紫色不含硫光合菌是一群代謝方式多樣的微生物，在不同的環境可進行不同之代謝模式：包括光合自營、光合異營、化學異營、及化學自營。由於這類細菌的特殊生理特質，近年來受到科學家高度的重視。它們廣泛地分佈於自然界，包括湖泊、水塘、海岸潟湖、水表淤泥或高濃度有機廢水池等。蓮池潭位於高雄市，是一優養化相當嚴重的人工湖泊，由於日照充足且富含有機質，其生態環境相當適合紫色不含硫光合菌的生長。本研究利用Winogradsky collumn，由蓮池潭分離出16株菌種。並利用PCR增幅分離菌株之16S-rDNA序列，依據16S-rDNA基因序列分析結果，確認篩選出的16株菌種皆為紫色不含硫光合菌。經由親緣關係比對結果發現，這16株紫色不含硫光合菌分屬Rhodopseudomonas palustris、Rubrivivax gelatinosus、及Rhodobacter sphaeroides三群。由菌種的特性得知，這三群菌株皆為革蘭氏陰性菌且都具有細菌葉綠素a。其中R. palustris及R. sphaeroides這二群菌株不僅可利用許多種短鏈有機酸作為碳源，也具有脫硝的能力。但僅R. palustris這群菌株可利用苯甲酸這個難分解的環狀化合物。對鹽的耐受度R. sphaeroides為3%，R. palustris及R. gelatinosus的菌株則為1%。

Purple nonsulfur bacteria are a group of extraordinary metabolic diverse bacteria. They can grow photoautotrophically, photoheterotrophically , chemoheterotrophically or chemoautotrophically. Under various conditions, they can enjoy exceptional flexibility within each of these modes of metabolism. Due to the special physical characteristics properties, they had attracted scientist’s attention in resent years. These bacteria are widely distributed in nature such as lakes, water ponds, coastal lagoons or high concentration organic waste lagoons. Lotus Pond, located in northern Kaohsiung City, is a serious eutrophied artificial lake. Because of receiving sufficient light and having been polluted by significant amounts of soluble organic matter, the ecology of the lake is suitable for the growth of purple nonsulfur bacteria. In the study, the lake water and sediments by using a Winograsdsky column, we successfully isolated 16 strains bacteria from the Lotus Pond. We also amplified the 16S-rDNA fragments of these strains by PCR and sequenced these PCR products, then aligned these sequences with the data of GeneBank. We affirmed that the 16 isolated strains belong to purple nonsulfur bacteria. From phylogenetic analysis, these 16 strains belong to the following three groups of bacteria: Rhodopseudomonas palustris, Rubrivivax gelatinosus, and Rhodobacter sphaeroides. Characteristic studies of these strains, we found that all isolated strains are Gram negative bacteria and contain bacteriochlorophyll a. The strains that belong to R. palustris and R. sphaeroides group can use several different types of short chain organic acid as their carbon source and have denitrification ability. However, only the strains belong to R. palustris group are able to use the aromatic compound benzoate. From salt tolerant studies, we found the strains in R. sphaeroides group can grow well in 3% NaCl, and both R. palustris and R. gelatinosus group can only grow in 1% NaCl.

圖目錄……………………………………………………………IX
表目錄……………………………………………………………XII
第一章 前言……………………………………………………1
第二章 材料與方法……………………………………………18
第三章 結果與討論……………………………………………26
參考文獻………………………………………………………39
附錄-各分離菌株之16S rDNA序列……………………………78

Albers, H., and Gottschalk, G. 1976. Acetate metabolism in Rhodopseudomonas gelatinosa and several other Rhodospirillaceae. Arch. Microbial. 111:45–49.

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, M., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res. 25:3389-3402.

Biebl, H., and Pfennig, N. 1981. Isolation of members of Rhodospirillaceae. In: The Prokaryotes. Starr, M. P., Stolp, H., Trüper, H. G., Balows, A., and Schlegel, H. G. (eds.). Springer-Verlag Berlin, pp. 267–273.

Buchanan, B. B., Evans, M. C. W., and Arnon, D. I. 1967. Ferredoxin-dependent carbon assimilation in Rhodospirillum rubrum. Arch. Microbiol. 59:32–40.

