以分子生物技術探討厭氧生物產氫程序之菌群結構

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：28

、訪客IP：18.188.91.223

姓名

黃俊霖(Chun-Lin Huang ) 查詢紙本館藏

畢業系所

環境工程研究所

論文名稱

以分子生物技術探討厭氧生物產氫程序之菌群結構
(Determining Microbial Community Structure of Hydrogen-Producing Anaerobic Sludge Processes with Molecular Techniques)

相關論文

★ 生物除磷反應槽中可分離微生物菌相多樣性的探討	★ 以葡萄糖為基質的生物除磷系統體積負荷與磷負荷對代謝行為與菌相影響之研究
★ 多氯聯苯厭氧馴養降解菌群微生物多樣性解析

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

本研究分為兩部份來進行。第一部份是利用快速分子生物檢測方法，確認預先經過自然酸篩處理的污泥中可以篩選出生成內孢子的高產氫菌群，且以Clostridium菌種為主。進一步利用各兩座體積分別為4升及1.5升的連續流式反應槽(CSTR)，進行不同污泥來源、基質馴養及水力停留時間(HRT)差異的操作，並比較其菌群結構。利用16S rRNA序列資料庫(clone library)建立法，成功地將其中三個樣品（LG：污水廠污泥，基質:glucose，HRT=10hrs和HRT=6hr，以及LS：污水廠污泥，基質:sucrose，HRT=5hrs）中菌群結構解析出，並與DGGE細菌菌群指紋譜進行比對相對位置。比對結果指出，於不同污泥來源(污水廠、酒廠及豆類污泥)、相同基質(蔗糖)與HRT (14.4小時)的操作下，厭氧生物產氫系統中的微生物菌群結構相似，與污泥來源關係不大，是屬於low G+C， gram-positive，與Clostridium pasteurianum親緣相近的菌群，產氫速率維持在107~137 (ml H2/g VSS-hr)左右。此外，相同污泥來源，不同基質(葡萄糖或蔗糖)及不同HRT操作下，隨著HRT的增加(2小時~10小時)，厭氧生物產氫系統中的微生物菌群多樣性顯著提高，比對位置結果顯示主要有三類菌群。其中兩類是屬於Gram-positive，low G+C bacteria，一類是與Clostridium pasteurianum親緣相近的菌群，而另一類是與Clostridium ramosum親緣相近的菌群。而第三類是屬於γ-Proteobacteria，與Pseudomonas親緣相近的菌群。在高HRT時，有較高的基質轉化率，各為1.65 mol H2/mol-glucose及3.94 mol H2/mol-sucrose；相對於低HRT時，則有較高的產氫速率，各為20.4 mmol H2/g VSS-hr (基質為glucose)及35.0 mmol H2/g VSS-hr (基質為sucrose)。進一步結合定量FISH以及水質分析結果，顯示屬於γ-Proteobacteria菌群幾乎可以忽略。另外LG反應器的菌群在不同HRT下有明顯的變動情形。其中，無法與Chis 150以及其他探針進行雜交的菌群，比例達到32.5~41.7%，甚至多到71%時，有較高的產氫速率，較低的基質轉化率，代謝產物主要為丙酸及乙醇，而乙酸和丁酸的濃度相對較低。相反地，屬於Gram-positive ，low G+C，與Chis150 probe可以進行雜交的菌群佔優勢時(94.5~97.5%)，代謝產物主要為乙酸及丁酸，而丙酸和乙醇濃度相對較低。故推論上述兩種菌群，也就是無法與Chis 150以及其他探針進行雜交的菌群，以及屬於Gram-positive ，low G+C，與Chis150 probe可以進行雜交的菌群，應是屬於基質競爭的關係，且這兩個族群應該是扮演主要產氫的角色。至於LS反應器，其菌群組成比例較為穩定。故基質種類、HRT、代謝產物和菌群種類皆會影響到厭氧產氫的效率。此外，探針測試結果顯示37℃、25% 的formamide濃度，是Lg10-6 probe的最佳雜交條件。進一步得到Lg10-6 probe與Chis150 probe 對於不同HRT操作下的污泥，其FISH偵測結果幾乎完全疊合(>99%)。
第二部份同樣是以16S rDNA為分析指標，來探討於另一個污泥來源(食品廠污泥)，更複雜的基質(peptone)，經過多次轉植馴養後之厭氧生物產氫微生物菌群多樣性。定量FISH結果顯示，主要有四大類菌群實際存在於peptone為基質之馴養試程，它們是屬於δ, α -Proteobacteria，以及屬於Gram-positive，low G+C，與Chis150 probe可以或不能進行雜交的菌群，分別佔細菌群菌群的30.4 ± 11.0 %、19.0 ± 10.1 %、11.0 ± 3.2 %和38 %。它們參與複雜的代謝peptone產氫反應。

