微生物多樣性 (microbial diversity) 研究是針對高屏溪流域支流-海拔分別為100~2204m的荖濃溪流域八個樣區及100~654m的楠梓仙溪流域八個樣區進行微生物相變化之探討。結合了微生物、化學及分子生物技術共同來探討兩條流域之微生物多樣性，並且提供高屏溪整體環境生態一個參考，作為環境保育實際落實前的基礎。結果發現：荖濃溪氣候溫度除天池較低外（16.3℃），其餘樣區土溫介於22~28℃之間，而楠梓仙溪氣候溫度除民生較低外（21.5℃），其餘樣區氣候溫度介於24~33℃之間；其他如pH值、總有機碳以及總有機氮方面，各樣區隨地點之不同各有差異。在微生物組成含量方面，所有的微生物當中以細菌含量最高，荖濃溪每克土壤約含有細菌106~109CFU；楠梓仙溪每克土壤約含有細菌105~109CFU，其他如放線菌、真菌則含量較低。以變性梯度凝膠電泳（denaturing gradient gel electrophoresis，DGGE）分析兩條流域的土壤細菌16s rDNA群落組成發現，楠梓仙溪流域的微生物群落組成較豐富，而荖濃溪之菌相群落則較單純。在季節性變化方面，以分子生物學技術探討微生物群落組成，發現和傳統分析相符合，亦即在溫暖季節時，微生物的含量較高且群落組成亦較豐富。在短時間的氣象變化，如豪雨及氣溫，會嚴重影響細菌數量；而大雨連續沖刷時，亦會造成菌相若干程度的改變；但大雨停止後，微生物菌數與群落組成會逐漸恢復為原狀。

The microbial diversity of branches of Kaoping River at Lao-Nong Basin with altitudes from 100-2204m and Nantzuhien Basin with altitudes from 100-654m were studied. We used combining methods of bacterial, chemical and biotechnological to explore microbial diversity at the two basins to provide a reference for the environmental ecology of Kapoing River. It is the foundation for practicable environmental care. The results show the following: (1) The temperature in Tianchin at Lao-Nong Basin was lower(16.3℃) than the rest of sampling sites(22-28℃). (2) The temperature in Minsheng at Nantzuhsien Basin is lower(21.5℃)than the of sampling sites(24-33℃); other factors such as pH value, total organic carbon, and total organic nitrogen, varied according to different locations. In the microbacterial composition quantitatively, among all microorganisms, the bacteria content was the highest. Each gram of soil contain about 106~109 CFU at Lao-Nong Basin and 105~109 CFU at Nantzuhsien Basin, where as others such as the contents of actinomycetes and fungi were lower. Using 16s rDNA DGGE (denaturing gradient gel electrophoresis)analysis, the soil bacterial community composition at the two basin districts had discoverd that the microbial diversites at Nantzuhsine Basin were more abundant than those at Lao-Nong Basin. In regard to seasonal changes, our data agreed with traditional data. In the warm season, the microbial content is higher, and the community composition is also more abundant. The weather change in a short time period, such as a rainstorm and a quick shift of temperature seriously affected the number of bacteria. When the rain pours steadily down, it caused some degree of drop in bacteria number. However, when the rain stop, the microbacterial content and community composition gradually retured to original forms.