Brunisholz, R., and Zuber, H. 1992. Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaceae. Eur. J. Photochem. Photobiol. B. Biol. 15:113–140.
Colbeau, A., and Vignais, P. M. 1992. Use of hupS:lacZ gene fusion to study the regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2. J. Bacteriol. 174:4258–4264.

Drews, G., and Imhoff, J. F. 1991. Phototrophic purple bacteria. In: Variations in Autotrophic Life. Shively, J. M. and Barton, L. L. (eds.). Academic Press London, UK, pp. 51–97.

Dutton, P. L., and Evans, W. C. 1969. The metabolism of aromatic compounds by Rhodopseudomonas palustris: a new reductive method of aromatic ring metabolism. Biochem. J. 113:525–536.

Dutton, P. L., and Evans, W. C. 1978. Metabolism of aromatic compounds by Rhodospirillaceae. In: The photosynthetic bacteria. Clayton, R. K. and Sistrom, W. R. (eds.). Plenum Press New York, NY, pp. 719–726.

Ensign, J. C. 1977. Biomass production from animal waste by photosynthetic bacteria. In: Microbiol energy conversion. Schlegel, H. G. and Barnea, J. (eds.). Pergamon Press Oxford, pp. 455–482.

Fascetti, E., D’addario, E., Todini, O., and Robertiello, A. 1998. Photosynthetic hydrogen evolution with volatile organic acids derived from the fermentation of source selected municipal solid wastes. Int. J. Hydrogen Energy 23:753-760.

Fuller, R. C. 1995. Polyesters and photosynthetic bacteria: from lipid cellular inclusions to microbial thermoplastics. In: R. Anoxygenic photosynthetic bacteria. Blankenship, E., Madigan, M. T., and Bauer, C. E. (eds.). Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 1245–1256.

Göbel, F. 1978. Quantum efficiencies of growth. In: The photosynthetic bacteria. Clayton, R. K., and Sistrom, W. R. (eds.). Plenum Press New York, NY, pp. 907–925.

Harwood, C. S., and Gibson, J. 1988. Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris. Appl. Environ. Microbiol. 54:712–717.

Hansen, T. A., and van Gemerden, H. 1972. Sulfide utilization by purple nonsulfur bacteria. Arch. Microbiol. 86:49–56.

Hansen, T. A., and Veldkamp, H. 1973. Rhodopseudomonas sulfidophila nov. spec., a new species of the purple nonsulfur bacteria. Arch. Microbiol. 92:45–58.

Hansen, T. A., and Imhoff, J. F. 1985. Rhodobacter veldkampii, a new species of phototrophic purple nonsulfur bacteria. Int. J. Syst. Bacteriol. 35:115–116.

Hiraishi, A., and Ueda, Y. 1994. Rhodoplanes gen. nov., a new genus of phototrophic bacteria including Rhodopseudomonas rosea as Rhodoplanes roseus comb. nov. and Rhodoplanes elegans sp. nov. Int. J. Syst. Bacteriol. 44:665–673.

Hiraishi, A., Urata, K., and Satoh, T. 1995. A new genus of marine budding phototrophic bacteria, Rhodobium gen. nov., which includes Rhodobium orientis sp. nov. and Rhodobium marinum comb. nov. Int. J. Syst. Bacteriol. 45:226–234.

Hiraishi, A., Muramatsu, K., and Ueda, Y. 1996. Molecular genetic analyses of Rhodobacter azotoformans sp. nov. and related species of phototrophic bacteria. Syst. Appl. Microbiol. 19:168–177.

Holm, H. W., and Vennes, J. W. 1971. Occurrence of purple sulfur bacteria in a sewage treatment lagoon. Appl. Microbiol. 19:988–996.

Imhoff, J. F., and Trüper, H. G. 1976. Marine sponges as habitats of anaerobic phototrophic bacteria. Microbial Ecol. 3:1–9.

Imhoff, J. F., and Trüper, H. G. 1977. Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch. Microbiol. 114:115–121.

Imhoff, J. F. 1982a. Response of photosynthetic bacteria to mineral nutrients. In: CRC handbook of biosolar resources. Mitsui, A., and Black, C. C. (eds.). Volume 1: Basic principles, part 1. CRC Press Boca Raton, FL, pp. 135–146.

