臺灣博碩士論文加值系統

本研究利用分子生物的方法來建立南仁山古湖底泥甲烷氧化菌的社會結構。針對甲烷氧化菌的功能性基因pmoA設計專一性的引子對A189Cf和mb661AC，選殖分析後共獲得120 clones，再經由變性梯度電泳比對分析，共獲得14種不同的OTU。親緣關係分析的結果顯示，此14條pmoA序列與Methylobacter和Methylomonas的關係較為相近。南仁山古湖底泥甲烷氧化菌之豐富度高於湖水，底泥層之種豐富度隨深度遞減。不同深度所出現的甲烷氧化菌的種類略有差異，但屬於Methylobacter之NJ M2、46、88和屬於Methylomonas之NJ M77在不同的深度均有出現，應屬於古湖底泥優勢的甲烷氧化菌。本研究亦結合MPN的方法和利用甲烷氧化菌功能性基因的引子，分析底泥表層甲烷氧化菌的含量，其含量為5 X 107 MPN/g soil，顯示底泥層甲烷氧化菌的含量相當豐富。將底泥分別以5%和10%的甲烷培養之後，分別抽取DNA和RNA，經DGGE分析之後比對甲烷氧化菌族群的變動，結果顯示以5%甲烷培養的呈現較大的微生物社會結構的改變。

Methane is an important greenhouse gas that significantly contributes to global warming. Methane-oxidizing bacteria ( MOB ) are considered to play a major role in regulating the fluxes of methane from important methane sources such as forest, wetlands and rice paddies. The MOB are obligately aerobic bacteria that can utilize methane as the sole source of carbon and energy for growth. They are important biological regulators of methane in nature. Very limited information is presently available on the levels of populations of MOB in Taiwan. The object of this study was to construct the community structure of methane-oxidizing bacteria in Nan-Jen lake. The methane oxidizing population was sampled by PCR amplification and cloning of particulate methane monooxygenase A ( pmoA ) gene from the total soil population. Primer set A189Cf and mb661AC were designed specific for pmoA gene of MOB. A total of 120 pmoA clones was screened by DGGE and fourteen OTUs were found. The similarity of the fourteen pmoA gene sequences with those of Methylobacter and Methylomonas were 85.6% and 91.3%. The diversity of MOB in lake sediment were higher than lake water. The MOB content in the surface layer of soil was about 5 X 107 MPN/g-soil determined by MPN-PCR method. The dynamic of MOB population was analyzed by DGGE cultivated with 5% and 20% methane. ARDRA also applied in this study for analysis MOB community in Nan-Jen lake.

中文摘要..................................................I
英文摘要.................................................II
致謝....................................................III
目錄.....................................................IV
表目錄..................................................VII
圖目錄...................................................IX
第一章 前言...............................................1
第二章 文獻回顧...........................................2
2.1 碳循環............................................3
2.2 甲烷氧化菌........................................4
2.3 甲烷氧化菌分解甲烷的機制..........................5
2.4 甲烷氧化菌的研究..................................7
2.5 甲烷氧化菌16S rDNA序列之核酸探針和引子...........9
2.6 甲烷氧化菌單氧氧化酵素序列之核酸探針和引子.......10
2.7 ARDRA在環境微生物生態研究的應用..................11
2.8 研究目的.........................................12
第三章 材料與方法........................................13
3.1 實驗材料.........................................13
3.1.1 採樣地點....................................13
3.1.2 甲烷氧化菌菌種來源..........................13
3.1.3 培養基......................................15
3.1.3.1 液體培養基.............................15
3.1.3.2 瓊脂培養基.............................15
3.1.4 化學藥品....................................15
3.1.5 主要儀器....................................17
3.2 實驗方法與步驟...................................19
3.2.1 底泥土壤的前處理............................19
3.2.2 甲烷氧化菌菌量計數..........................19
3.2.3 DNA萃取.....................................19
3.2.4 RNA萃取.....................................22
