Microbiological Mechanism Of Methane Oxidation Coupled To Denitrification Based On Leachate Sediment

Sediment in leachate collection ponds is an extreme environment with considerably high of ammonium and organic matter concentration. Therefore, there might be some specific microognaisms which metabolize in differential pathway in this special habitat, especially carbon and nitrogen transforming microorganisms which could bear high amounts of organic matters and ammonium. Recently, methane oxidation coupled to denitrification (MOD) was attracted much attention due to less energy consumption and simple design reactor. However, the mechanisms and microbial community of this process have not yet been confirmed. In this thesis, sediment (0-8cm) in the16years old leachate collection pond of Dongyang Landfill in Zhejiang Province was taken as the research object. Prokaryotic diversity, composition structure and the potential microbial metabolic processes in leachate sediment ecosystems were analysed by modern molecular biotechnology. With the leachate sediment as inoculum, a consortium involved in MOD was enriched under micro-aerobic condition. Methane oxidation activity and denitrification acitivity of the enrichment reactor were determined.16S rRNA gene phylogeny combined with pmoA gene-based phylogeny of methanotrophs and nirK gene-based phylogeny of denitrifiers were analyzed to reveal the pivotal microbial populations and functional microorganisms. Functional genes combined with molecular cloning technique were performed to identify the primary functional microorganisms and coupling mechanism involved in MOD system. Results of this study would be significant in exploring microbial resources and new technologies for biological wastewater treatment. Main results of the study were summaried as follows:(1) Sediment (0-8cm) in the16years old leachate collection pond of Dongyang Landfill in Zhejiang Province was taken as the research object. Archeael diversity and community structure was illuminated using PCR (polymerase chain reaction)-molecular cloning technique combined with phylogenetic analysis of16S rRNA gene. It was suggested that ammonium-oxidizing crenarchaeote might exist in leachate sediment. A total of59phylotypes affiliated with two phyla Euryarchaeota and Crenarchaeota were examined among425randomly selected archaeal rDNA clones. Among them,45phylotypes were identified as Euryarhaeota, representing397out of the total425clones, accounting for93.41%of the total archaeal clones. All of the euryarhaeotic clones were closely related to methanogens, mainly belonging to Methanomicrobiales, Methanosarcinales and Methanobacteriales. Methanosaeta spp. was the dominant spcies. Fourteen crenarchaeotic phylotypes, representing28clones, had relatively high level (>95%) of similarity with unclassified environmental clones, except that clone B9was98.3%indentical to a moderately thermophilic ammonium-oxidizing crenarchaeote "Candidatus Nitrososphaera gargensis"(2) Phylogenetic analysis of the bacterial16S rRNA gene clone library revealed the population of organic matter degrading and ammonium transforming microorganisms. Analysis of the diversity indexes indicated that compared to archaeal community, the bacterial community associated with the landfill leachate sediment was much more diverse and complicated. Two hundred and forty four phylotypes were affiliated with18distinct phyla, representing313bacterial clones. Bacteria belonging to Firmicute and Proteobacteria were the dominant populations, accounting for21.60%and19.20%of the total bacterial clones, respectively. More than50%of bacterial clones had high levels of similarity with unculure bacteria. A substantial fraction of bacterial clones showed low levels of similarity with any previously documented sequences and thus might be taxonomically new. Some phylotypes belonging to Alpha-, Beta-and Gammaproteobacteria were affiliated with chemoorganoheterotroph denitrifiers. It was inferred that these bacteria played an important role in carbon and nitrogen transforming precess in the leachate sediment. Chemoorganotrophy bacteria, such as fermentative bacteria, acetogenic bacteria, and hydrogen producing bacteria, which belonged to phylum Firmicuteand order Clostridia, would provide methanogens with substrates, including acetate, H2/CO2and methylated compounds. These collections of different microbial species were referred to as a methanogenic consortium. Ammonium-oxidizing archaea and ammonium utilizing microorganisms might consume surrounding ammonium to form a relatively low ammonium microenvironment and to alleviate or avoid negative influence on other microorganisms. There were a degree of resemblances in the microbial populations between leachate sediment of current study and leachate of other references. However, distinct landfill circumstances, various climate conditions, and different physicochemical properties would result in discrepant microbial communities in leachate and leachate sediment.(3) Taking the leachate sediment (0-8cm) of16years old in Dongyang Landfill as inoculum, a consortium which was mainly gram-negative bacteria, involved in MOD was successfully enriched under micro-aerobic condition, with methane as external carbon source, nitrite and nitrate as electron acceptor. The denitrification rate of the MOD reactor was around4.5mmol N L-1d-1at the steady period. The batch experiment suggested that more oxygen providing in the reactor would favor a more efficient denitrification, which indicated that nitrite and nitrate depletions might be associated with aerobic denitrification. GC-MS analysis of the influent and effluent liquid of enrichment and batch experiment cultures demonstrated that methanotrophs produced high amount of formaldehyde, citrate, and acetate during the methane oxidation process.(4) Composition and abundance of primary functional microbial communities in the MOD ecosystem were investigated by pmoA gene-based phylogeny of methanotrophs and nirK gene-based phylogeny of denitrifiers conbined with real-time PCR. There were amount of methanotrophs and denitrifiers in the enriched sludge, and the copy number of pmoA gene were almost3times more than that of nirK gene. Microbial community in the enrichment culture was dominated by Type I methanotrophs, which belonged to Methylococcaceae, Gammaproteobacteria, comprising37.5%of the total bacterial16S rRNA gene clone library. The Methylobacter spp. and Methylocaldum spp. were the dominant population. Aerobic methylotrophic denitrifiers of Methylophilaceae, Betaproteobacteria, might make great contribution in denitrification in our MOD ecosystems, comprising36.76%of the total bacterial16S rRNA gene clone library.In conclusion, the microbiological mechanism of the MOD system in this study was that type I methanotrophs, which belonged to Methylobacter spp. and Methylocaldum spp., Methylococcaceae, Gammaproteobacteria, oxidized methane, employed the ribulose monophosphate (RuMP) pathway for assimilation and released organic intermediates (i.e. formaldehyde, citrate, and acetate) under mireo-aerobic condition. Aerobic methylotrophic denitrifiers of Methylophilaceae, Betaproteobacteria, utilizing organic intermediates released from the methanotrophs as electron donor, played a vital role in aerobic denitrification.