摘要(英)

By using molecular biological techniques, the microbial community structures of two hydrogen producing CSTRs fed with glucose and sucrose respectively were investigated. In the reactor fed with glucose, cloning results indicated that the ecology contained members of Gram Positive Low G+C (LGC) group and beta-Proteobacteria. Otherwise, the microbial community of sucrose feeding reactor was composed of bacteria affiliated with LGC group and gamma-Proteobacteria. Fluorescence in situ hybridization further revealed that three morphotypes of bacteria existed in both microbial communities. One is long rod bacteria which can be targeted by Chis150 probe designed to hybridize LGC bacteria (Franks ei al., 1998). Another type is curved rod bacterium not detected by Chis150. The other morphotype is short rod bacteria phylogenetically familiar with Pseudomonas spp. Moreover, the quantitative results of FISH clearly showed that Chis150 targeted cells and curved rod bacteria dominated both microbial community but in different ratio, whereas the amount of gamma-Proteobacteria rods were negligible. Interestingly, the population ratios of the two predominant groups reflected different operational performance of the two reactors such as hydrogen producing rates, substrate turnover rates and metabolites compositions. Therefore, a competition mode of the two dominant bacteria groups was hypothesized.
On the other hand, microbial community of hydrogen producing batch reactor fed with peptone was also studied. FISH indicated that the community was more diversified than those of reactors fed with glucose or sucrose. Short rods akin to the δ-Proteobacteria subdivision and bacteria untargeted by probes used in this study accounted for 30.4% and 38% of EUB338 stained cells respectively. Besides, members of α-Proteobacteria and rods can be targeted by Chis150 probe were also abundant in the microbial community (19.0 ± 10.1 % and 11.0 ± 3.2 % each). The diversity of the society structure may be due to the complex components of peptone substrate. In general, feeding substrate played an important role in determining the bacterial community composition.

關鍵字(中)

★ 基質
★ 探針
★ 水力停留時間
★ 污泥來源
★ 產氫
★ 酸篩

關鍵字(英)

