiently respire As（Ⅴ）,NO₃^-and Fe（Ⅲ）coupled with the oxidation of lactate.We observed that nitrate/nitrite significantly inhibited the DARPs-catalyzed solubilization of As from the sterile arsenic contaminated soils.After 14 days,As（Ⅲ）decreased by24.5-71.9%.Meanwhile nitrate is quickly reduced to nitrite by GL90,while the nitrite is slowly reduced to ammonium.RT-q PCR analyses indicated that the presence of nitrate/nitrite significantly decreasing the transcriptions of the gene of the respiratory arsenate reductase in GL90 cells.In order to further explore the effect of nitrate/nitrite on the reductive dissolution of arsenic-bearing minerals mediated by dissimilatory arsenate-respiring strain GL90,scorodite was selected for the microcosm assay.The reduced scorodite catalyzed by GL90 revealed that the mineral particles were altered dramatically,and nitrate/nitrite inhibited the reductive dissolution of solid arsenic in scorodite.It was found that nitrate/nitrite significantly reduced the reductive dissolution of arsenic by dissimilatory arsenate-respiring microorganisms by inhibiting the expression of arsenate-resipring reductase（Arr）genes.（2）Anaerobic As（Ⅲ）oxidation coupled to nitrate reduction in different arsenic-contaminated enviroments.In order to know anaerobic As（Ⅲ）oxidation coupled to nitrate reduction in different arsenic-contaminated enviroments,samples were collected from three different arsenic contaminated environments,including the deep underground of Jianghan Plain,the realgar mining area in Shimen and the hot springs in Tibet.Microcosm assay indicated that the microbial communities of the three environments possessed significant anaerobic As（Ⅲ）oxidation activitiest.The carbon source preferences of microorganisms in different environments are diverse.The anaerobic As（Ⅲ）oxidation efficiencies are higher when microorganisms in the Jianghan Plain utilize the inorganic carbon sources,and the anaerobic As（Ⅲ）oxidation efficiencies are more stable when microorganism in the Shimen Mining area utilize the inorganic carbon sources.In the presence of inorganic and organic carbon sources,the microbes from hot springs in Tibet have high As（Ⅲ）oxidation activities,and can still catalyze As（Ⅲ）anaerobic oxidation at 80°C.In addition,the autotrophic nitrate reduction coupled with As（Ⅲ）oxidation system using HCO₃^-as carbon source was constructed.It was found that the nitrate reduction products catalyzed by microorganisms in different arsenic-rich environments were also different.NO and N₂ were the main reduction products driven by microorganisms in Jianghan Plain sediments,and N₂O and NO₂^-were the main products in Shimen,the reduction products in hot spring are mainly NO₂^-.The microbial-catalyzed autotrophic nitrate reduction coupled with As（Ⅲ）oxidation in the above three high arsenic contaminated environments can significantly reduce the total soluble arsenic content in the system.The results show that the microbes in different arsenic contaminated environments have the activity of nitrate reduction coupled with arsenic oxidation,but the activity is significantly different.（3）Isolation of different anaerobic As（Ⅲ）-oxidizing strains and their coupling nitrate reduction process.In order to investigate the of nitrate reduction kinetics coupled with different anaerobic As（Ⅲ）oxidation strains,ten strains capable of anaerobic arsenite oxidation were obtained from Jianghan plain sediments and Shimen soils by enrichment and separation technique.These 10strains all showed strong arsenic oxidation ability,the oxidation efficiency in 60 minutes was as high as 80-100%.Moreover,we found that there are many different types of nitrate reduction products catalyzed by the different anaerobic As（Ⅲ）-oxidizing strains.The 16S r RNA gene and Aioa gene were amplified,cloned and sequenced.The results showed that the 10 strains were distributed in different Genera.The functional expriements showed the iron transformation function of different arsenic-oxidizing bacteria is also different.JH-4 strain is a NO₃^-reducing Fe（II）oxidizer.JH-5 can reduce 1.0 m M ferric citrate in the presence of 0.2%yeast.We also found the strains of JH-1、JH-2、JH-3、JH-4、SM-3、SM-4 can catalyze the reaction of nitrate reduction couple with sulfide oxidation.The results showed the coupling nitrate reduction kinetics of different anaerobic As（Ⅲ）oxidation strains and the diversity of their functions.（4）As（Ⅴ）catalyzes the single bacterium Escherichia sp.GA1-mediated dissimilatory nitrate reduction to ammonium.In order to investigate the effect of As（Ⅴ）on microbial-catalyzed nitrate reduction,we isolated a arsenate reducing bacterium Escherichia sp.GA1.It was found that the addition of As（Ⅴ）catalyzes the single bacterium Escherichia sp.GA1-mediated dissimilatory nitrate reduction to ammonium by increasing the growth of GA1.Three arsenic resistance reductase（Ars C）and three different types of nitrate reductase,including respiratory nitrate reductase（Nar G）,periplasmic nitrate reductase（Nap A）and cytochrome C nitrite reductase（Nrf A）were identified by analyzing the bacterial genomic data.The strain of GA1 can efficiently reduce As（Ⅴ）,Fe（Ⅲ）,S₂O₃^2-and SO₄^2-by using 10.0 m M sodium lactate as the only electron donor under anaerobic condition.The results showed As（Ⅴ）catalyzes the single bacterium Escherichia sp.GA1-mediated dissimilatory nitrate reduction to ammonium.