3.2.5 引子的設計..................................22
3.2.6 聚合酵素連鎖反應(PCR).......................23
3.2.7 反轉錄(RT)..................................24
3.2.8 瓊脂膠體電泳................................24
3.2.9 變性梯度電泳................................25
3.2.10 選殖分析...................................27
3.2.10.1 選殖步驟..............................27
3.2.10.2 選殖樣品的製備........................28
3.2.11 定序.......................................30
3.2.12 親緣關係分析...............................30
3.2.13 利用ARDRA分析甲烷氧化菌pmoA序列..........30
第四章 結果..............................................31
4.1 PCR鍊合的溫度....................................31
4.1.1 甲烷氧化菌pmoA序列PCR引子.................31
4.1.2甲烷氧化菌pmoA序列DGGE之PCR引子...........31
4.2 DNA萃取方法的比較................................34
4.2.1 環境樣本未經過培養直接萃取DNA...............34
4.2.2 環境樣本經過培養之後萃取DNA.................34
4.3 RNA的萃取........................................37
4.4 甲烷氧化菌pmoA序列選殖分析(cloning).............39
4.5 親緣分析.........................................41
4.5.1 甲烷氧化菌pmoA序列親緣關係.................41
4.5.2甲烷氧化菌pmoA胺基酸序列親緣關係............44
4.6 ARDRA結果比較....................................48
4.7 甲烷氧化菌的計數.................................54
4.8 南仁山古湖樣區不同深度樣本之比較.................55
4.8.1 10公分之南仁山古湖底泥......................55
4.8.2 20公分之南仁山古湖底泥......................55
4.8.3 30公分之南仁山古湖底泥......................56
4.8.4 南仁山古湖湖水樣本..........................56
第五章 討論..............................................65
第六章 結論..............................................73
第七章 參考文獻..........................................74

參考文獻
謝建智，1998。南仁湖之甲烷釋放與甲烷氧化的研究。碩士論文，國立成功大學生物學研究所，台南市。
Auman, A. J., C. C. Speake, and M. E. Lidstrom. 2001. nifH Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs. Appl. Envir. Microbiol. 67: 4009-4016.
Auman, A. J., S. Stolyar, A. M. Costello, and M. E. Lidstrom. 2000. Molecular Characterization of Methanotrophic Isolates from Freshwater Lake Sediment. Appl. Envir. Microbiol. 66: 5259-5266.
Baere D. T., R. D. Mendonca, G. Claeys, G. Verschraegen, W. Mijs, R. Verhelst, S. Rottiers, L. V. Simaey, C. D. Ganck, and M. Vaneechoutte. 2002. Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory. BMC Microbiol. 1:2-4.
Bodegom, P. F. Stams, L. Mollema, S. Boeke, and P. Leffelaar. 2001. Methane Oxidation and the Competition for Oxygen in the Rice Rhizosphere. Appl. Envir. Microbiol. 67: 3586-3597.
Bourne, D. G., I. R. McDonald, and J. C. Murrell. 2000. Comparison of pmoA PCR Primer Sets as Tools for Investigating Methanotroph Diversity in Three Danish Soils Appl. Envir. Microbiol. 67: 3802-3809.
Brusseau, G. A., E. S. Bulygina, and R. S. Hanson. 1994. Phylogenetic analysis and development of probes for differentiating methylotrophic bacteria. Appl. Environ. Microbiol. 60: 626-636.
Brusseau, G. A., H. C. Tsien, R. S. Hanson, and L. P. Wackett. 1990. Optimization of trichloroethylene oxidation by methanotrophs and use of a ecolorimetric assay to detect solube methane monooxygenase activity. Biodegradation. 1: 19-29.
Bull, I. D., N. R. Parekh, G. H. Hall, P. Ineson, and R. P. Evershed. 2000. Detection and classification of atmospheric methane oxidizing bacteria in soil. Nature. 405:175-178.
Calhoun, A., and G. M. Hing. 1998. Characterization of root- associated methanotrophs from three freshwater macrophytes: pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia. Appl. Environ. Microbiol. 64:1099-1105.