★ hydrogen
★ microbial community structures

論文目次

第一章前言
1-1 研究緣起1
1-2 研究目的2
第二章文獻回顧
2-1 生物產氫原理3
2-1-1 機制種類及應用3
2-1-1-1 The light side 3
2-1-1-2 The dark side 4
2-1-2 厭氧發酵產氫的影響因子5
2-1-2-1 基質6
2-1-2-2 污泥來源6
2-1-2-3 HRT (SRT)6
2-2 產氫菌8
2-2-1 產氫菌的界定8
2-2-2 傳統培養及分類方法8
2-2-3 分子生物學10
2-3 利用分子生物技術探討微生物生態及菌群結構13
2-3-1 聚合酵素鏈鎖反應14
2-3-2 變性梯度明膠電泳14
2-3-3 16S rDNA clone library approach 15
2-3-4 限制酵素斷片法15
2-3-5 螢光原位雜交法16
2-3-5-1 探針的標示16
2-3-5-2 螢光染料17
2-3-5-3 細胞固定18
2-3-5-4 標本製備及預處理18
2-3-5-5 雜交反應18
2-3-5-6 上機操作19
第三章實驗材料與方法
3-1 污泥來源20
3-1-1 污泥處理20
3-1-2 以Glucose及Sucrose為基質之馴養試程21
3-1-3 以Peptone為基質之馴養試程22
3-2 分析方法22
3-2-1 GC-TCD22
3-2-2 GC-FID22
3-2-3 一般水質分析項目23
3-2-4 DNA萃取23
3-2-5 DNA萃取產物檢視23
3-2-6 聚合酵素鏈鎖反應23
3-2-7 16S rDNA 分子選殖25
3-2-8 變性梯度明膠電泳法25
3-2-9 變性梯度明膠電泳之快速篩選26
3-2-10 限制酵素斷片法27
3-2-11 親緣分析27
3-2-12 螢光原位雜交的影像觀察28
3-2-13 FISH結合DAPI staining 28
3-2-14 掃描式電子顯微鏡(SEM)觀察28
第四章結果與討論
4-1 污泥來源之反應槽操作條件及操作結果31
4-1-1 以Glucose及Sucrose為基質之馴養試程31
4-1-2 以Peptone為基質之馴養試程37
4-2 利用分子生物技術分析以Glucose及Sucrose為基質的馴養試程39
4-2-1 不同污泥來源(污水廠、酒廠及豆類污泥)、相同基質(蔗糖)與HRT (14.4小時)下的操作39
4-2-2 相同污泥來源，不同基質(葡萄糖或蔗糖)及不同HRT下的操作40
4-2-3 微生物種類16S rDNA基因資料庫初步的建立41
4-2-3-1 DGGE快速篩選法41
4-2-3-2 以16rDNA為基礎並針對LG(HRT=10hrs)和LS(HRT=5hrs)污泥樣品製作之親源樹41
4-2-3-3 限制酵素斷片法45
4-2-4 螢光原位雜交法(FISH)的影像觀察47
4-2-5 定量FISH結合水質分析結果54
4-2-6 掃描式電子顯微鏡(SEM)觀察58
4-2-7 微生物種類16S rRNA基因資料庫60
4-2-7-1 以16rDNA為基礎並針對LG(HRT=10hrs)、LS(HRT=5hrs)和LG(HRT=6hrs)污泥樣品製作之親源樹60
4-2-7-2 設計探針62
4-2-8 以16S rRNA基因資料庫的結果與DGGE細菌菌群指紋譜進行相對位置的比對63
4-2-8-1 不同污泥來源(污水廠、酒廠及豆類污泥)、相同基質(蔗糖)與HRT (14.4小時)下的操作63
4-2-8-2 相同污泥來源，不同基質(葡萄糖或蔗糖)及不同HRT下的操作64
4-2-9 專屬寡核甘酸探針Lg10-6 probe測試65
4-3 利用分子生物技術分析以Peptone為基質的馴養試程68
4-3-1 以peptone為基質的菌群結構68
4-3-2 微生物種類16S rRNA基因資料庫的建立70
4-3-3 螢光原位雜交法(FISH)的影像觀察72
4-3-4 綜合討論76
第五章結論與建議
5-1 結論77
5-2 建議79

