Enrichment Of Archaeal Anaerobic Methanotrophs And Anaerobic Oxidation Of Methane Driven By Manganese Minerals

Anerobic oxidation of methane?AOM?is an important biochemical process.As a key step in the natural CH₄ metabolism,it cuts down more than 90%of CH₄ from ocean floor and about 50%of CH₄ from freshwater wetland.As a result,only limited amount of CH₄ eventually emitted into the atmosphere.Therefore,AOM plays a very important role in the Earth's carbon cycle.Researchers have observed metal-driven AOM in both marine and freshwater systems at rates up to 15%of the maximum methanogenic rate in natural habitats.Metal-driven AOM is important to biogeochemical cycles and geological evolution on early and modern Earth.Manganese is widely present in various marine and freshwater environments.However,the functional microbes and processes involved in the AOM by manganese minerals have rarely been studied so far.In this thesis,sequencing batch reactors were constructed to enrich microorganisms.The inoculum of the reactors was either sludge from constructed wetland using manganese ore as substrate?wetland sludge?,or mixed sludge from different nature wetlands?mixed sludge?.Both natural manganese powder and?-MnO₂ were used as electron acceptor to enrich manganese-driven AOM?Mn-AOM?microbes.Combined with ¹³CH₄ stable isotope tracing,high-throughput sequencing,fluorescence absolute quantification?qPCR?,fluorescence in situ hybridization?FISH?,phylogenetic analysis and RDA correlation,etc.,this thesis analyzed the performance of AOM by the Mn-AOM microbes during the long-term enrichment procedure.This thesis also explored the key microbial community and its role in mediating AOM,and analyzed the effects of manganese mineral types and inoculum sources on AOM.The correlation between AOM rate and functional microbial abundance clarified the Mn-AOM process.Below are key research findings:After more than 700 days of enrichment,all four reactors showed significant reduction of methane under anaerobic conditions.During the enrichment process?T=150d⁷00d?,the average CH₄ oxidation rates of reactor R1?wetland sludge+natural manganese powder?,R2?mixed sludge+natural manganese powder?,R3?wetland sludge+?-MnO₂?,and R4?mixed sludge+?-MnO₂ group?were:0.1270mmol·L^-1·d^-1,0.1598 mmol·L^-1·d^-1,0.2118 mmol·L^-1·d^-1,and 0.1749 mmol·L^-1·d^-1,respectively.In the long-term operation,both reactors using?-MnO₂ as electron acceptor?R3and R4?have higher enrichment efficiency of AOM bacteria than the reactors using natural Manganese power?R1 and R2??p<0.05?.The AOM rate of R3 and R4 is also higher,indicating that the morphology of the tetravalent manganese can affect the enrichment of Archaeal Anaerobic Methanotrophs.The number of ANME-2d in R3 and R4 were higher than that in R1 and R2 as well.The results also found that the inoculated sludge significantly affected the community structure of the enriched culture,and the microbial community structure of the two different inoculated sludges was significantly different.Stable isotope experiments demonstrated the Mn-AOM used CH₄ as the sole electron donor.When supplied the ¹³CH₄ in the system,the ¹³C of total inorganic carbon(¹³C_TIC)increased significantly with the cultivation time.The electron conservation experiment verified the reduction of manganese minerals accompanied by the methane oxidation process,and the molar ratio between the total production of Mn?II?and the total reduction of CH₄ in the experiment was 3.85±0.25,close to the theorical stoichiometric ratio of reaction between CH₄ and Mn?IV?,which was 4.32±0.36.The ratio between the reduced Mn?IV?or produced Mn?II?,and the oxidized CH₄ were close to 4:1,which is consistent with the stoichiometric ratio of AOM driven by manganese minerals.This results further confirmed that the AOM in the reactor was using Mn?IV?as the sole electron acceptor.High-throughput sequencing,qPCR,FISH and other molecular biological techniques were used to identify the types of ANME-2d,methanogens and two manganese-reducing bacteria?Geobacter and Anaeromyxobacter?.The coexistence of ANME-2d,methanogens,and manganese-reducing bacteria were found in the enrichment reactors.The number of ANME-2d in the four reactors after enrichment for 700 days was obtained via qPCR,which was R1:4.57×10⁶ copies·g^-1?dry sludge?,R2:6.90×10⁶copies·g^-1?dry sludge?,R3:1.07×10⁷copies·g^-1?dry sludge?and R4:8.85×10⁶ copies·g^-1?dry sludge?.Correlation analysis showed that the oxidation of methane was increased accompanied by the growth of ANME-2d cluster.There was a significant positive correlation between the change of AOM rate and the increase of ANME-2d cluster number during the cultivation.The correlation coefficient R² was 0.95.For the other microorganisms like methanogens,the bacillus,the dissimilated metal reducing bacteria,their correlation with AOM was less obvious.This result illustrated that the Mn-AOM was dominated by ANME-2d.