Cheng, Y. S., J. L. Halsey, K. A. Fode, C. C. Remsen, and M. L. P. Collins. 1999. Detection of methanotroph in groundwater by PCR. Appl. Environ. Microbiol. 65: 648-651.
Christensen, H., M. Hansen, and J. S�wrensen. 1999.Counting and size classification of active soil bacteria by Flurorescence In Situ Hybridization with an rRNA oligonucleotide probe. Appl. Environ. Microbiol. 65:1753-1761.
Chu, K. Hui, and L. A. Cohen. 1999. Evaluation of toxic effects of aeration and trichloroethylene oxidation on methanotrophic bacteria grown with different nitrogen Sources. Appl. Envir. Microbiol. 65: 766-772.
Costello, A. M., and M. E. Lidstrom. 1999. Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. Appl. Envir. Microbiol. 65: 5066-5074.
Davenport, R. J., T. P. Curtis, M. Goodfellow, F. M. Stainsby, and M. Bingley. 2000. Quantitative use of Fluroscent In Situ Hybridization to examina relationships between Mycolic acid-containing actinomycetes and foaming in activated sludge plants. Appl. Environ. Microbiol. 66:1158-1166.
Dedysh, S. N., M. Derakshani, and W. Liesack. 2000. Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris. Appl. Envir. Microbiol. 67: 4850-4857.
Dedysh, S. N., N. S. Panikov, and J. M. Tiedje. 1998. Acidophilic methanotrophic communities from Sphagnum peat bogs. Appl. Environ. Microbiol. 64:922-929.
Dedysh, S. N., N. S. Panikov, W. Liesack, R. Grobkopf, J. Zhou, and J. M. Tiedje. 1998. Isolation of acidophilic methane-oxidizing bacteria from northern peat wetlands. Science 282:281-284.
Delong, E. F., L. T. Taylor, T. L. Marsh, and C. M. Preston. 1999. Visualization and enumeration of marine planktonic archaea and bacteria by using polyribonucleotide probes and Fluorescent In Situ Hybridation. Appl. Environ. Microbiol. 65:5554-5563.
Dumestre J. F., J. Guézennec, C. Galy-Lacaux, R. Delmas, S. Richard, and L. Labroue. 1999. Influence of Light Intensity on Methanotrophic Bacterial Activity in Petit Saut Reservoir, French Guiana. Appl. Envir. Microbiol. 65:534-539.
Dunfield, P. F., W. Liesack, T. Henckel, R. Knowles, and R. Conrad. 1999. High-Affinity Methane Oxidation by a Soil Enrichment Culture Containing a Type II Methanotroph. Appl. Envir. Microbiol. 65: 1009-1014.
Dunfield, P. F., W. Liesack, T. Henckel, R. Knowles, and R. Conras. 1999. High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph. Appl. Environ. Microbiol. 65: 1009-1014.
Edgcomb, V. P., J. H. McDonald, R. Devereux, and D. W. Smith. 1999. Estimation of bacterial cell numbers in humic acid-rich salt marsh sediments with probes directed to 16S ribosomal DNA. Appl. Environ. Microbiol. 65: 1516-1523.
Eller, G., and P. Frenzel. 2001. Changes in Activity and Community Structure of Methane-Oxidizing Bacteria over the Growth Period of Rice. Appl. Envir. Microbiol. 67: 2395-2403.
Fang, J., M. J. Barcelona, and J. D. Semrau. 2000. Characterization of methanotrophic bacteria on the basis of intact phospholipid profiles. FEMS Microbiol. Letters. 189:67-72.
Gilbert, B. I., R. McDonald, R. Finch, G. P. Stafford, A. K. Nielsen, and J. C. Murrell. 2000. Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs. Appl. Envir. Microbiol. 66: 966-975.
Glockner, F. O., B. M. Fuchs, and R. Amann. 1999. Bacterioplankton compositions of lakes and oceans: a first comparsion based on Fluorescence In Situ Hybridation. Appl. Environ. Microbiol. 65:3721-3726.