參考文獻

中文參考資料
1.鄭幸雄,曾怡禎,白明德. 2000.“厭氧生物乙酸化分解蛋白質有機物的生物產氫行為”, 第廿五屆廢水處理技術研討會論文集, 299-304頁。
英文參考資料
2.Abdul, R.M., F. Yoshinori, N. Yutaka, K. Toshihide, and N. Naomichi. 1997. Enhanced hydrogen production in altered mixed acid fermentation of glucose by Enterobacter aerogenes. Journal of Fermentation and Bioengineering. 83(4)： 358-363.
3.Adams, M.W.W., L.E. Mortenson, and J.-S. Chen. 1980. Hydrogenase. Biochimica et biophysica acta. 594：105-176.
4.Akano, T., Y. Miura, K. Fukatsu, H. Miyasaka, Y. Ikuta, and H. Matsumoto. 1996. App. Biochem Biotechnol. 57/58：677-688.
5.Albracht, S.P.J. 1994. Nickel hydrogenases：in search of the active site. Biochim. Biophys. Acta. 1188：167-204.
6.Alm, E.W., D.B. Oerther, N. Larsen, D.A. Stahl, and L. Raskin. 1996. The oligonucleotide probe database. Appl. Environ. Microbiol. 62:3557-3559.
7.Amann, R., W. Ludwig, and K.H. Schleifer. 1988. β-subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria. J. Gen. Microbiol. 134：2815-2821.
8.Amann, R. I., B. J. Binder, R. J. Olson, S.W. Chisholm, R. Devereux, and D.A. Stahl. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial population. Appl. Environ. Microbiol. 56:1919-1925.
9.Amann, R., L. Krumholz, and D.A. Stahl. 1990b. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172：762-770.
10.Amann, R. I., W. Ludwig, and K. H. Schliefer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. Mar.：143-169.
11.Bailey, J.E., and D.F. Ollis. 1986. Biochemical engineering fundamentals, 2nd edition, McGraw-Hill Book Co., N. Y.
Beneman, J.R., K. Miyamoto, and P.C. Hallenbeck. 1980. Enzyme Microb. Technol. 2：103-111.
12.Beneman, J. 1996. Hydrogen biotechnology-progress and prospect. Nature Biotechnol. 14：1101.
13.Bredholt, S., J. Sonnehansen, P. Nielsen, I.M. Mathrani, and B.K. Ahring. 1999. Caldicellulosiruptor Kristjanssonii sp Nov., a cellulolytic extremely thermophilic, anaerobic bacterium. Int. J. Syst. Bacteriol. 49(JUL)：991-996.
14.Brosius, J., T. J. Dull, D.D. Sleeter, and H. F. Noller. 1981. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148: 107-127.
15.Brown-Howland, E.B., S.A. Danielsen, and S.A. Nierzwicki-Bauer. 1992. Development of a rapid method for detecting bacterial cells in situ using 16S-targeted probes. BioTechniques. 13：928-933.
16.Busse, H.J., E.M.B. Denner, and W. Lubitz. 1996. Classfication and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics. Journal of Biotechnology. 47：3-38.
17.Chen, C.C., and C.Y. Lin. 2000. Using Sewage Sludge as seed in an Anaerobic Hydrogen Producing Reactor. 第廿五屆廢水處理技術研討會，p.368-372.
18.Chen, C.C., and C.Y. Lin. 2001. Using sucrose as a substrate in an anaerobic hydrogen producing reactor，sludge management entering the 3rd millennium- industrial, combined, water and wastewater residues. Taipei, Taiwan, March 25-28, p. 610-615.
19.Chen, C.C., C.Y. Lin., and J.S. Chang. 2001. Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate. Appl. Microbiol. Biotechnol. (submission)
20.Choi, B.-K., B.J. Paster, F.E. Dewhirst, and U.B. Gobel. 1994. Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis. Infect. Immun. 62：1889-1895.
21.Deere, D., G. Vesey, N. Ashbold, K.A. Davies, K.L. Williams, and D. Veal. 1998. Evaluation of fluorochromes for flow cytomeric detection of Cryptosporidium parvum oocysts labeled by fluorescence in situ hybridization. Lett. Appl. Microbiol. 27：352-356.
22.Ferris, M.J., G. Muyzer, and D.M. Ward. 1996. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 62：340-346.
23.Franks, A. H., H. J. M. Harmsen, G. C. Raangs, G. J. Jansen, F. Schut, and G. W. Welling. 1998. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64:3336-3345.
Ghirardi, M.L., S.P. Toon, and M. Seibert. 1995. Proceedings of the Annual Review Meeting of DOE Office of Utility Technologies Hydrogen Program Review. Miami. FL.