謝誌…………………………………………………………I
中文摘要 …………………………………………………Ⅱ
Abstract……………………………………………………Ⅲ
目錄 ………………………………………………………Ⅳ
表目錄 ……………………………………………………Ⅵ
圖目錄 ……………………………………………………Ⅶ
一、緒論 …………………………………………………1
1.1 微生物多樣性研究之背景介紹及重要性……………1
1.1.1 土壤生物多樣性概述………………………………1
1.1.2 不同土壤微生物族群的功能………………………6
1.1.3 土壤微生物多樣性研究的重要性…………………9
1.1.4 影響土壤微生物多樣性的因素……………………12
1.1.5 目前土壤微生物多樣性的研究……………………12
1.1.6 土壤微生物多樣性的未來展望……………………14
1.2 土壤微生物多樣性研究上的困難與限制……………15
1.2.1 採樣的依據………………………………………15
1.2.2 傳統分離培養技術的瓶頸………………………16
1.3 16S rDNA在微生物鑑定、分類與多樣性研究上的應用…17
1.4 變性梯度電泳膠 (DGGE) 的應用……………………17
1.5 本論文研究目的 ……………………………………18
二、材料與方法 …………………………………………20
2.1 研究策略流程圖……………………………………20
2.2 研究樣區概述 ……………………………………21
2.2.1 高屏溪流域概述………………………………21
2.2.2 荖濃溪流域概述………………………………22
2.2.3 楠梓仙溪流域概述……………………………22
2.2.4 樣區採樣………………………………………23
2.3 土壤環境因子檢測…………………………………24
2.3.1 土壤溫度的測定方法…………………………24
2.3.2 土壤pH值的測定方法 ………………………24
2.3.3 土壤含水量的測定方法 ……………………24
2.3.4 土壤總有機碳的測定方法……………………24
2.3.5 土壤總有機氮的測定方法……………………25
2.4 菌種培養...…………………………………………26
2.4.1 細菌菌數測定…………………………………26
2.4.2 放線菌菌數測定………………………………27
2.4.3 真菌菌數測定…………………………………27
2.4.4 固氮微生物菌數測定…………………………27
2.4.5 溶磷微生物菌數測定…………………………27
2.4.6 纖維分解微生物菌數測定……………………28
2.5 資料分析……………………………………………28
2.6 以分生技術分析細菌相……………………………28
2.6.1 微生物群落之去氧核糖核酸萃取方法………28
2.6.2 以PCR方法放大16S rDNA…………………29
2.6.3 清洗PCR產物 ………………………………31
2.6.4 瓊脂膠體電泳… ……………………………31
2.6.5 以DGGE電泳分離PCR產物………………31
三、結果與討論…………………………………………34
3.1 樣區環境條件與土壤特性.………………………34
3.1.1氣溫與土溫……………………………………34
3.1.2土壤pH值………………………………………36
3.1.3土壤含水量……………………………………37
3.1.4土壤總有機碳含量……………………………41
3.1.5土壤總有機氮含量……………………………42
3.1.6土壤碳氮比…………………………………………44
3.2 各樣區土壤微生物組成…………………………46
3.2.1 細菌含量……………………………………46
3.2.2 放線菌含量……………………………………48
3.2.3 真菌含量………………………………………50
3.2.4 固氮菌含量……………………………………51
3.2.5 溶磷菌含量……………………………………55
3.2.6 纖維分解菌含量………………………………57
3.2.7 樣區微生物組成及環境因子之綜合討論……58
3.3以DGGE建立土壤菌相分析………………………61
3.3.1 以單一流域在單一季節的比較………………62
3.3.2 不同樣區之不同季節比較……………………63
3.3.3 同一流域不同季節之比較……………………64
3.3.4 未來展望………………………………………64
四、結論…………………………………………………66
五、參考文獻……………………………………………109
六、附錄…………………………………………………115

高屏溪河川水質變化趨勢分析，1999~2000，環保署水資源單位，台北，台灣。
水中氨氮檢測方法-鈉氏比色法 (NIEA W416.50A)，1993，行政院環境保護署環境檢驗
所，台北，台灣。
水中凱氏氮檢測方法-分光光度計法 (NIEA W420.51B)，2004，行政院環境保護署環境
檢驗所，台北，台灣。
林良平。1987。土壤微生物(上/下冊)，初版。國立編譯館，南山堂出版社。台北，台灣。
河北氣象。2005。瑞雪兆豐年之解。（http://www.cdlh.heagri.gov.cn/default3.aspx?id=2429）
卓昕岑。2002。高海拔塔塔加及低海拔福山森林土壤微生物族群研究。碩士論文。國立台灣
大學農業化學研究所。台北，台灣。
何春蓀。1994。台灣地質概論，經濟部中央地質調查所。
洪富文。1999。全球變遷：福山森林生態系研究—地景階層的土壤氮動態。行政院國家
科學委員會專題研究計畫成果報告。台北，台灣。
洪維鍊。2001。塔塔加土壤微生物多樣性及族群之探討。碩士論文。國立台灣大學農業
化學研究所。台北，台灣。
胡弘道。1993。森林土壤學，修訂版。國立編譯館，茂昌圖書有限公司。台北，台灣。
陳明杰。1999。福山森林生態系研究一集水區坡面土壤水分變化之研究。行政院國家科
學委員會專題研究計畫成果報告。台北，台灣。
陳華癸等編著。1981。土壤微生物學，上海科學技術出版社﹐上海。
陳尊賢、許正一合著。2002。台灣的土壤。遠足文化出版社。新店，台灣。
黃山內。1995。土壤的生物性質。（http://www.tnfd.gov.tw/upload/farmlook/1_473.pdf）。
趙榮台。2004。生物多樣性。(www.tmue.edu.tw/~envir2/bitdiv/2004715/07.pdf)。
楊盛行、賴朝明、孫瓏月、羅陽青、范曉雲、魏嘉碧。1998。塔塔加高山土壤微生物生
態。中國農業化學會誌。36(3):229-238。
劉有美。2001。森林土壤與肥料。(www.heyuan.gov.cn/nongye/lingxue)。
劉淑惠。2004。泥火山景觀考察。(www.wxp.ks.edu.tw/nature/theme1/badlandt/han/400.htm)。
薛達元等譯。J.A.麥克尼利等著。1997。保護世界的生物多樣性。地景企業股份有限公
司。台北，台灣。
齊士崢、陳文山、龔琪嵐、黃美璇、吳唐竹。2004。荖濃溪與楠梓仙溪流域的河階地與其新構造運動意義。中國地理學會彙刊。
Alexander M. 1977. Introduction to soil microbiology. p.1-554. John Wiley & Sons, New
York.
