Impacts Of Joint H₂ And O₂ On Microbial Community Structure And Function In Chlorinated Hydrocarbons Contaminated Groundwater

Chlorinated hydrocarbons are widely used in various industrial processes,such as the production of plastics,rubber,silicone resin,adhesives,pharmaceuticals,cleaning agents,refrigerants,metal degreasing,etc.Due to its widespread use and improper treatment methods,it often causes severe pollutants to soil and groundwater.Most chlorinated hydrocarbons are carcinogens,significantly harming the ecological environment and human health.The most common chlorinated hydrocarbons in the environment include chlorinated alkanes and chlorinated alkenes,usually in the form of combined pollution in groundwater,mainly including tetrachloroethylene（PCE）,trichloroethylene（TCE）,carbon tetrachloride（CT）,chloroform（CF）and so on.Remediating chlorinated hydrocarbon-contaminated groundwater is a hot issue in environmental science and engineering at home and abroad.Functional microorganisms can degrade chlorinated hydrocarbons in contaminated groundwater by reductive dechlorination and bio-metabolism or cometabolism.However,electron donors and acceptors often limit the biodegradation of chlorinated hydrocarbons in the remediation process.It is necessary to inject hydrogen（H₂）or organic substrates such as lactate,acetate,and minor molecular alcohols into groundwater to enhance the reductive dechlorination of chlorinated hydrocarbons or provide O₂ by injecting air,pure oxygen（O₂）,hydrogen peroxide to promote the aerobic biodegradation of chlorinated hydrocarbons.Compared to injecting other electron donors/acceptors,in situ supply H₂ and O₂ do not produce secondary pollution and are more environmentally friendly.The direct injection of H₂ and O₂ has high costs and poor safety and has rarely been used before.However,the electrochemical technology developed in recent years can provide H₂ and O₂ to the aquifer by electrolysis of water,forming a coexistence area of H₂ and O₂ in the aquifer.However,the effect of joint H₂and O₂（H₂/O₂ coexistence）on microbial community structure and function is still unclear,and the degradation mechanism of complex chlorinated hydrocarbons under H₂/O₂ coexistence needs to be explored.Because of the above scientific problems,the research contents of this thesis include（1）Response of microbial functional groups to H₂/O₂ injection in contaminated site aquifer;（2）Microbial community mechanism of complex chlorinated hydrocarbons degradation in H₂/O₂ coexistence system;（3）Identification and cooperation mechanism of H₂,O₂ and TCE metabolic microorganisms in the H₂/O₂ coexistence system;（4）The molecular biological mechanism of TCE degradation by functional strain Acinetobacter soli HO-1 in the H₂/O₂ coexistence system.The main conclusions are as follows:（1）Response of microbial functional groups to H₂/O₂ injection in contaminated site aquiferIn a typical chlorinated hydrocarbon contaminated site,different H₂ and O₂conditions were provided to the monitoring wells by water electrolysis.A self-made bio-trap device obtained in-situ sediment samples at different reaction times.The evolution process of microbial functional groups was tracked and monitored.The effects of varying H₂ and O₂ conditions on microbial functional groups and their chlorinated hydrocarbon degradation potential were explored.The applicability of the bio-trap method to study the dynamic changes in microbial community structure during the remediation process was verified.Through KEGG functional annotation,it was found that under O₂ conditions,the electron transfer activity,carbon fixation ability,and D-type amino acid metabolism function of the microbial community were enhanced.Still,most of the carbon source utilization and pollutant degradation functions were inhibited,and the functional genes related to phenol monooxygenase,nitrogen fixation,nitrification,aerobic nitrate reductase（catalyzing NO₃^-to NO₂^-）and sulfur oxidation were enriched;The input of H₂ inhibited electron transport activity,most carbon source utilization and pollutant degradation activity of the microbial community,but promoted sulfur metabolism,and enriched genes related to anaerobic pollutant degradation and sulfur reduction;The input of H₂/O₂ promoted oxidative phosphorylation,metabolism of most carbon sources,degradation of various pollutants,and N and S metabolism.Functional genes encoding phenol monooxygenase,dioxygenase,nitrogen fixation,nitrification,aerobic and anaerobic nitrate reductase（catalyzing NO₃^-to NO₂^-）,nitrite reductase（catalyzing NO₂^-to NH₄⁺）,nitric oxide reductase（catalyzing NO to N₂）and sulfur oxidation increased.The results showed that H₂/O₂ coexistence is more conducive to the C,N and S cycle and the degradation of organic pollutants,including chlorinated alkanes and chlorinated alkenes.At the same time,it was verified that the bio-trap method was suitable for studying the dynamic change process of microbial community structure in the actual remediation process,which solves the problem that in-situ sediments are difficult to obtain in the field experiment.