Imhoff, J. F. 1982b. Occurrence and evolutionary significance of two sulfate assimilation pathways in the Rhodospirillaceae. Arch. Microbiol. 132:197-203.

Imhoff, J. F. 1983. Rhodopseudomonas marina sp. nov., a new marine phototrophic purple bacterium. Syst. Appl. Microbiol. 4:512–521.

Imhoff, J. F., Trüper, H. G., and Trüper, N. 1984. Rearrangements of the species and genera of the phototrophic "purple nonsulfur bacteria". Int. J. Syst. Bacteriol. 34:340–343.

Imhoff, J. F. 1988. Anoxygenic phototrophic bacteria. In: Methods in Aquatic Bacteriology. Austin, B. (ed.). Wiley and Sons Chichester, UK

Imhoff, J. F. 1989a. Genus Rhodobacter. In: Bergey's Manual of Systematic Bacteriology. Staley, J. T., Bryant, M. P., Pfennig, N., and Holt, J. G. (eds.). Williams and Wilkins Baltimore, M. D. 3:1668–1672.

Imhoff, J. F., and Trüper, H. G. 1989b. The purple nonsulfur bacteria. In: Bergey's Manual of Systematic Bacteriology. Staley, J. T., Bryant, M. P., Pfennig, N., and Holt, J. G. (eds.). Williams and Wilkins Baltimore, M. D. 3:1658–1661.

Imhoff, J. F., and Trüper, H.G. 1991. The genus Rhodospirillum and related genera. In: The prokaryotes. Balows, A., Trüper, H.G., Dworkin, M., Harder, W., and Schleifer, K. H. (eds). 2nd edn, vol 3. Springer, Berlin Heidelberg New York, pp. 2141–2155.

Janssen, P. H., and Harfoot, C. G. 1987. Phototrophic growth on n-fatty acids by members of the family Rhodospirillaceae. Syst. Appl. Microbiol. 9:9–11.

Kaiser, P. 1966. Contribution a l'étude de l'écologie des bacteries photosynthetiques. Ann. Inst. Pasteur. 111:733–749.

Kamen, M. D., and Gest, H. 1949. Evidence for a nitrogenase system in the photosynthetic bacterium Rhodospirillum rubrum. Science 109:560.

Kelley, B. C., Meyer, C. M., Gandy, C., and Vignais, P. M. 1977. Hydrogen recycling by Rhodopseudomonas capsulate. FEBS Lett. 81:281–285.

Klemme, J. H. 1968. Untersuchungen zur photoautotrophie mit molekularem wasserstoff bei neuisolierten schwefelfreien purpurbakterien. Arch. Microbiol. 64:29–42.

Kobayashi, M., and Tchan, Y. T. 1973. Treatment of industrial waste solutions and production of useful byproducts using photosynthetic bacterial method. Water Res. 7:1219–1224.

Kobayashi, M. 1977. Utilization and disposal of wastes by photosynthetic bacteria. In: Microbial Energy Conversion . Schlegel, H. G., and Barnea, J. (eds.). Pergamon Press Oxford. pp. 443–453.

Kobayashi, M., and Kobayashi, M. 1995. Waste remediation and treatment using anoxygenic phototrophic bacteria. In: Anoxygenic Photosynthetic Bacteria. Blankenship, R. E., Madigan, M. T., and Bauer, C. E. (eds.). Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 1269–1282.

Kompantseva, E. I. 1989. A new species of budding purple bacteria: Rhodopseudomonas julia sp. nov. Mikrobiologiya. 58:254–259.

Kondratieva, E. N. 1979. Interrelation between modes of carbon assimilation and energy production in phototrophic purple and green bacteria. In: Microbial Biochemistry: International Review of Biochemistry. Quale, J. R. (ed.). University Park Press Baltimore, M.D. 21:117–175.

Kumar, S., Tamura, K., Jakobsen, I. B., and Masatoshi, N. 2001. MEGA2: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA.

