Microbial Roles Of Methane Sinks In Southern Karst Caves From China

Methane（CH₄）,as the second largest greenhouse gas,is considered to be the research focus on rapidly mitigating the greenhouse effect due to its residence time in the atmospheric is shorter than that of carbon dioxide.Rencently,subterranean caves are proposed as an important but overlooked methane sink for atmoapheric in the terrestrial ecosystem apart from soil ecosystem.However,many issues are still needed to be addressed in order to incorporate cave ecosystems into the global methane budget.Despite of the detection of methane-oxidizing bacteria（MOB）in caves,we still know little about their distribution,environmental driving mechanism,ecological function,and methane oxidation potential in these ecosystems.Besides,the other methane microbial functional group,methanogenic archaea,are poorly investigated in karst caves.We know little about their methane production potential,their interaction with methanotrophs and the impact of the interaction on the ecological functions of caves.These unknowns severely hampered our understanding of the microbial-mediated CH₄ cycle in caves and the incorporation of caves into models of the global CH₄ budget.Therefore,it is of great significance to elucidate the composition,distribution,environmental driving mechanism,potential of CH₄ production and CH₄ oxidation,and the interaction between methane microbial functional groups to explore the key research link of the missing sink of methane in cave.Water plays the pivotal role in the development of karst ecosystems.Recently,extreme precipitation events such as rainstorms and droughts occurre frequently,which will shift the hydrological-redox conditions in caves,subsequently affect the composition and function of microbial functional taxa.However,many knowledge gaps exist in understanding the impacts of hydrological variation in caves on anaerobic methanogenic archaea and aerobic methanotrophs and further on the CH₄ cycle.These unresolved issues become a bottleneck for the size and dynamics of caves atmospheric methane sinks.Furthermore,methane cycle is also a pivotal component of the carbon cycle,which should couple with other elemental cycles theretically.MOB and methanogenic archaea also need major nutrient elements to maintain their own growth,such as nitrogen and phosphorus.Therefore,the microbial-mediated CH₄ cycle is not an isolated metabolic process,it should couple with metabolic processes such as N and P cycles,thus playing an important role in the maintenance and stability of cave ecosystems.To address the above unsubstantial scientific questions,samples of the weathered rocks and loose sediments were selected from three caves in Guilin City（the Panlong Cave,the Luohandu Cave,and the Xincuntun Cave）and the Heshang Cave in Yichang City,which subsequently subjected to amplicon sequencing of pmo A and mcr A gene,q PCR quantification,CH₄ oxidiation and production potential measurement,metagenomic sequencing,high-throughput gene chips of elements,outdoor and indoor microcosm experiments,stable isotope probe（DNA-SIP）of pmo A gene.The community structure of cave MOB and methanogens,survival strategies,and microbial element cycle coupling with methane oxidation under low CH₄ concentration were systematacially explored.The results will provide biological evidences to support cave as atmospheric methane sinks,discern the biological methanism and the dynamics of methane sink under extreme precipitation events.The main achivements of this research are as follows:1)The methane oxidation potential of caves is comparable to that of soil,and there are abundant atmospheric methane-oxidizing bacteria USCγin cave.Temperature and CH₄ concentration signigicantly shaped the MOB community.Atmospheric CH₄ concentrtaion decreased with the increasing distance from the entrances into karst caves.The potential methane oxidization rates in the weathered rock and sediment(0.46±0.03-6.92±0.28 ng CH₄·g^-1DW·h^-1)were comparable to those reported in soils(1.35 ng CH₄·g^-1 DW·h^-1),supporting the karst cave serves as an important atmospheric CH₄ sink.The absolute abundance of USC taxa is the highest in the weathered rock of the inner zone of the cave with higher temperature.High affinity atmospheric MOB with high gene abundance dominated methanotrophs particularly the USCγ（Upland soil clusterγ）,and the absolute abundance of USCγis 4.85×10⁷ copies·g^-1 DW,which may result from the alkaline conditions.In addition to temperature,cave atmospheric MOB also showed different preferences for CH₄ concentrations:USCγpreferred CH₄ concentrations of 10 ppm or lower,whereas USCαpreferred CH₄concentrations in the range of 50-100 ppm.Homogeneous selection and homogeneous dispersal were the main ecological processes responsible for the community assembly of atmospheric MOB in caves,which reulsulted in the convergence of the community structure of the atmospheric MOB.2)MOB predominated methanogens in caves as confirmed by higher methane oxidation potential,higher number,and the dominance in the keystone taxa in the networks with methanogens,which revealed the biogical mechanism of caves as methane sinks.The potential methane oxidation rate of weathered rock(2.78-21.89 ng CH₄·g^-1DW·h^-1)was higher than those in sediment(0.29-1.13 ng CH4·g^-1 DW·h^-1),indicating cave MOB would preferentially comsume CH₄ from the atmosphere.Atmosphericδ¹³C-CH₄ values andδ¹³C-CH₄ negatively correlated withδ¹³C-CO₂,indicating microbial consumption of methane in the karst cave.In contrast,although