24.Gottschalk, G. 1986. Bacterial metabolism. p.208-282. Spring-Verlag. New York.
Heuer, H., M. Krsek, P. Baker, K. Smalla, and E.M.H. Wellington. 1997. Analysis of actinomycete communities by specific amplification of genes encoding 16S Rrna and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63：3233-3241.
25.Heyndrickx, M., P. Devos, and J. Deley. 1991. Fermentation characteristics of Clostridium pasteurianum LMG-3285 grown on glucose and mannitol. J. Appl. Bacteriol. 52-58.
26.Hugenholtz, P., B.M. Goebel, and N.R. Pace. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180：4765-4774.
27.Ike, A., N. Toda, K. Hirata, and K. Miyamoto. 1997. Hydrogen photoproduction from CO2-fixing microalgal biomass - application of lactic-acid fermentation by lactobacillus-amylovorus. Journal of Fermentation and Bioengineering. 84(5)：428-433.
28.Innis, M.A., and D.H. Gelfand. 1990. Optimization of PCRs. In：PCR protocols：A guide in methods and applications. Edited by Inns M.A., Gelfand D.H., Sninsky J.J. and White T.J. Academic Press, INC. p.3-12.
29.Jurtshuk, R.J., M. Blick, J. Bresser, G.E. Pox, and P. Jurtshuk. 1992. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans. Appl. Environ. Microbiol. 58：2571-2578.
30.Kataoka, N., A. Miya, and K. Kiriyama. 1997. Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria. Wat. Sci. Tech. 36：41-47.
31.Kataoka, N., and Y. Tokiwa. 1998. Isolation and characterization of an active mannanase producing anaerobic bacterium, Clostridium tertium Kt-5A, from Lotus Soil. Journal of Applied Microbiology. 84(3)：357-367.
32.Kim, B. H., P. Bellows, R. Datta, and J. G. Zeikus. 1984. Control of carbon and electron flow in Clostridium acetobutylicum fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. Appl. Environ. Microbiol. 48:764-770.
33.Krimmer, V., H. Merkert, C. von Eiff, M. Prosch, J. Eulert, J.F. Lohr, J. Hacker, and W. Ziehbur. 1999. Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization. J. Clin. Microbiol. 37：2667-2673.
34.Kumar, S., K. Tamura, and M. Nei. 1993. MEGA：molecular evolutionary genetics analysis, version 1.0. University Park, PA：Pennsylvania State University.
35.Laguerre, G., M. Allard, F. Revoy, and N. Amarger. 1994. Rapid identification of Rhizobia by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 60：56-63.
36.Lane, D.J., B. Pace, G.J. Olsen, D.A. Stahl, M.L. Sogin, and N.R. Pace. 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses, Proc. Natl. Acad. Sci. U.S.A. 82：6955.
37.Lathe, R. 1985. Synthetic oligonucleotide probes deduces from amino acid sequence data. Theoretical and practical considerations. J. Mol. Biol. 183：11-12.
38.Lawier, A. 1995. Walker Bill to boost hydrogen sparks democratic grumbling. Science 267：613.
Lay, J.J. 2000. Modeling and optimization of anaerobic digested-sludge converting starch to hydrogen. Biotechnol. Bioeng. 68(3)：269-278.
39.Lee, J.W., and E. Greenbaum. 1995. Bioelectronics and biometakko-catalysis for production of fuels and chemicals by photosynthetic water splitting. Appl. Biochem. Biotechnol. 51/52：295.
40.Lee, N., P.H. Nielsen, K.H. Andreasen, S. Juretschko, J.L. Nielsen, K.-H. Schleifer, and M. Wagner. 1999. Combination of fluorescent in situ hybridization and microautoradiography — a new tool for structure — function analyses in microbial ecology. Appl. Environ. Microbiol. 65：1289-1297.
41.Li, S., R.N. Spear, and J.H. Andrews. 1997a. Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscopic slides and leaf surfaces. Appl. Environ. Microbiol. 63：3261-3267.
42.Lin, C.Y., and R.C. Chang. 1999. Hydrogen-production during the anaerobic acidogenic conversion of glucose. J. chem. technol. biotechnol. 74(6)：498-500.
43.Lin, C.Y., C.C. Chen, and M.C. Lin. 2000a. Hydrogen production in anaerobic acidogenesis process－Influences of thermal isolation and acclimation environment. J. Chinese Institute of Environ. Eng. 10: 163-168.
