甲烷為重要溫室效應氣體之一，大氣中每年約以0.5-0.8 %的速率在增加中，甲烷是極端厭氧之甲烷生成菌於還原環境下分解簡單有機分子所產生，掩埋場是大氣甲烷的重要來源，甲烷的分解主要就是依賴甲烷氧化菌。國內針對掩埋場土壤之甲烷氧化菌社會結構之研究甚為缺乏，本研究擬利用分子生物的方法來建立我國掩埋場土壤甲烷氧化菌的社會結構。針對甲烷氧化菌之16S rDNA與弁鄔夆穧]mxaF片段設計專一性的引子對，本研究以聚合酶連鎖反應放大此二DNA片段，分析不同掩埋齡土壤之甲烷氧化菌親緣關係，並於實驗室培養條件下變化不同甲烷濃度、土壤水分含量與溫度等條件，利用 MPN 的方法比較各土壤環境條件對甲烷氧化菌族群之變動與甲烷氧化速率之影響。本研究選擇台南的南沙崙掩埋場之不同掩埋齡，進行篩選菌種及研究影響甲烷氧化之環境因子。本研究分別從現齡掩埋區中篩出七株菌，分別為SA2、SA4、SA6、SA9、SA14、SA15及一株現齡掩埋區中甲烷排氣口旁的SD1菌株，還有一菌株-SC2來自5~7年之掩埋齡區，分別進行革蘭氏染色及氧化酶與催化酶試驗，由結果顯示SA6、SA9、SA15為革蘭氏陽性菌，SA2、SA4、SA14、SC2、SD1則為革蘭氏陰性菌，且均具有氧化酶，其中SA6與SA14亦具有催化酶。利用NCBI及MEGA等軟體分析，分別製作出16S rDNA及mxaF特定弁鈰穧]之親緣樹狀圖，由16SrDNA親緣樹狀圖中顯示SA2、SA4及SC2為甲烷氧化菌Type I及Type II之外的單系群，由mxaF特定弁鈰穧]之親緣樹狀圖佐證SA6、SA9、SA14、SA15及SD1為甲烷氧化菌Type II之Methanobacterium sp.。 另外針對會影響甲烷氧化之因子及添加菌株後的影響加以探討，本研究分別對甲烷濃度變化、溫度變化及含水率之變化作測試，結果得知當溫度30℃、低甲烷濃度5%及含水率為20%時，其甲烷氧化效果較佳；而於最佳環境因子下植種研究中所分離之甲烷氧化菌，也可發現其甲烷氧化速率較未植種(僅有掩埋場覆土)之空白組顯著快2~5天。 Methane is an important greenhouse gas, and the concentrations of methane increase at the rate of 0.5-0.8 % year-1 recently. Most of methane is produced biologically by a group of strictly anaerobic bacteria in highly reduced environments. Landfill is one of the important biogenic methane sources. Methanotrophs are important regulators of methane fluxes from the landfill to atmosphere. There is very limited information presently available on the community structure of methanotrophs in landfills in Taiwan. The study is an investigation on the community structure of methanotrophs as revealed by molecular method in Nan-Sa-Lun landfills in Tainan. The methanotrophic strains will be isolated by enrichment culture and thereby subsequent loss of a variety of other methanotroph. Furthermore, methanotrophic isolates will be sampled by PCR amplification and cloning of particulate methane dehydrogenase (mxaF) gene and 16S rDNA from the total soil population. The isolated will be selected from different cover-age (0-2, 3-5 and 5-7 year-old) landfill soils. The number of methanotrophs in the surface layer of landfill soils will also determined by most-probable number (MPN) method. The 7 test strains (SA2、SA4、SA6、SA9、SA14、SA15 and SD1) obtained from in the 0-2 year-old, one strain (SC2) from 5-7 year-old landfill. SA6, SA9 and SA15 are Gram-positive strains；SA2, SA4, SA14, SC2 and SD1 are Gram-negative strains. All test strains could be detected by oxidase test. Only SA6 and SA14 could be detected by catalase test. Phylogenic analysis of the test strains were identified by 2 primers specific for the 16S rDNA and mxaF gene by NCBI database and MEGA software. Database searches indicated that SA2, SA4 andSC2 are monophyly from the phylogenic tree constructed by 16 S rDNA and would not hybridize to the Type I and Type II methanotrophs. SA6, SA9, SA14, SA15 and SD1 are most closely related to a group Methanobacterium sp.(Type II methanotroph) by phylogenic analysis of partial DNA sequence of mxaF. Changes in various factors of methane concentration, soil water content and incubation temperature will also be estimated based on incubation experiments. The maximum oxidation rates for methane were found in 30℃, 5 % CH4 and moisture content of 20% . Inoculation of methanotrophic isolates would also promote the methane oxidation rates more than 2-5 days.