Altieri M. 1999, The ecological role of biodiversity in agroecosystems. Agriculture Ecosystems & Enoironment. 74:19-31.
Akkermans ADL, Van Elsas JD, De Bruijn FJ.1995. Molecular Microbial Ecology Manual.” Kluwer Academic Publishers, Dordrecht.
Alef K and Nannipieri P.1995. Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London.
Bater JE.1996. Methods for the examination of organismal diversity in solis and sediments. Micro- and macro-arthropods. In: G.S. Hall（ed.）pp. 163-174.
Bremner JM, Mulvaney CS, Miller RH, Keeney DR., Miller RH, Keenry DR. 1982 .Methods of soil analysis. American Society of Agronorny and Soil Science Society of Amenica pp.595-622.
Brookes PC. 1995. The use of microbial parameters in monitoring soil pollution by heavy metals. Biology and Fertility of Soils 19:269-279.
Biovin-Jahns V, Bianchi A, Ruimy R, Garcin J, Daumas S, Christen R. 1995. Comparison of phenotypical and Molecular Methods for the Identification of Bacterial Strains Isolated from a Deep Subsurface Environment. Applied and Environmental microbiology. 61(11):4140.
Bloem J, Schouten T, Kidden W, Akkerhuis GJ, Keidel H, Rutgers M, Breure T. 2003. Measuring soil biodiversity: experiences, impediments and research needs, OECD expert meeting on soil erosion and soil biodiversity indicators, 25-28 March, Rome, Italy.
Barlow J,Carlos A. 2004. Ecological responses to E1 Nino-induced surface fires in central Brazilian Amazonia: management implications for flammable tropical forests. Philosophical Transactions of the Royal Society B: Biological Sciences. 359:367-380.
Brussaard L, Behan-Pelletier VM, Bignell DE, Brown VK, Didden W, P. Folgarait C, Fragoso D, Wall-Freckman VVSR, Gupta T, Hattori DL, Hawksworth C, Klopatek P, Lavelle DW, Malloch J, Rusek B, Soderstrom JM, Tiedje RA. 1997. Biodiversity and ecosystem functioning in soil. Ambio. 26: 563-570.
Curnutt JL, Pimm S. 1995. Biodiversity, Managing nature when there are no ill winds. Curr Biol. 5(7): 713-5.
Davis J. 1999. Millennium bugs. Trends Cell Biol. 9:M2-5.
Demain AL ,Solomon NA. 1986. Manual of Industrial Microbiology and Biotechnology. American Society for Microbiology, USA.
Dobereiner Jo, Urquiaga S and Boddey RM. 1995, Alternatives for nitrogen nutrition of crops in tropical agriculture. Nutrient Cycling in Agroecosystems. 42(1-3):339-346.
Dobereiner J, Pedrosa FO.1987. Nitrogen fixing bacteria in nonleguminous crop plants.. Science and Technology publishers. p 57-62.
FAO/AGL.2000.Soil Biodiversity Portal (www.fao.org/AG/AGL/agll/soilbiod/index_en.stm)
Giller KE, Bear MH, Lavelle P, Izac A.-M.N, Swift MJ.1997.Agricultural intensification, soil biodicersity and agroecosystem function. Soli Ecol. 6:3-16.
Hågvar S. 1998.The relevance of the Rio-Convention on biodiversity to conserving the biodiversity of soils. Soil Ecol. 9:1-7.
Hawksworth DL. 1991. The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol. Res. 95:641-655.
Hawksworth KL, Mound LA. 1991. The biodiversity of microorganisms and invertebrates: Its orle in sustainable agriculture. Biodiversity database: The crucial significance of collections. In: D.L. HAWKS WORTH(ed.). pp.17-29.
Hawksworth DL, Kalin-Arroyo MT. 1995. Golbal biodiversity assessment. Cambridge University Press. Magnitude and distribution of biodiversity. In: V.H. Heywood. (ed.).pp.107-191.