44.Lin, C.Y., C.C. Chen, and M.C. Lin. 2000b. Enhancements of acid-base isolation on hydrogen production in anaerobic process. Biotechnology 2000 the World Congress on Biotechnology. Sept. 3-8. 3：184-185. Berlin. Germany.
45.Liu, W.-T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63：4516-4522.
46.Ludwig, W., G. Kirchhof, N. Klugbauer, M. Weizenegger, D. Betzl, M. Ehrmann, C. Hertel, S. Jilg, R. Tatzel, H. Zitzelsberger, S. Liebl, M. Hochberger, J. Shah, D. Lane, P.R. Wallnofer, and K.H. Schleifer. 1992. Complete 23S ribosomal RNA sequences of Gram-positive bacteria with low DNA G+C content. Syst. Appl. Microbiol. 15：487-501.
47.Madigan, M.T., J.M. Martinko, and J. Parker. 1997. Biology of Microorganisms. Prentice-Hall. Inc. Upper Saddle River. NJ. USA.
48.Maidak, B.L., G.L. Olsen, N. Larsen, R. Overbeek, M.J. McCaughey, and C.R. Woese. 1997. The RDP (Ribosomal Database Project). Nucleic Acids Res. 25：109-110.
49.Majizat, A., Y. Mitsunori, W. Mitsunori, N. Michimasa, and M. Junichiro. 1997. Hydrogen gas-production from glucose and its microbial kinetics in anaerobic systems. Water Science and Technology. 36：279-286.
50.Manz, W., R. Amann, W. Ludwig, M. Wagner, and K.-H. Schleifer. 1992. Phylogenetic oligonucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15:593-600.
51.Manz, W., R. Amann, W. Ludwig, M. Vancanneyt, and K.-H. Schleifer. 1996. Application of a siute of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment. Microbiology.142:1097-1106.
52.Markov, S.A., M. Bazin, and D.O. Hall. 1995. Adv. Biochem. Eng./Biotechnol. 52：61-86.
53.Markov, S.A., R. Weaver, and M. Seibert. 1996. Presentation at the Hydrogen 96 Meeting. Stuttgart. Germany. June.
54.Markov, S.A., and P.F. Weaver, and M. Seibert. 1997. Spiral tubular bioreactors for hydrogen-production by photosynthetic microorganisms - design and operation. Appl. Biochem. Biotechnol. 63-5(SPR)：577-584.
55.Mechichi, T., M. Labat, B.K.C Patel, T.H.S. Woo, P. Thomas, and J.L. Garcia. 1999. Clostridium methoxybenzovorans sp. Nov., a new aromatic O-demethylating homoacetogen from an olive mill waste-water treatment digester. Int. J. Syst. Bacteriol. 49：1201-1209.
56.More, M., J.B. Herrick, M.O. Silva, W.C. Ghiorse, and E.J. Madsen. 1994. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment, Appl. Environ. Microbiol. 60：1572.
57.Moter, A., C. Hoeing, B.-K. Choi, B. Riep, and U.B. Gobel. 1988a. Molecular epidemiology of oral treponemes associated with periodonyal disease. J. Clin. Microbiol. 36：1399-1403.
58.Moter, A., G. Leist, R. Rudolph, K. Schrank, B.-K. Choi, M. Wagner, and U.B. Gobel. 1998b. Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions. Microbiology. 144：2459-2467.
59.Moyer, C.L., F.C. Dobbs, and D.M. Karl. 1994. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi seamount, Hawaii. Appl. Environ. Microbiol. 60：871-879.
60.Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich. 1986. Specific enzymatic amplification of DNA in vitro：the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51：263-273.
61.Mullis, K.B., and F.A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155：335-350.
62.Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700.
63.Muyzer, G., and N. B. Ramsing. 1995. Molecular methods to study the organization of microbial communities. Wat. Sci. Tech. 32(8)：1-9.
64.Muyzer, G., A. Teske, and C. O. Wirsen. 1995. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164:165-172.
65.Nakamura, M., H. Kanbe, and J.I. Matsumoto. 1993. Fundamental-studies on hydrogen-production in the acid-forming phase and its bacteria in anaerobic treatment processes - the effects of solids retention time. Water Sci. Technol. 28(7)：81-88.
66.Nandi, R., and S. Sengupta. 1998. Microbial-production of hydrogen - an overview. Crit. Rev. Microbiol. 24：61-84.