mcr A gene abundances were detected in the weathered rock and sediment,CH₄ production was not detected in the experiment,which might result from the oligotrophic conditions in the cave.The absolute abundance of methanotrophic pmo A gene in the Heshang Cave was 1-3 orders of magnitude higher than that of mcr A gene of methanogenic archaea.MOBs in karst cave were dominated by high affinity MOB,upland soil cluster（USC）,with USCγpmo A gene abundance within the range of 1.57×10⁴ to 1.88×10⁷ copies·g^-1 DW.The range of mcr A gene copies were 7.21×10³ to 8.31×10⁴ copies·g^-1 DW.The inter-domain ecological network（IDEN）analysis indicated that MOB and methanogens cooperated more inward to the cave revealing that MOB would cooperate with methanogens to survive the extreme conditions.Althouogh methanogens occupied more nodes in the networks,MOB served as the keystone nodes in the ecological network of cave methane functional microbes,indicating a leading function in methane cycling.Collectively,methanotrophs dominated methanogens in activity,quantity,and interaction with methanogens,whch implied the biological mechanism of karst caves as natural methane sinks.3)Despite of the changes in composition and abundance of methane func tional groups due to the extreme precipitation event,cave showed stable methane oxidizing capability as confirmed by filed samples and microcosom experiments.These results strongly suggested the stability of ecological function of caves as atmospheric CH₄sinks.The abundance of MOBs in the sediments during drougnt events was higher by comparing the effects of extreme rainfall events（drought events and rainstorm events）on methane functional microbial communities in the Heshang Cave,which were domianted by USCγwith the range of pmo A gene abundance from 2.83×10⁴ copies·g^-1 DW to 6.50×10⁸ copies·g^-1 DW.The relative abundance of USCγ（affiliated to clones from open patch soil）and USCα（affiliated to clones from soil）were dominant in the sediments during the drought events,and the absolute abundance of USC ranged from 2.05×10⁵copies·g^-1 DW to 6.22×10⁸ copies·g^-1 DW.Methanogens affecting by rainstorm events were affiliated to clones from soil,petroleum reservoir,and pond sediment,whereas methanogens were affiliated to clones from sediment,paddy soil,and wetland were dominant in the sediments of drought events.The modules of the drought IDEN were closely connected and the network structure was more complex than those of rainstorm events,whereas the rainstorm event network was more stable,indicating microbial different feedback mechanisms under different extreme climate events.The results of microcosm experiments confirmed that increasing moisture content reduced the absolute abundance of pmo A genes of USC taxa in caves by an order of magnitude,and increased the relative abundance of type I methanotrophs（increased from 78.10±18.56%to 86.30±5.30%）in sediments.In most cases,the samples still maintained methane oxidation ability(0.01±0.02-0.12±0.01 ng CH₄·g^-1 DW·h^-1),whereas methane production(0.03ng CH₄·g^-1 DW·h^-1)was only detected in the high moisture content（200%）treatments of the W3 and W5 samples.Therefore,the ecological function of caves as CH₄ sinks was stable.4)Energy acquisition was the main survival strategy for microbes living in oligotrophic karst caves as indicated by metagenomic analysis.Proteobacteria and Actinobacteria jointly participate in cave methane oxidation.The atmospheric methane oxidation rate in weathered rock(0.65 ng CH₄ g^-1 DW h^-1)was higher than that in sediments(0.32 ng CH₄ g^-1 DW h^-1)as measured in the in-situ experiments in the Heshang Cave.Actinobacteria dominated the microbial Bins in the weathered rock reaction group,which survived in the oligotrphic caves via A strategy.When the continuous supply of atomospheric methane was blocked,they might rely on the degradation of other types of carbon,such as glucose,cellulose and mannan,to obtain energy and fix CO₂ to obtain necessary carbon sources.Nevertherless atmospheric CH₄was still their main carbon source.MAGs with pmo A genes also contained key genes of HP/HD cycle（dark carbon fixation）and denitrification in the weathered rock,indicating their chemoautotrophy characteristics.The protein encoded by the pmo A gene of these MAGs showed close phylogenetic relationship with the high-affinity TUSC.Proteobacteria dominated in microbial Bins dominated by in sediments.In the case of limited methane input,the absolute abundance of genes related to carbon degradation and carbon fixation in the reaction treatment significantly increased.MOB MAGs were affiliated with Pseudonocardia,which contained soluble methane monooxygenase,and the complete pathway related to nitrate transporter and nitrogen cycle to maintain the stability of the cave microenvironment.The above results play important roles in promoting the understanding of the microbial-driven CH₄ cycle and its underlying environmental driving mechanism.The innovation of this dessertation includes:（1）systematically revealing the distribution,niche differentiation and environmental driving mechanism of methanotrophs in southern karst caves,and clarifying the biological mechanism of karst caves as atmospheric CH₄sinks.（2）The unique resource-acquisition-based survival strategy of cave microbes and the differentiated element coupling process of methanotrophs in different habitats were firtly discovered.It shed a light on the subsequent evaluation of the ecological role of caves as methane sinks and response to the national“double carbon”policy.