Ludden, P. W., and Roberts, G. P. 1995. The biochemistry and genetics of nitrogen fixation by photosynthetic bacteria. In: Anoxygenic Photosynthetic Bacteria. Blankenship, R. E., Madigan, M. T., and Bauer, C. E. (eds.). Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 929–947.

Madigan, M. T. 1995. Microbiology of nitrogen fixation by anoxygenic photosynthetic bacteria. In: Anoxygenic Photosynthetic Bacteria. Blankenship, R. E., Madigan, M. T., and Bauer, C. E. (eds.). Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 915–928.

Madigan, M. T., and Gest, H. 1979. Growth of the photosynthetic bacterium Rhodopseudomonas capsulata chemoautotrophically in the darkness with H2 as energy source. J. Bacteriol. 137:524-530.

Madigan, M. T., Jung, D. O., Woese, C. R., and Achenbach, L. A. 2000. Rhodoferax antarcticus, sp. nov. a moderately psychrophilic purple nonsulfur bacterium from an Antarctic microbial mat. Arch. Microbiol. 173:269–277.

Meyer, J., Kelley, B. C., and Vignais, P. M. 1978. Effect of light on nitrogenase function and synthesis in Rhodopseudomonas capsulata. J. Bacteriol. 136:201-208.

Michalski, W. P., and Nicholas, D. J. D. 1988. Identification of two new denitrifying strains of Rhodobacter sphaeroides. FEMS Microbiol. Lett. 52:239–244.

Mitsui, A. 1979. Biosaline research. In: The use of photosynthetic marine organisms in food and feed production. Hollaender, A., Aller, J. C., Epstein, E., San Pietro, A., and Zaborsky, O. (eds.). Plenum Press New York, NY, pp. 177–215.

Neutzling, O., Imhoff, J. F., and Trüper, H. G. 1984. Rhodopseudomomas adriatica sp. nov., a new species of the Rhodospirillaceae, dependent on reduced sulfur compounds. Arch. Microbiol. 137:256–261.

Neutzling, O., Pfleidever, C., and Truper, H. G. 1985. Dissimilatory sulfur metabolism in phototrophic 'non-sulfur' bacteria. J. Gen. Microbiol. 131:791-798.

Pfennig, N. 1967. Photosynthetic bacteria. Ann. Rev. Microbiol. 21:285–324.

Pfennig, N. 1969. Rhodospeudomonas acidophila, sp. n., a new species of the budding purple nonsulfur bacteria. J. Bacteriol. 99:597–602.

Qadri, S. M. H., and Hoare, D. S. 1968. Formic hydrogenlyase and the photoassimilation of formate by a strain of Rhodopseudomonas palustris. J. Bacteriol. 95:2344–2357.

Sasaki, K., Noparatnaraporn, N., Hayashi, M., Nishizawa, Y., and Nagai, S. 1981. Single-cell protein production by treatment of soybean wastes with Rhodopseudomonas gelatinosa. J. Ferm. Technol. 59:471–477.

Sasaki, K., Tanaka, T., Nishizawa, Y., and Hayashi, M. 1990. Production of a herbicide, 5-aminolevulinic acid, by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor. Appl. Microbiol. Biotechnol. 32:727–731.

Sasikala, K., Ramana, C. H. V., Roa, P. R. 1992. Photoproduction of hydrogen from the waste water of a distillary by Rhodobacter sphaeroides OU 001. Int. J. Hydrogen Energy 17:23–27.

Sasikala, K., Ramana, C. V., Rao, P. R., and Kovacs, K. L. 1993. Anoxygenic phototrophic bacteria: Physiology and advances in hydrogen production technology. Adv. Appl. Microbiol. 38:211–295.

Schick, H. J. 1971. Interrelationship of nitrogen fixation, hydrogen evolution and photoreduction in Rhodospirillum rubrum. Arch. Microbiol. 75:102–109.

Schön, G., and Biedermann, M. 1973. Growth and adaptive hydrogen production of Rhodospirillum rubrum (F1) in anaerobic dark cultures. Biochim. Biophys. Acta 304:65–75.

Shipman, R. H., Kao, I. C., and Fan, L. T. 1975. Single-cell protein production by photosynthetic bacteria cultivation in agricultural by-products. Biotechnol. Bioeng. 17:1561–1570.