Nielsen, A.T., W.-T. Liu, C. Flilipe, L. Grady, Jr., S. Molin, and D.A. Stahl. 1999. Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Appl. Environ. Microbiol. 65：1251-1258.
67.Olsen, G.J., D.J. Lane, S.J. Giovannoni, N.R. Pace, and D.A. Stahl. 1987. Microbial ecology and evolution: a ribosomal RNA approach. Annu. Rev. Microbiol. 40：337.
68.Owen, W.F., D.C. Stuckey, Jr. J. B. Herly, L.Y. Young, and P.L. McCarty. 1979. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Wat. Res. 13：485-492.
69.Pakes, W.C.C., and W.H. Jollyman. 1901. The bacterial decomposition of formic acid into CO2 and H2. J. Chem. Soc. 79：386.
70.Peck, H.D., and H. Gest. 1957. Formic dehydrogenase and the hydrogenlyase enzyme complex in coli-aerogenes bacteria. J. Bacteriol. 73：706.
71.Poulsen, L.K., G. Ballard, and D.A. Stahl. 1993. Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms. Appl. Environ. Microbiol. 59：1354-1360.
72.Reysenbach, A.-L., L.J. Giver, G.S. Wickham, and N.R. Pace. 1992. Differential amplification of rRNA genes by polymerase chain reaction. Appl. Environ. Microbiol. 58：3417-3418.
73.Riesner, D., G. Steger, R. Zimmat, R. A. Owens, M. Wagenhofer, W. Hillen, S. Vollbach, and K. Henco. 1989. Temperature-gradient gel electrophoresis of nucleic acids： analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10：377-389.
74.Roller, C., M. Wagner, R. Amann, W. Ludwig, K.-H. Schleifer. 1994. In situ probing of Gram-positive bacteria with high DNA G+C content using 23S rRNA-targeted oligonucleotides. Microbiology. 140：2849-2858.
75.Roychowdhury, S., D. Cox, and M. Levandowsky. 1988. Production of hydrogen by microbial fermentation. Int. J. Hydrogen Energy. 13：407.
76.Saiki, R.K., S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, and N. Arnheim. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis diagnosis of sickle cell anemia. Science 230：1350-1354.
77.Saiki, R.K., D.H. Gelfand, S. Scharf, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis, and H.A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239：487-491.
78.Sasikala, K., C.V. Ramana, P.R. Rao, and K.L. Kovacs. 1993. Adv. Appl. Microbiol. 38：221-295.
79.Schonhuber, W., B. Puchs, S. Juretschko, and R. Amann. 1997. Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl. Environ. Microbiol. 63：3268-3273.
80.Shuman, S. 1994. Noval approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J. Biol. Chem. 269：32678-32684.
81.Smith, B.E., and R.R. Eady. 1992. Metalloclusters of the nitrogenases. Eur. J. Biochem. 205：1-15.
82.Snaidr, J., R. Amann, I. Huber, W. Ludwig, and K.-H. Schleifer. 1997. Phlogenetic analysis and in situ identification of bacteria in activated sludge. Appl. Environ. Microbiol. 63：2884-2896.
83.Solomon, B.O., A.P. Zeng, H. Biebl, H. Schlieker, C. Posten, and W.D. Deckwer. 1995. J. Biotechnol. 39：107-117.
84.Spear, R.N., S. Li., E.V. Nordheim, and J.H. Andrews. 1999. Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans. J. Microbiol. Methods. 35：101-110.
85.Stahl, D.A., D.J. Lane, G.J. Olsen, and N.R. Pace. 1984. Analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences. Science 224：409.
86.Stahl, D.A., D.J. Lane, G.J. Olsen, and N.R. Pace. 1985. Characterization of a Yellowstone hot spring microbial community by 5S ribosomal RNA sequences. Appl. Environ. Microbiol. 49：1379.
87.Stahl, D.A., and R. Amann. 1991. Development and application of nucleic acid probes in bacterial systematics, p. 205-248. In E. Stackebrandt and M. Goodfellow (cd.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons Ltd., Chichester, England.
88.Taguchi, F., J. D. Hang, N. Mizukami, T. Saito-Taki, K. Hasegawa, and M. Morimoto. 1993. Isolation of a hydrogen-producing bacterium, Clostridium beijerinchii strain Am21B, from termites. Can. J. Microbiol. 39：726-730.