Hassan AA, Vossen A, Lammler C, Siebert U, Fernandez-Garayzabal JF. 2006.PCR amplification of species specific sequences of 16s rDNA and 16S-23S rDNA intergenic spacer region for identification of strptococcus phocae. Microbiological Research.
Hayashi H, Sakamoto M, Kitahara M, BennoY. 2003. Molecular analysis of fecal microbiota in elderly individuals using 16s rDNA library and T-RFLP. Microbiology and immunology. 47(8):557-70.
Isenegger DA, Taylor PWJ, Mullins K, McGregor GR, Barlass M, Hutchinson JF. 2003. Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures. Plant cell peports 21(8).
Muyzer G. 1999. DGGE/TGGE a method for identifying genes from natural ecosystems. Microbiol. 2:317-322.
Motta FC, Rosado AS, Sigueird MM. 2006. Comparison between denaturing gradient gel electrophoresis and phylogenetic analysis for characterization of A/H3N2 influenza samples detected during the 1999-2004 epidemics in Brazil. Journal of virological methods 135(1):76-82.
Nubel U, Ferran GP, Kuhl M, and Muyzer, G. 1999. Quantifying microbial diverity:
morphotypes,16S rRNA genes,and carotenoids of oxygenic phototrophs in microbial mats. Appl. Environ. Microbiol. 65:4803-4809.
Palys T, Nakamura LK, and Cohan FM. 1997. Discovery and classification of
ecological diversity in the bacteria world: the role of DNA sequence data. Int. J. Syst. Bacteriol. 47:1145-1156.
Pimentel. 1995. Enviromental and economic Environment costs of soil erosion and conservation benefits. Science. 267:1117-1123.
Pimente D, Wilson C, McCullam C, Huang R, Dwen P, Flack J, Traw Q, Saltman T, Cliff B. 1997,.Economic and Environmental Benefits of Biodiversity. Bio Science 47(11):747-757.
Santoni D,Romano-Spica V. 2006. A gzip-based algorithm ot identify bacterial families by 16s rRNA. Lett Appl Microbiol. 42(4)312-4.
SchaeferM, Schauermann J. 1990. The soil fauna of beech forests:comparison between a mull and a moder soil. Pedobiologia 34:299-314.
Schnurer J, Clarholm S, Bastrom S, and Rosswell T. 1986.Effect of moisture on soil
microorganisms and nematodes. A field experiment. Microb. Ecosyst. 12:217-230.
Smalla K, Wieland G, Buchner A, Zock A, Parzy J, Kaiser S, Roskot N, Heuer H, and Berg G. 2001. Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed. Appl. Environ. Microbiol. 67: 4742-4751.
Swift MJ, Heal OW, Aderson JM. 1979.Decomposition in terrestrial ecosystems. Blackwell Scientific, Oxford, U.K.
Torsvik VJ, Goksoyr FL , Daae R,Sorheim J, Michalsen, Salte K. 1994. Use of DNA analysis to determine the diversity of microbial communities. Beyond the biomass: communities pp. 39-48.
Van Elsas JD and Van Overbeek LS. 1993. Bacterial repones to soil stimuli. In:
Kjelleberg S(ed.), Starvation in Bateria. Plenum Press, New York, p:55-79.
Walkley A, Black TA. 1934. An examination of the Degtjareff methods for determining of soil organic matter, and e proposed modification of the chromincacid titration method. Soil sci. 37:29-38.
Warith, M.A.; Ferehner. R. Fernandes. L. 1992. Bioremediation of organic contaminated soil. Hazardous Waste and Hazardous Matenials. 9:27:137-147.
Woese CR. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271.
Wallwork JA. 1970. Ecology of soil animals. McGraw Hill, London, U.K.
Walter DE, Proctor HC. 1999. Life at the microscale: Mites and the study of ecology, evolution and behaviour, New South Wales Press, Sydney, Australia.
Warith MA., Ferehner R, Fernancles L. 1992. Bioremediation of organic contaminated soil. Hazardous. Waste and Hazardous Materials 9(2):137-147.
Watanabe K , Teramoto M, Futamata H, and Harayama S. 1998.Molecular detection,
isolation, and physiological characterization of functionally dominant phenol-degrading
bacteria. Appl. Environ. Microbiol. 64:4396-4402.
Wawer C, Jetten MSM and Muyzer G. 1997. Genetic diversity and expression of the NiFe
hydrogenase large-subunit gene of desulfovibrio spp. in environmental samples.
Appl. Environ. Microbiol. 63:4360-4369.
WARD DM, Weller R, Bateson MM. 1990. 16s rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 345:63-65.