Goldberg, I., and J. S. Rokem. 1991. Biology of methylotrophs. Butterworth-Heinemann, Bpston.
Gulledge, J., A. Ahmad, P. A. Steudler, W. J. Pomerantz, and C. M. Cavanaugh. 2001. Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria. Appl. Envir. Microbiol. 67: 4726-4733.
Halda, A. L., S. P. Hendricks, and T. C. Johnston. 2001. Spatial and Temporal Variation of Enterobacter Genotypes in Sediments and the Underlying Hyporheic Zone of an Agricultural Stream. Microb. Ecol. 42: 286-294
Henckel, T., M. Friedrich, and R. Conrad. 1999. Molecular Analyses of the Methane-Oxidizing Microbial Community in Rice Field Soil by Targeting the Genes of the 16S rRNA, Particulate Methane Monooxygenase, and Methanol Dehydrogenase . Appl. Envir. Microbiol. 65: 1980-1990.
Henckel, T., U. Jäckel, S. Schnell, and R. Conrad. 2000. Molecular Analyses of Novel Methanotrophic Communities in Forest Soil That Oxidize Atmospheric Methane. Appl. Envir. Microbiol. 66: 1801-1808.
Henckel, T., M. Friedrich, and R. Conrad. 1999. Molecular analysis of the methane-oxidizing microbial community in rice field soil by targeting the gene of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl. Environ. Microbiol. 65:1980-1990.
Henckel, T., U. Jackel, and R. Conrad. 2001. Vertical distribution of the methanotrophic community after drainage of rice field soil. FEMS Microbiol. Ecol. 34:279-291.
Holmes, A. J., A. Costello, M. E. Lidstorm, and J. C. Murrell. 1995. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol. Lett. 132: 203-208.
Holmes, A. J., P. Roslev, I. R. McDonald, N. Iversen, K. Henriksen, and J. C. Murrell. 1999. Characterization of Methanotrophic Bacterial Populations in Soils Showing Atmospheric Methane Uptake. Appl. Envir. Microbiol. 65: 3312-3318.
Horz, H. Peter, M. T. Yimga, and W. Liesack. 2001. Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling. Appl. Envir. Microbiol. 67: 4177-4185.
Iwamoto, T., K. Nakamura, Y. Suzuki, and M. Nasu. 2000. Monitoring impact of in situ biostimulation treatment on groundwater bacteria community by DGGE. FEMS Microbiol. Ecol. 32: 129-141.
Jensen, S., A. J. Holmes, R. A. Olsen, and J. C. Murrell. 2000. Detection of methane oxidizing bacteria in forest soil by monooxygenase PCR amplification. Microb. Ecol. 39: 282-289.
Jensen, S., L. Ovreas, and V. Torsvik. 1998. Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16S rDNA fragments. FEMS Microbiol. Ecol. 26:17-26.
King G. M., 1999. Attributes of Atmospheric Carbon Monoxide Oxidation by Maine Forest Soils. Appl. Envir. Microbiol. 65: 5257-5264.
Lawlor, K., B. P. Knight, V. L. Barbosa-Jefferson, P. W. Lane, A. K. Lilley, G. I. Paton, S. P. McGrath, S. M. O’Flaherty, and P. R. Hirsch. 2000. Comparison of methods to investigate microbial populations in soils under different agricultural management. FEMS Microbiol. Eco. 33:129-137.
Lidstrom, M. E. 1990. Methylotrophs: Genetics and commercial applications. Annu. Rev. Microbiol. 44: 27-58.
Llobet-Brossa, E., R. Rossello-mora, and R. Amann. 1998. Microbial community composition of wadden sea sediments as revealed by Fluorescence In Situ Hybridation. Appl. Environ. Microbiol. 64:2691-2696.
McDonald, I. R. E. M. Kenna, and J. C. Murrell. 1995. Detection of methanotrophs bacteria in environment samples with the PCR. Appl. Environ. Microbiol. 61: 116-121.
