臺灣博碩士論文加值系統

針對Bacillus licheniformis (BL) 和Bacillus megaterium (BM)進行特性分析與可利用性評估。藉由觀察細菌生理型態及利用细菌16S rRNA gene、16S-23S Internal Transcribed Space region（ITS）與專一性功能基因進行PCR增幅及鑑定。本實驗分別測試這兩株菌的CMCase、Avicelase及β-glucosidase在不同培養溫度下的酵素活性，發現在60℃與50℃各有最大酵素活性。針對單一菌株進行紫外光存活影響之試驗，BL經改良MISP培養，紫外光照射第60分鐘的存活率降至60％，第180分鐘BL的存活率已降至54％；BM經改良MISP培養第60分鐘的存活率降至85％，第180分鐘BM的存活率已降至73％。經LB培養的BL和BM，在化學藥劑之耐受性試驗中，影響較大為殺真菌劑的聚合二硫代胺基甲酸鹽類、胺基甲酸鹽類、三唑類；對殺細菌劑的銅類、抗生素類；對殺蟲劑的吡啶有機硫代磷酸鹽類；對除草劑的醯胺類，存活率皆低於60％，展著劑部分則無顯著影響，而經MISP培養的BL和BM，在以上敘述之化學農藥的存活率，幾乎都高於81％以上。在耐鹽測試方面BL可在17%氯化鈉濃度環境生長，而BM可耐至10%之鹽濃度。在溶磷試驗與纖維降解試驗中皆有透化圈，其中又BL效果最好。此外在抗旱迫害試驗中，BL可提高芥藍菜之存活率，而BL與BM可有效提高包心芥菜之產量，惟BL效果最好。

In this study we isolated two bacteria from swamp by using polymerase chain reaction (PCR) and DNA sequencing to identify the 16S rRNA gene, 16S-23S internal transcribed spa (ITS) region and specific genes. Bacillus licheniformis (BL) and Bacillus megaterium (BM) were isolated to perform further analysis. We detected the enzyme activities of CMCase, Avicelase and β-glucosidase of BL and BM. The result indicated that BL can be applied to cellulose degradation, the result also showed that optimal temperature for the enzymes activities is 60℃, 50℃ and 50℃, respectively. We investigated the survival rate of BL and BM after exposed to UV radiation. The survival rate of BL was lower than 60% and 54% after 60 minutes and 180 minutes of UV-light exposure, respectively. The survival rate of BM was over 87% after 60 minutes and was below 73% after 180 minutes exposure of the UV-light. During the chemical pesticides test, the BL and BM survival rate show that in poly-dithiocarbamate, carbamates, riazole, bactericides indicated copper compounds antibiotic pesticide, and the of insecticides indicated that pyridine organo thiophosphate are all lower than 60％. Expertly the weedicides indicated that Amides had worst survival rate of BL, but the surfactant had no effect during the experiment. In addition, we tested the salt stress tolerance of BL and BM, BL can survive at 17% NaCl concentration that was significantly higher than BM. Furthermore BL and BM both showed high phosphorus solubilizing ability. In field test, BL of strengthens drought tolerance of survival rates. Both BL and BM promote the growth of Brassica juncea compared with control group.

壹、前言 1
一、 磷肥 1
二、 微生物肥料 2
三、 溶磷微生物肥料 2
四、 纖維分解酵素（cellulase） 3
五、 Bacillus spp.簡介 5
六、 研究目的 5
貳、材料與步驟 7
一、菌種來源、純化與保存 7
（一）來源 7
（二）純化 7
（三）保存 7
二、以分子技術進行菌種鑑定 7
（一）Genomic DNA之萃取 7
（二）16S rRNA 核苷酸序列聚合酶連鎖反應、定序與序列比對 9
（三）16S-23S internal transcribed spacer (ITS)區域之核苷酸序列聚合酶連鎖反應、定序與比對 10
（四）Keratinaes（kerA）之核苷酸序列聚合酶連鎖反應、定序與序列比對 11
（五）Beta-amylase（amyG）之核苷酸序列聚合酶連鎖反應、定序與序列比對 12
三、基本生理特性試驗 13
（一）CFU測定活菌數 13
（二）CFU測定活孢數 14
（三）內生孢子染色試驗 15
（四）pH值測定 15
四、紫外光(UV-B)照射試驗 15
五、對化學農藥之耐受性試驗 16
六、耐鹽性試驗 17
七、溶磷試驗 17
（一）溶磷菌篩選試驗 17
（二）BL與BM對PR27、PR30溶磷試驗 18
（三）BL與BM對作物生長勢及其產量影響試驗 18
八、纖維降解試驗 19
（一）固態纖維素分解酵素活性測試 19
（二）粗酵素液製備 20
（三）分解纖維酵素活性分析-DNS（附錄十）還原糖試驗 20
（四）粗酵素液之特性分析 21
（五）棕櫚泥纖維降解試驗 21
（六）咖啡渣纖維降解試驗 22
九、乾旱迫害存活率評估 22
參、結果 24
一、菌種來源、純化與保存結果 24
二、以分子技術進行菌種鑑定結果 24
（一）Genomic DNA之萃取結果 24
（二）16S rRNA 核苷酸序列聚合酶連鎖反應、定序與序列比對結果 24
（三）16S-23S internal transcribed spacer (ITS)區域之核苷酸序列聚合酶連鎖反應、定序與比對結果 25
（四）Keratinaes（kerA）之核苷酸序列聚合酶連鎖反應、定序與序列比對結果 25
（五）Beta-amylase（amyG）之核苷酸序列聚合酶連鎖反應、定序與序列比對 26
三、基本生理特性試驗結果 26
（一）活菌數測定結果 26
（二）測定活孢數結果 27
（三）內生孢子染色試驗結果 27
（四）pH值測定結果 28
四、紫外光(UV-B)照射試驗結果 28
五、對化學農藥之耐受性試驗結果 30
（一）殺真菌劑結果 30
（二）殺細菌劑結果 30
（三）殺蟲劑結果 31
（四）除草劑結果 32
（五）展著劑結果 32
六、耐鹽性試驗結果 32
七、溶磷試驗結果 33
（一）溶磷篩選試驗結果 33
（二）BL與BM對PR27、PR30溶磷試驗結果 33
（三）BL與BM對作物生長及其產量影響試驗結果 33
八、纖維降解試驗結果 34
（一）固態纖維素分解酵素活性測試結果 34
（二）分解纖維酵素活性分析-DNS還原糖試驗結果 34
（三）棕櫚泥降解試驗結果 35
（四）咖啡渣降解試驗結果 36
九、乾旱迫害存活率評估結果 36
肆、討論 37
伍、參考文獻 44
陸、圖表 51
柒、附錄 103

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