89.Taguchi, F., N. Mizukami, K. Yamada, K. Hasegawa and T. Saito-Taki. 1995. Direct conversion of cellulosic materials to hydrogen by Clostridium sp. strain no.2, Enzyme Microbiol. Technol. 17：147.
90.Taguchi, F., K. Hasegawa, T. Saitotaki, and K. Hara. 1996. Simultaneous production of xylanase and hydrogen using xylan in batch culture of Clostridium sp. strain X53. Journal of Fermentation and Bioengineering. 81(2)：178-180.
91.Tarlera, S., L. Muxi, M. Soubes, and A.J.M. Stams. 1997. Caloramator proteoclasticus sp. Nov, a new moderately thermophilic anaerobic proteolytic bacterium. Int. J. Syst. Bacteriol. 47(3)：651-656.
92.Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W：improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22：4673-4680.
93.Ueno, Y., M. Morimoto, S. Ootsuka, T. Kawai, and S. Satous. 1995. US Patent. 5：464-539.
94.Ueno, Y., S. Otsuka, and M. Morimoto. 1996. Hydrogen-production from industrial waste-water by anaerobic microflora in chemostat culture. Journal of Fermentation and Bioengineering. 82(2)：194-197.
95.Van Haandel , A.C., and G. Lettinga. 1994. Anaerobic sewage treatment- a practical guide for regions with a hot climate. Chapter 2. John Wiley & Sons Ltd. New York.
96.Viale, A.M., A.K. Arakaki, F.C. Soncini, and R.G. Ferreyra. 1994. Evolutionary relationships among eubacterial groups as inferred from GroEL (chaperonin) sequence comparisons. Int. J. Syst. Bacteriol. 44：527-533.
97.Wagner, M., R. Amann, H. Lemmer, and K.H. Schleifer. 1993. Probing activated sludge with proteobacteria-specific oligonucleotides：inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbial. 59：1520-1525.
98.Wagner, M., R. Erhart, W. Manz, R. Amann, H. Lemmer, D. Wedi, and K.-H. Schleifer. 1994. Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol. 60：792-800.
99.Wagner, M., M. Schmid, S. Juretschko, K.-H. Trebesius, A. Bubert, W. Goebel, and K.-H. Schleifer. 1998. In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes. FEMS Microbiol. Lett. 160：159-168.
100.Weaver, P.F., S. Lien, and M. Seibert. 1980. Solar Energy. 24：3-45.
101.Wessendorf, M.W., and T.C. Brelje. 1992. Which fluorophore is the brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18. Histochemistry. 98： 81-85.
102.Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51：221-271.
103.Wu., L.F., and L.A. Mandrand. 1993. Microbial hydrogenase-primary structure classification, signature and phylogeny. FEMS Microbial. Rev. 104：243.
104.Yigit, D.O., U. Gunduz, L. Turker, M. Yucel, and I. Eroglu. 1999. Identification of by-products in hydrogen producing bacteria - Rhodobacter-sphaeroides Ou-001 grown in the waste-water of a sugar refinery. J. Biotechnol. 70(1-3)：125-131.
105.Yokoi, H., T. Ohkawara, J. Hirose, S. Hayashi, and Y. Takasaki. 1995. Characteristics of hydrogen-production by aciduric Enterobacter-aerogenes strain Ho-39. J. Ferm. Bioeng. 80(6)：571-574.
106.Yokoi, H., T. Tokushige, J. Hirose, S. Hayashi, and Y. Takasaki. 1997. Hydrogen production by immobilized cells of aciduric Enterobacter aerogenes strain HO-39. J. Ferm. Bioeng. 83：481-484.
107.Yokoi, H., S. Mori, J. Hirose, S. Hayashi, and Y. Takasaki. 1998. H-2 production from starch by a mixed culture of Clostridium-butyricum and Rhodobacter Sp. M-19. Biotechnol. Lett. 20(9)：895-899.
108.Zajic, J.E., N. Kosaric, and J.D. Brosseau. 1978. Microbial production of hydrogen. Adv. Biochem. Eng. 9：57.
109.Zarda, B., R. Amann, W. Wallner, and K.-H. Schleifer. 1991. Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. J. Gen. Microbiol. 137：2823-2830.
110.Zimmer, C., and U. Wahnert. 1986. Nonintercalating DNA-binding ligands： specificity of the interaction and their use as tools in biophysical, biochemical and biological investigations of the genetic materials. Prog. Biophys. Mol. Biol. 47：31-112.

指導教授

劉文佐

審核日期

2001-6-25

推文