McDonald, I. R., and J. C. Murrell. 1997 The methanol dehydrogenase structural gene mxaF and its use as a function gene probe for methanotrophs and methylomethylotrophs. Appl. Environ. Microbiol. 63: 3218-3224.
McDonald, I. R., M. Upton, G. Hall, R. W. Pickup, C. Edwards, J. R. Saunders, D. A. Ritchie, and J. C. Murrell. 1999. Mplecular ecological analysis of methanogenase and methanotrophs in blanket bog peat. Microb. Ecol. 38: 225-233.
Miguez, C. B., C. F. Shen, D. Bourque, S. R. Guiot, and D. Groleau. 1999. Monitoring Methanotrophic Bacteria in Hybrid Anaerobic-Aerobic Reactors with PCR and a Catabolic Gene Probe. Appl. Envir. Microbiol. 65: 381-388.
Miguez, C. B., D. Bourque, J. A. Sealy, C. W. Greer, and D. Groleau. 1997. Detection and isolation of methanotrophic bacteria possessing soluble methane monooxygenase (sMMO) genes using the Polymerase Chain Reaction (PCR). Microb. Ecol. 33: 21-31.
Miller, D. N., J. E. Bryant, E. L. Madsen, and W. C. Ghiorse. 1999. Evalution and Optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol. 65: 4715-4724.
Morton, J. D., K. F. Hayes, and J. D. Semrau. 2000. Effect of Copper Speciation on Whole-Cell Soluble Methane Monooxygenase Activity in Methylosinus trichosporium OB3b. Appl. Envir. Microbiol. 66: 1730-1733.
Murrell, J. C., I. R. McDonald, and D. G. Bourne. 1998. Molecular methods for the study of methylotroph ecology. FEMS Microbiology Ecology. 27: 103-114.
Neufeld, J. D., and R. Knowles. 1999. Inhibition of nitrifiers and methanotrophs from an agricultural humisol by allylsulfide and its implications for environmental studies. Appl.Environ. Microbiol. 65:2461-2465.
Nold, S. C., H. T. S. Boschker, R. pel, and H. J. Laanbroek. 1999. Ammonium addition inhibits 13C-methane incorporation into methanotroph membrane lipids in a freshwater sediment. FEMS Microbiol. Ecol. 29:81-89.
Paul L. E. Bodelier and Peter Frenzel. 1999. Contribution of Methanotrophic and Nitrifying Bacteria to CH4 and NH4+ Oxidation in the Rhizosphere of Rice Plants as Determined by New Methods of Discrimination. Appl. Envir. Microbiol. 65: 1826-1833.
Peters, S., S. Koschinsky, F. Schwieger, and C. C. Tebbe. 2000. Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Appl. Environ. Microbiol. 66:930-936.
Purdy, K. J., T. M. Embley, S. Takii, and D. B. Nedwell. 1996. Rapid extraction of DNA and rDNA from sediments by a novel hydroxyapatite spin-column method. Appl. Environ. Microbiol. 62: 3905-3907.
Reichardt, W., G. Mascarina, B. Padre, and J. Doll. 1998. Microbial communities of continuously cropped, irrigated rice fields. Appl. Environ. Microbiol. 63:233-238.
Ren, T., R. Roy, and R. Knowles. 2000. Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria. Appl. Envir. Microbiol. 66: 3891-3897.
Richard, S. H. and T. E. Hanson. 1996. Methylotrophic Bacteria. Microbiol. Rev. 60: 439-471.
Rocheleau, S., C. W. Greer, J. R. Lawrence, C. Cantin, L. Laramee, and S. R. Guiot. 1999. Differentiation of Methanosaeta concilii and Methanosarcina Barkeri in anaerobic mesophilic granular sludge by Fluorescent In Situ Hybridation and confocal scanning laser microscopy. Appl. Environ. Microbiol. 65: 2222-2229.