（2）Microbial community response mechanism of complex chlorinated hydrocarbons degradation in H₂/O₂ coexistence systemThe field experiment suggested that the microbial community function changed significantly after the simultaneous introduction of H₂ and O₂,and the microbial chlorinated hydrocarbon degradation function was enhanced.However,due to the complex environment of the field site and the influence of groundwater flow,the degradation of chlorinated hydrocarbons cannot be determined by detecting the concentrations.Therefore,an indoor batch experiment was designed to simulate the groundwater environment.Nine kinds of mixed chlorinated hydrocarbons（PCE,TCE,trans-1,2-dichloroethylene,CT,CF,dichloromethane,1,1,2,2-tetrachloroethane,1,1,2-trichloroethane,1,2-dichloroethane）were selected as representative chlorinated pollutants.Different H₂ and O₂ conditions were created to analyze the kinetic processes of various chlorinated hydrocarbon degradation.High-throughput sequencing and functional gene q PCR quantitative methods were used to analyze the microbial community structure and functional changes.To further analyze the mechanism of microbial transformation of compound chlorinated hydrocarbons from the community level.It was found that joint H₂/O₂ could promote the conversion of 2-chloro and 3-chloro substituted chlorinated hydrocarbons that could be degraded under anaerobic or aerobic conditions,and formed a unique microbial community with high biodiversity;The specific microbial species mainly exist in phyla Proteobacteria and Bacteroidetes,including Methyloversatilis,Dechloromonas,Sediminibacterium,Pseudomonas,Acinetobacter,Curvibacter,Comamonas and Acidovorax;Compared with H₂ and O₂ conditions,the consumption of H₂ and O₂ was faster under the condition of H₂/O₂ coexistence,indicating that microorganisms using H₂ or O₂ coexisted in the microbial community,or there were microorganisms that consumed both H₂ and O₂;The relative abundance of aerobic functional genes phe,sox B and anaerobic functional gene tce A increased simultaneously in the H₂/O₂coexistence system,indicating that aerobic and anaerobic degradation of chlorinated hydrocarbons existed simultaneously under the condition of H₂/O₂ coexistence.The presence of H₂ can reduce the toxicity of O₂ to anaerobic or facultative anaerobic microorganisms and may also be one of the reasons why H₂/O₂ coexistence promotes the degradation of chlorinated hydrocarbons.（3）Identification and cooperation mechanism of H₂,O₂ and TCE metabolic microorganisms in the H₂/O₂ coexistence systemTo further determine the degradation process of chlorinated hydrocarbons and the types of functional microorganisms in the H₂/O₂ coexistence system,the degradation kinetics of TCE were studied by simulating the actual groundwater environment.The TCE,H₂ and O₂ in the H₂/O₂ coexistence system were labelled with ¹³C,²H and ¹⁶O isotopes.Stable isotope probes and high-throughput sequencing technology（DNA-SIP）were used to detect the microbial composition of heavy isotope DNA,identify the microbial species that metabolize TCE,H₂ and O₂ under the condition of H₂/O₂coexistence,and explore the synergistic mechanism of functional microorganisms on TCE degradation.The results showed that in the H₂/O₂ coexistence system,the degradation amount of TCE was 13.00μM within 40 days,and a small amount of 1,1-dichloroethylene（less than 0.53μM）was detected on the 25th day;Through DNA-SIP,72 OTUs of TCE metabolic functional microorganisms,112 OTUs involved in H₂metabolism and 116 OTUs involved in O₂ metabolism were screened out;Among them,8 OTUs can metabolize TCE and O₂ simultaneously,6 OTUs can metabolize TCE and H₂ simultaneously,9 OTUs can metabolize H₂ and O₂ simultaneously,and 3 OTUs can metabolize TCE,H₂ and O₂ simultaneously,belonging to Methyloversatilis and SH-PL14,respectively;Geobacter and Clostridium capable of anaerobic reductive dechlorination TCE were found in the H₂/O₂ coexistence system,and enriched in the heavy layer of the ²H-H₂ labelled group.Therefore,reductive dechlorination of TCE and aerobic oxidative degradation of TCE and reductive dechlorination product 1,1-DCE existed in the H₂/O₂ coexistence system,and there were functional microorganisms that use H₂ and O₂ as energy sources to grow and metabolize TCE.（4）The molecular biological mechanism of TCE degradation by functional strain Acinetobacter soli HO-1 in the H₂/O₂ coexistence systemTo further explore the degradation mechanism of TCE under H₂/O₂ coexistence conditions,Acinetobacter soli HO-1,which was screened from the complex chlorinated hydrocarbon contaminated sediments treated with H₂/O₂ coexistence,was used as a representative dominant strain to explore the degradation kinetics of TCE.Using ¹³C,²H and ¹⁶O isotope labelling combined with Nano secondary ion mass spectroscopy technology（Nano SIMS）,the metabolism of ¹³C-TCE,²H-H₂ and ¹⁸O-O₂ by the strain was investigated from a microscopic view,and the molecular biological mechanism of TCE degradation by the strain under the condition of H₂/O₂ coexistence was studied using genomic framework technology.It was found that the isolated strain Acinetobacter soli HO-1 could degrade TCE in an H₂/O₂ coexisting system,and the degradation efficiency of TCE was the highest when the molar ratio of H₂ to O₂ was 2:1.The results of Nano SIMS showed that after cultured with ¹³C-TCE,²H-H₂ and ¹⁸O-O₂,the ²H/¹H ratio of the bacteria was about 3.4 times that of the unlabeled bacteria.The¹⁸O/¹⁶O ratio was about 2.6 times that of the unlabeled bacteria.The ¹³C/¹²C isotope ratio increased slightly.These results indicated that the strain could utilize both H₂ and O₂,and metabolize a small amount of TCE.Through the analysis of functional genes in the genome framework,the molecular biological mechanism of TCE degradation was revealed as follows:Reductase encoded by the Que G gene catalyzes the reductive dechlorination of TCE,hydrogenase was responsible for directing electrons from H₂ to the TCE respiratory chain.glutathione S transferase catalyzes the initial oxidation of TCE,which is converted to S-（1,2-dichloroethylene）glutathione,further converted to S-（1,2-dichloroethylene）L-cysteine,and then to chloroacetic acid under the catalysis ofβ-lyase;H₂,O₂ and CO₂ were used for cell growth and accumulation of poly-β-hydroxybutyrate（PHB）with the participation of Pha C gene,PHB as carbon source,or endogenous reducing energy,regenerated NADH for TCE degradation.In summary,this study found that the coexistence of H₂/O₂ could promote the degradation of chlorinated hydrocarbons.The influence mechanism of H₂/O₂ coexistence on microbial functional community structure and chlorinated hydrocarbon degradation was revealed.