The Mechanism Of Perchlorate Reduction In Methane Based Biofilm

The pollution of perchlorate（ClO₄^-）in water environment,especially in groundwater,has becoming more and more serious.The latest research showed that microorganisms can reduce ClO₄^-to non-toxic Cl-using the greenhouse gas methane（CH₄）as an electron donor,which can achieve the potential greenhouse gas emission reduction while removing pollutants.However,the microbiological mechanism of this process has not been explored.In order to solve these key scientific questions,methane oxidation coupled with perchlorate reduction（MO-PR）microorganisms was used in this study.Constructing the methane-based membrane biofilm reactor,we studied the inter-specific interaction,methane metabolic pathway and electron competition mechanism,as well as optimized the operation conditions of the reactor.The conclusions were as follows:In MBBR,0.56 mM ClO₄^-was reduced to below the detection limit within 31 days,and the highest reduction rate was 2.34 m M/m²·d.The dominant bacteria in the reactor were PRBs Denitratisoma,Azospira,Methylococcus,Methylomonas and Methanosarcina.The abundance of perchlorate reductase gene pcr A,key enzyme gene mcr A in reverse methanogenesis process and the functional bacteria Denitratisoma increased with the increase of ClO₄^-load.Since no O₂ was detected during the operation,we speculated that the archaea Methanosarcina carried out the reverse methanogenesis to activate methane,and generated electrons for PRBs Denitratisoma and Azospira to degrade ClO₄^-.Isotope tracing and enzyme inhibition tests showed different electron acceptors lead to different carbon metabolic pathways of methane.When ClO₃^-acted as electron acceptor,the dechlorination rate of biofilm can reach 17.5 μM/d.77.6% ¹3CH₄ consumed by microorganisms was converted into ¹3CO₂,while the rest was assimilated into DOC.However,no DOC was detected when ClO₄^-acted as electron donor.Metagenomics analysis confirmed that NC10 bacteria participated methane oxidation coupled to chlorate reduction（MO-CR）,and used O₂ produced by ClO₂^- disproportionation to activate methane.PRBs can also utilize NO₃^-as electron acceptor,when NO₃^-and ClO₄^-coexisted,the biofilm will preferentially consume NO₃^-,and NO₃^-will reversibly inhibit the reduction of ClO₄^-.The analysis of metabolic kinetics showed both Nar and Pcr catalyzed NO₃^-faster than ClO₄^-.Therefore,dominant bacteria Denitratisoma and Azospirillum consumed NO₃^-first when NO₃^-and ClO₄^-coexist.Density functional analysis showed that compared with ClO₄^-reduction,the energy barrier required for proton coupled electron transfer in NO₃^-reduction was lower,which leads to NO₃^- has advantage over ClO₄^-in competing for enzyme activity sites.Pilot scale MBfR could completely reduce 2 mg/L ClO₄^-in simulated wastewater. The maximum pollutant removal flux was 2.18 g/m²·d.The results showed that the pollutant removal flux decreased during the scale-up process,mainly due to the increase of dissolved oxygen in the influent,which inhibited the dechlorination efficiency of the biofilm.Batch experiments confirmed that a limited amount of oxygen（0.5 1.0 mg/L）can effectively enhance the aerobic oxidation of methane and promote dechlorination,while excessive oxygen concentration（≥2.0 mg/L）could inhibit the reduction of perchlorate.The above results enriched people’s understanding of microbial dechlorination.It also provided theoretical supports for perchlorate bioremediation,and have theoretical and practical significance for the removal of oxyanions and greenhouse gas emission reduction.