Species Composition And Function Of Aerobic Methane Oxidizing Microbial Communities

The group of methanotrophic microorganisms in natural and artificial habitats can not only use methane as the only carbon source and energy source for growth and reproduction,reduce the emission of greenhouse gas methane,but also completely reduce the nitrate and nitrite in the environment.It is nitrogen to alleviate the problem of carbon and nitrogen pollution in the environment.This study took the methane-oxidizing microorganisms in the sediments of Lake Fuxian in Yunnan Province as the research object.Through long-term domestication in the laboratory,a large number of microbial groups that can efficiently oxidize methane were obtained,and the temperature and oxygen concentration were studied to oxidize methane.The impact of ability;using metagenomics and macrotranscriptome sequencing to reveal the species composition and metabolic characteristics of the methanotrophic group;by separating and purifying the microorganisms in the group,methylotrophic bacteria such as Methylophilus and Methylotenera and heterotrophic Bacterial strain resources;explore the microbial interaction mechanism of the methane oxidation coupled denitrification process in the group.The specific research carried out in this paper and the results obtained are as follows:(1)Long-term domestication of microbial groups and influencing factors of methane oxidation process.At the deepest point of Lake Fuxian,a sediment column with a depth of 27 cm was obtained,with one layer per 1 cm,for long-term domestication of methanotrophic microorganisms(currently more than 100 passages).The methane oxidizing ability of methane oxidizing microorganism groups at different depths of sediments is different.The methane oxidizing ability of the group of 1-18cm shallow sediments is stronger than that of the 18-27cm deep layer and can stabilize at a higher level faster.The species in the group was stream Lined through flow cytometric sorting,and 99 groups of methane-oxidizing microorganisms that can stably use methane were further obtained.The group methane oxidation rate was measured under different temperature(10,20,30,40,45?)and oxygen concentration(20%,10%,2%,V/V)conditions.The results showed that the rate of methane oxidation and denitrification of the microbial group was the highest at 30?.Cultivation at 40?45?slowed the growth of the microbial group and decreased the rate of methane oxidation.It was found that#22 is more resistant to high temperature than#91 methane oxidizing microbial group.Under the condition of 20%oxygen concentration,the CH₄ oxidation rate of the methanotrophic microbial group is the highest,but under the condition of 2%oxygen concentration,it promotes the coupling of methane oxidation and denitrification and increases the production of N₂O,an intermediate product of denitrification.(2)Metagenomic sequencing of the methanotrophic group of microorganisms and the isolation and purification of species.Through the metagenomic sequencing of two culture systems(#22 and#91),it was found that the microorganisms in the group have a rich diversity,where the number of species in the#22 group is?20,and where the microorganisms number of species in the#91 group is?25.The species composition at the phylum level includes?and?Proteobacteria,Bacteroidetes,Verrucomicrobia and Actinobacteria.By the dilution coating method and the plate streaking method,20 strains of heterotrophic bacteria(mainly including Aquidulcibacter,Flavobacterium,Hydrogenophaga,Acidovorax and Pseudomonas)and 11 methylotrophic bacteria(belonging to Methylophilus)were isolated and purified from the#22 group.25 strains of heterotrophic bacteria(mainly including Pelomonas and Pseudomonas)and 7 strains of methylotrophic bacteria(belonging to Methylophilus)were isolated and purified from the#91 group.(3)Methane oxidation coupled with denitrification function(AME-D)of microbial groups.Through the dual isotope labeling of ¹³CH₄/¹⁵NO₃^-,explore the conditions for the occurrence of group AME-D function.The results show that the methanotrophic microbial group can reduce nitrate or nitrite while oxidizing methane under the condition of 2%oxygen concentration.The denitrification end products ²⁹N₂ and ³⁰N₂are detected in the system.There were significant differences in the methane oxidation rate of groups under different oxygen concentrations.The pmoA gene abundance of methane-oxidizing microorganisms in ¹³C-DNA in the high-oxygen treatment group was significantly higher than that in the low-oxygen treatment group.The number of copies of the denitrification nirS gene in the group was significantly higher than that of the pmoA gene(p<0.05),indicating that a variety of microorganisms are involved in the denitrification process.The macrotranscriptome analysis of the methanotrophic microbial group shows that the species in the group can be further divided into 6 functional groups.Divide methane and methylotrophic bacteria into G1 functional group,whose metabolism consumes methane and methanol;divide microorganisms such as heterotrophic bacteria into G2-G6functional group,which can utilize methane oxidation intermediate metabolites such as formaldehyde and formic acid.Many functional groups have the transfer of metabolites between species in N metabolism.In summary,there are differences in the microbial composition and methane oxidation capacity of the methane-oxidizing microbial groups in lake sediments at different depths.Temperature and oxygen concentration are the factors affecting the growth and reproduction,methane oxidation,and denitrification capabilities of the methane-oxidizing microbial groups.The strains obtained by separating and purifying the group of methanooxidizing bacteria belong to the most proteobacteria.The methanotrophic microbial group cultured under aerobic conditions for a long time can reduce nitrate to obtain energy under hypoxic conditions and maintain the survival of the group under hypoxic conditions.