Isolation And Identification Of Photosynthetic Bacteria And Study Of Arsenic Metabolism Mechanism

Arsenic(As)is a toxic and carcinogenic metalloid,which is widely distributed in the environment.In recent years,arsenic contamination in paddy soil is seriously.Arsenic contamination not only reduce crop yield,but also increase As accumulation in rice grain,causing the potential threat to human health.Phyllosphere of duckweeds in paddy soil environments is a hotspot in arsenite(As(?))oxidation which may have a substantial impact on As biogeochemistry in water environments.Previous work has shown that As(?)oxidizing bacterial communities in the phyllosphere of duckweeds were dominated by Rhodobacter species.There are many microorganisms on the surface of duckweed,which can oxdize As(?).Gene clone library indicate that the arsenite oxidase gene(aioA)is mainly derived from photosynthetic bacteria(Rhodobacter).Therefore,we suppose that there might be light-dependent As(?)oxidation pathway in the process of arsenic cycling in paddy soil.We aim to find light-dependent As(?)oxidation bacteria in duckweeds surface.And a photosynthetic bacterium named Rhodobacter sp.CZR27 was successfully isolated from duckweeds,which collected from an As-contaminated paddy soil in Hunan province.The As transformation and resistance mechanism in Rhodobacter sp.CZR27 was studied in this project.The main results are as follows:(1)16S rRNA and aioA gene abundance in duckweeds,water,surface soil and rhizosphere soil in As-contaminated paddy soil were quantified.It showed that As(?)-oxidizing bacteria mainly distribute on duckeeds surface.Therefore,we enriched microorganisms on duckeeds and a photosynthetic As-oxidizing bacteria named Rhodobacter sp.CZR27 was successfully isolated.Scanning electron microscope showed that CZR27 is round and without flagella.The diameter of CZR27 is about 1-1.?m.The photosynthetic pigment in this bacterium is chlorophyll a.The optimal carbon source for CZR27 growth is malate under anoxic light,anoxic denitrification or aerobic darkness conditions.We also quantified CZR27 distribution indifferent samples,Rhodobacter sp.CZR27 had a larger proportion on the surface of duckweeds.(2)The highest tolerance concentration of As(?)and As(V)for strain CZR27 were 200 ?M and 10 mM,respectively.Strain CZR27 can oxidize As(?)under anoxic light,anoxic denitrification and aerobic darkness conditions.CZR27 has higher As(?)oxidation efficiency under anoxic contions in the light,which can completely oxidize 50 ?M As(?)within 96 hours.Oxidation of As(?)by the strain under anoxic phototrophic conditions coincided with bacterial expression of aioA,indicating participation of the gene in the As(?)oxidation process.(3)Expression of ars genes after exposure to As(?)and As(?)was analyzed.Both As(?)and As(?)were effective inducers.There was no detection of As(?)in cell cultures incubated with As(?).Gene analysis of Rhodobacter sp.CZR27 showed that arsRl(arsRC)was involved in the reduction process of As(?).The results showed arsRC sequence is homologous with As(?)reductase gene arsC.We expressed arsRC in E.coli WC3110 and found that arsRC can confer E.coli resistance and reduction to As(?).