Roslev, P. and N. Iversen. 1999. Radioactive Fingerprinting of Microorganisms That Oxidize Atmospheric Methane in Different Soils. Appl. Envir. Microbiol. 65: 4064-4070.
Roslev, P., and N. Iversen. 1999. Radioactive Fingerprinting of Microorganisms that oxidize atmospheric methane in different soils. Appl. Environ. Microbiol. 65:4064-4070.
Semrau, J. D., A. Chistoserdov, J. Lebron, A. Costello, J. Davagnino, E. Kenna, A. J. Holmes, R. Finch, J. C. Murrell, and M. E. Lidstorm. 1995. Particulate methane monooxygenase genes in methanotrophs. J. Bacteriol. 177: 3071-3079.
Shigematsu, T., S. Hanada, M. Eguchi, Y. Kamagata, T. Kanagawa, and R. Kurane. 1999. Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading Methylomonas sp. Strains and Detection of Methanotrophs during In Situ Bioremediation. Appl. Envir. Microbiol. 65: 5198-5206.
Spring, S., R. Schulze, J. Overmann, and K. Schleifer. 2000. Identification and characterization of ecologically significant prokaryotes in the sediment of freshwater lakes: molecular and cultivation studies. FEMS Nicrobiol. Res. 24: 573-590.
Steingrube, V. A., J. L. Gibson, B. A. Brown, Y. Zhang, R. W. Wilson, M. Rajagopalan, and R. J. Wallace. 1995. PCR amplification and restriction endonuclease analysis of a 65-Kilodalton heat schock protein gene sequence for taxonomic separation of rapidly growing mycobacteria. J Clin Microbiol. 33: 149-153
Steingrube, V. A., J. L. Gibson, B. A. Brown, Y. Zhang, R. W. Wilson, M. Rajagopalan, and R. J. Wallace. 1995. PCR amplification and restriction endonuclease analysis of a 65-Kilodalton heat schock protein gene sequence for taxonomic separation of rapidly growing mycobacteria. J Clin Microbiol. 33: 149-153.
Sreinkamp, R., W. Zimmer, and H. Papen. 2001. Improved method for detection of methanotrophic bacteria in forest soils by PCR. Current Microbiol. 42: 316-322.
Stephan, G., L. Laramee, K. Wendlandt, I. R. McDonald, C. B. Miguez, and H. Kleber. 1999. Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium Methylocystis sp. Strain WI 14. Appl. Envir. Microbiol. 65: 3929-3935.
Stolyar, S., A. M. Costello, T. L. Peeples, and M. E. Lidstrom. 1999. Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath. Microbiol. 145:1235-1244.
Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bottger, and T. Bodmer. 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 31: 175-178.
Tsien, H. C., B. J. Bratina, K. Tsuji, and R. S. Henson. 1990. Use of oligodeoxynucleotide signature probe for identification of physiological groups of methylotrophic bacteria. Appl. Environ. Microbiol. 56: 2858 -2865.
Tsuji, K., H. C. Tsien, R. S. Hanson, S. R. DePalma, R. Scholtz, and S. LaRoche. 1990. 16S ribosomal RNA sequence analysis for determination for phylogenetic relationship among methylotrophs. J. Gen. Microbiol. 136: 1-10.
Vaneechoutte, M., H. D. Beenhouwer, G. Claeys, G. Verschraegen, A. D. Rouck, N. Paepe, A. Elaichouni, and F. Portaels. 1993. Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 31: 2061-2065.
Whittenbury, R.. 1981. The methylotrophs in The Prokaryote,.. Springer-verlag, New York. 894-902.
Wise, M. G., J. V. McArthur, and L. J. Shimkets. 1999. Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis. Appl. Envir. Microbiol. 65: 4887-4897.
Worden, A. Z., S. W. Chisholm, and B. J. Binder. 2000. In Situ Hybridation of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-targeted peptide nucleic acid probes. Appl. Environ. Microbiol. 66: 284-289.
Zhou, J., M. A. Bruns, and J. M. Tiedje. 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62: 316-322.