Functional and Unique Diversities of Genes and Microorganism Involved in Redox Reactions of Arsenic from the Severely-Contaminated Soils


Supervisor:zeng xian chun


Degree Year:2019





The soils near the tailings of Shiman Relgar mine are severely contaminated by arsenic.To determine the microbial reactions and environmental factors responsible for the mobilization and release of arsenic from soils into pore water,we collected 24 soils samples from the representative sites close to the tailings of Shiman Realgar mine.The soluble arsenic in the samples show significant correlations with theThis study focused on the functional and molecular characterizations of microbial communities from the Shimen town.We fully explored,for the first time,the arsenate-respiring reduction,and arsenite-oxidizing activities,the functional gene diversities of microorganisms from the Shimen town,as well as the correlation of the microbial activities/diversities with environmental factors.The findings of this study help us to better understand the diversities of the arsenite-oxidizing bacteria and the geochemical cycle of arsenic in the arsenic severely contaminated soil of the Shimen.This research gains an insight into the microbial mechanisms by which the microorganisms drive from oxidation and reduction in our arsenic-severely contaminated soils.However,diversity and functions of the genes and microorganism involve in arsenite-oxidation and arsenate-reduction,with their biogeochemical process in Shimen town remains to be characterized,but the issues were addressed,by having arsenic-severely contaminated soil.We critically look at the roles of microbial community,with their functions and release of the arsenic from the arsenic-severely contaminated soil under aerobic and anaerobic conditions.This work also offers a set of unique arsenite-oxidizing bacteria for basic research of the molecular functions of arsenite-oxidation in a bacterial and arsenate-respiring reduction for the environmentally friendly bioremediation of arsenic-contaminated groundwater.Our research is based on microbiology and molecular biology point of views,a systematical analysis of the diversity and functions of the genes and microorganisms involved in the redox from the arsenic-severely contaminated soil.Based on our specific objective’s geochemical feature analysis was detected followed by extraction of genomic DNA.Microcosm strategy was used to evaluate arsenite-oxidation of the microbial communities from our arsenic-severely contaminated soils.For the clear understanding of the molecular basis of the microorganisms involved in arsenite-oxidation and arsenate reduction,we detected the diversity of the aioA and arrA genes from our total genomic DNA for each sample.To determine if the microbial communities form our arsenic severely contaminated soils,are able to promote arsenic release from the soil into an aqueous phase,both aerobic and anaerobic condition were used.Geochemical analyses indicated that maj ority of the soil samples contained extremely high concentrations of total and soluble arsenic.As shown in Table 2,among the all the 25 samples,7 contained>10,000 mg/kg total As(TA)(S1,S2,S15,S11,S7,S10,and S12,)8 contained 1000-10,000 mg/kg TA(S6,S13,S8,S3,S20,S6,and S15,)6 contained 100-1000 mg/kg(S19,S22,S5,S21,S16,and Sl1)and 2 contained 80-100 mg/kg,while 9 contained 1000-5000 mg/kg soluble As(SA),(S7,S8,S1,S20,S13,S11,and S12)8 contained 100-1000 mg/kg SA 8 contained 1-100 mg/kg SA(S4,S25,S15,S23,S7,S6,and S11)and only 2 contained less than 1 mg/kg SA(S25,and S7).Considering that the general As-contaminated soils contained approximately 20-100 mg/kg total As and less than 0.1 mg/kg soluble As,we can say that the soils near the abandoned Shiman Realgar Mine are terribly contaminated.Arsenite-oxidation is one of the major biogeochemical processes that occur in the town.However,little is known about the functional and molecular aspects of the microbial community involved in arsenite-oxidation.Here,we fully explored the functional and molecular features of the microbial communities from the Shimen town,all the 50 bacteria colonies belong to the different phyla among them the most abundant phyla was Proteobacteria(30.63%,),Chloroflexi(17.59%),Acidobacteria(13.60%),Nitrospiran(5.36%,).The microbial communities of the six sites contain 50 phyla of bacteria and 18 subs new family that show extremely high diversity.Our microbial communities’ structures were significantly shaped,by some key environmental factors such as NO3-,NH4 TOC and total As also has an impact on the structure of the communities.The arsenite-oxidation of microbial communities indicated that the microbial community from site S7 and S11 organic sample was oxidized fully from As(Ⅲ)into As(Ⅴ)at 24hrs,while the inorganic and non-carbon sources were fully oxidized at 48hrs.The 0.2%yeast extract(organic),has strong oxidization activities,10.OmM NaHCO3,respectively.The arsenic release assay of the microbial communities from the arsenic-severely contaminated soil indicated that the concentration of soluble arsenic from S7,S11,S15,S15,and S9,,after incubation for 16,days in the slurry reached the maximum 2.499107 mM,3.335108 mM,3.00413 mM,2.61786 mM,and 3.08129 mM,respectively.This suggests that significant aerobic oxidation and re-absorption or re-immobilization of arsenic occurred in the microcosm.It was shown that under anaerobic conditions,severely-arsenic contamination from the soil from Shimen town was severely contaminated with arsenic,however,little is known about the mechanism by which the arrA genes arsenic was mobilized and released into groundwater from the arsenic severely contaminated sediments in this area.Although all of the samples contain high contents of total arsenic,the soluble arsenic was only detectable in our different site,with different concentration’s,ranging from(2.499197 to 3.335108mM)respectively,of the soil,but was readily detectable in all of the soil at the Analysis of the genes of arsenate-respiring reductases indicated all our site has different level of concentration of arsenate releases,all of the identified reductase genes are new or new-type,and they display unique diversity.Microcosm assay indicated that the microbial communities from our sites of each soil were able to reduce As(V)into As(Ⅲ)using lactate,formate,pyruvate or acetate as an electron donor under anaerobic condition.Arsenic release assay demonstrated that these microbial communalities efficiently catalyzed the mobilization and release of the soil arsenic into an aqueous phase.We also clone Arr genes from the soil.It is able to completely reduce 2.0 mM As(Ⅴ)into As(Ⅲ)in 72 h,and efficiently promote the reduction and release of the mineral arsenic into an aqueous phase.In comparison,on the 5th,13th,and 21st day of incubation,the un-autoclaved slurry contained 0.40,1.39,and 1.73 and 2.0mM of soluble arsenic,respectively;however,the autoclaved slurry still contained no significant amount of soluble arsenic.This suggests that microorganisms from the S4 of significantly promoted the mobilization and release of insoluble arsenic into an aqueous phase.