徐源泰Shyu, Yuan-Tay臺灣大學:園藝學研究所王鐘毅Wang, Chung-YiChung-YiWang2010-05-052018-06-292010-05-052018-06-292009U0001-0508200922134900http://ntur.lib.ntu.edu.tw//handle/246246/180956微生物之多樣性是由各種複雜的環境因子與長時間演化所形成的，而台灣的海洋環境造就了台南沿海地區經日照蒸發而成的鹽田，其中含有極為豐富的耐鹽菌有待發掘。本研究第一部份先以變性梯度膠體電泳 (Denaturing gradient gel electrophoresis，DGGE)針對微生物的16S rDNA片段進行分群，再以生物多樣性指數加以描述，另輔以螢光原位雜交技術 (Fluorescence in situ hybridization，FISH) 調查本區域之微生物多樣性。經由DGGE分析得出24個分群中，有19屬的細菌和5個屬的古生菌，此外有8.7%的菌群是未知菌種；多樣性指數演算結果指出，七股鹽田具有最低的微生物多樣性。在 FISH 的部分，利用螢光標定之專一性引子針對鹽田中不同族群之微生物進行雜交，發現此區域有37.2–52.9%的細菌和2.7–6.6%的古生菌，顯示本區域存在相當豐富之微生物族群，其中以四草鹽田具有最高的生物多樣性。本研究亦從鹽田分離出兩株極端耐鹽菌，經由型態、生理、生化及分子演化等多重分析比對，確認這兩株菌株為新菌種，分別依其分離之鹽田命名為七股菌 (Virgibacillus chiguensis)與四草菌 (Marinobacter szutsaonensis)，並寄存於菌種保存研究中心。本研究最後一部分，自鹽田分離出之耐鹽菌中，進行聚醣水解酵素的篩選，藉此發現具耐鹽性質的聚醣水解酵素，以因應不同使用領域之需求，經篩選後，分離出可產生纖維水解酵素的Salinivibrio sp. NTU-05，與可產生木糖水解酵素的Bacillus sp. NTU-06，對於農產廢棄物之再循環利用與生質能源的開發極具有發展潛力。In the coastal and marine areas of southern Taiwan, various solar salt fields have been established. Since ancient time, Tainan has already known as key salt production site. Discovery of these microbial information in these unique ecosystems is rather encouraging for further novel species discovery, exploration and conservation of useful microbes. In the first section, the microbiota diversity of the former salterns was investigated by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Soil samples from three salterns were analyzed using DGGE representing 5 archaea and 18 bacteria taxonomic groups, whereas 8.7% of microbes were unclassified microorganisms. During FISH analysis, several taxonomic-specific probes were used. Archaea occupied 2.7–6.6% whereas bacteria accounted for 37.2–52.9% of total microbial population at the three sites. Among these sampling sites, the Szutsao saltern had the highest diversity in halophilic microbial composition, as indicated by DGGE and FISH. In the second section, we describe two novel species that were isolated from the Chigu and Szutsao saltern. On the basis of phenotypic, phylogenetic and genetic analyses, these two strains were considered to represent a novel species of the genus Virgibacillus and Marinobacter, and were named Virgibacillus chiguensis and Marinobacter szutsaonensis according to Chigu and Szutsao saltern. These two strains were deposited in culture collection center. In the last section, we reported the characteristics of the two polyglucan hydrolytic enzymes (cellulase and xylanase) were purified from the halophilic bacterium Salinivibrio sp. NTU-05 and Bacillus sp. NTU-06. These enzymes were active over a range of salt concentration (0–25% NaCl) when examined in a culture broth. Some of the novel characteristics of these halostable enzymes indicate that they may have potential applications in treating agricultural waste and in the bioremediation of cellulose materials.中文摘要...................................................1bstract...................................................2hapter 1: Introduction....................................4hapter 2: Microbial diversity analysis of former salterns in southern Taiwan by 16S rRNA-based methods..............11bstract..................................................11ndroduction..............................................12aterials and Methods.....................................14esults...................................................20iscussion................................................23able 1. Properties of soil samples.......................27able 2. Oligouncleotide probes...........................28able 3. Total DAPI cell count and relative percentages of hybridized cells with specific probes.....................29able 4. Genus and groups at three sampling sites.........30able 5. Similarity matrix based on 16S rRNA gene sequence comparisons...............................................31igure 1. DGGE band patterns obtained from soil samples...32igure 2. Expected number of species against number of individuals...............................................33hapter 3: Virgibacillus chiguensis sp. nov., isolated from Chigu, a previously commercial saltern located in southern Taiwan....................................................34bstract..................................................34ntroduction..............................................35ethods...................................................36esults...................................................40able 1. Differential phenotypic characteristics of NTU-101T and other Virgibacillus species......................44able 2. Major composition of fatty acid in strain NTU-101T and related Virgibacillus species.........................45igure 1. Neighbour-joining tree showing phylogenetic topology of strain NTU-101T with other Virgibacillus species...................................................46hapter 4: Marinobacter szutsaonensis sp. nov., isolated from Szutsao, a solar saltern in southern Taiwan..........47bstract..................................................47ntroduction..............................................48ethods...................................................49esults...................................................53able 1. Differantial phenotypic characteristics of NTU-104T and other Marinobacter species.......................57able 2. Cellular fatty acid of strain NTU-104T and related Marinobacter Species......................................58igure 1. Neighbour-joining tree showing phylogenetic topology of strain NTU-104T with other Marinobacter species...................................................59hapter 5: Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05..60bstract..................................................60ntroduction..............................................61aterials and Methods.....................................64esults...................................................72iscussion................................................76able 1. Morphological, physiological and biochemical characteristics of Salinivibrio sp. NTU-05................81able 2. Purification steps of cellulase enzyme...........82able 3. Effect of various metal ions on cellulase activity..................................................83igure 1. Effect of various carbon sources on the cellulase production................................................84igure 2. Purification of cellulase from Salinivibrio sp. NTU-05....................................................85igure 3. SDS-PAGE and Zymogram analysis..................86igure 4. Effect of temperature on the enzyme activity....87igure 5. Thermal stability of cellulase residual activity88igure 6. Effect of pH on the activity of cellulase.......89igure 7. Effect of pH on cellulase stability.............90igure 8. Effect of NaCl on cellulase activity............91igure 9. Effect of NaCl on cellulase stability...........92igure 10. Kinetic assayed of the cellulase...............93hapter 6: Production, purification and characterization of halotolerant xylanase from Bacillus sp. NTU-06............94bstract..................................................94ntroduction..............................................95aterials and Methods.....................................98esults and Discussion...................................106onclusions..............................................112able 1. Morphological, physiological and biochemical characteristics of Bacillus sp. NTU-06...................113able 2. Effect of various carbon sources on the xylanase production...............................................114able 3. Purification steps of xylanase..................115igure 1. Purification of xylanase from Bacillus sp. NTU-06.......................................................116igure 2. SDS-PAGE and Zymogram analysis.................117igure 3. Thermal stability of xylanase residual activity118igure 4. Effect of pH on xylanase stability.............119igure 5. Effect of NaCl on xylanase stability...........120igure 6. Kinetic assayed of the xylanase................121eference................................................122application/pdf1908915 bytesapplication/pdfen-US微生物多樣性極端耐鹽菌聚醣水解酵素Microbial diversityextremely halotolerant bacteriapolyglucan hydrolytic enzymes[SDGs]SDG14thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/180956/1/ntu-98-F93628210-1.pdf