Siefert, E., Irgens, R. L., and Pfennig, N. 1978. Phototrophic purple and green bacteria in a sewage treatment plant. Appl. Environ. Microbiol. 35:38–44.

Siefert, E., and Pfennig, N. 1979. Chemoautotrophic growth of Rhodopseudomonas species with hydrogen and chemotrophic utilization of methanol and formate. Arch. Microbiol. 122:177–182.

Sockett, R. E., Donohue, T. J., Varga, A. R., and Kaplan, S. 1989. Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences. J. Bacteriol. 171:436-446.

Stackebrandt, E., Murray, R. G. E., and Trüper, H. G. 1988. Proteobacteria classis nov., a name for the phylogenetic taxon that includes the "purple bacteria and their relatives." Int. J. Syst. Bacteriol. 38:321–325.

Tabita, F. R. 1995. The biochemistry and metabolic regulation of carbon metabolism and CO2 fixation in purple bacteria. In: Anoxygenic photosynthetic bacteria. Blankenship, R. E., Madigan, M. T., and Bauer, C. E. (eds.). Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 885–914.

Thompson, J. D., Higgins, D. G., and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence aliment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acid Res. 22:4673-4680

Trüper, H. G. and Imhoff, J. F. (1991) The genera Rhodocyclus and Rubrivivax. In: The prokaryotes. Balows, A., Trüper, H. G., Dworkin, M., Harder, W., and Schleifer, K. H. (eds.). 2nd edn, vol 3. Springer, Berlin Heidelberg New York, pp. 2556–2561.

Van Niel, C. B. 1944. The culture, general physiology, morphology and classification of the nonsulfur purple and brown bacteria. Bacteriol. Rev. 8:1–118.

Van Niel, C. B. 1971. Techniques for the enrichment, isolation, and maintenance of photosynthetic bacteria. In: Methods in enzymology. Collowick, S. P., and Kaplan, N. V. (eds.). Volume 23, Part A, Academic Press New York, NY, pp. 3–28.

Vrati, S. 1984. Single cell protein production by photosynthetic bacteria grown on the clarified effluents of a biogas plant. Appl. Microbiol. Biotechnol. 19:199–202.

Uffen, R. L. 1973. Growth properties of Rhodospirillum rubrum mutants and fermentation of pyruvate in anaerobic, dark conditions. J. Bacteriol. 116:874–884.

Uffen, R. L. 1978. Fermentative metabolism and growth of photosynthetic bacteria. In: The photosynthetic bacteria. Clayton, R. K., and Sistrom, W. R. (eds.). Plenum Press New York, NY, pp. 857–872.

Widdell, F., Schnell, S., Heising, S., Ehrenreich, A., Assmus, B., and Schink, B. 1993. Ferrous iron oxidation by anoxygenic phototrophic bacteria. Nature 362:834-836.

Winogradsky, S. 1888. Beiträge zur Morphologie und Physiologie der Bakterien. Volume 1: Zur Morphologie und Physiologie der Schwefelbakterien.

Woese, C. R, 1987. Bacterial evolution. Microbiol. Rev. 51:221–271.

Wong, D. K., Collins, W. J., Harmer, A., Lilburn, T. G., and Beatty, J. T. 1996. Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes. J. Bacteriol. 178:2334-2342.

Yildiz, F. H., Gest, H., and Bauer, C. E. 1991. Attenuated effect of oxygen on photopigment synthesis in Rhodospirillum centenum. J. Bacteriol. 173:5502–5506.

Yokoi, H., Maki, R., Hirose, J., and Hayashi, S. 2002. Microbial production of hydrogen from starch-manufacturing wastes. Biomass and Bioenergy 22:389-395.

Zhu, H., Suzuki, T., Tsygankov, A. T., Asada, Y., and Miyake, J. 1999. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. Int. J. Hydrogen Energy 24:305-310.

Zuber, H., and Cogdell, R. J. 1995. Structure and organization of purple bacterial antenna complexes. In: Anoxygenic photosynthetic bacteria. Blankenship, R. E., Madigan, M. T., and Bauer, C. E. (eds.). Kluwer Academic Publishers Dordrecht, The Netherlands, pp. 315–348.