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研究生:林宥甯
研究生(外文):LIN, YOU-NING
論文名稱:光合菌Marichromatium purpuratum和Rhodovulum sulfidophilum的使用對虱目魚養殖的影響
論文名稱(外文):The effects of phototrophic bacteria Marichromatium purpuratum and Rhodovulum sulfidophilum supplementation to the Chanos chanos milkfish aquaculture
指導教授:應靜雯
指導教授(外文):YING, JHING-WEN
口試委員:趙維良葉信利應靜雯
口試委員(外文):CHAO, WEI-LIANGYEH, SHINN-LIHYING, JHING-WEN
口試日期:2019-12-19
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:123
中文關鍵詞:光合菌虱目魚
外文關鍵詞:Marichromatium purpuratumRhodovulum sulfidophilumChanos chanos
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在不使用抗生素的前提下,為了使養殖物能夠預防疾病,並增加產值,水產益生菌的開發與使用越來越受重視。在本篇研究中,我們自製水槽並養殖虱目魚(Chanos chanos),探討紫硫菌Marichromatium purpuratum和紫非硫菌Rhodovulum sulfidophilum兩株光合菌在個別以及混合施用的情況下,對養殖環境和虱目魚帶來什麼樣的效益。從水和底泥的物化性質檢測結果看到加入光合菌能夠協助穩定水中的總氮變化;加入M. purpuratum組別的硫氧化菌增加,同時也增加了硫酸根離子的含量;加入R. sulfidophilum能夠降低水中的COD,但會使養殖環境中的銨氮增加;加入混和光合菌能夠協助降低環境中的銨氮和COD的含量,並維持水中總磷的含量。抗藥基因的偵測結果顯示加入光合菌能夠降低水中磺胺類抗藥基因(sul1、sul2)的存在量,M. purpuratum能有效降低sul1的發生率,混合施用兩種光合菌能更加快速降低sul2的發生率。
虱目魚在養殖八週後,對照組沒有虱目魚死亡,加入單一種光合菌處理的虱目魚大量的死亡,死亡率分別是33% (M. purpuratum)和29% (R. sulfidophilum),混合光合菌處理的虱目魚有10%的死亡率。R. sulfidophilum和混合光合菌處理的組別,虱目魚在長度和重量方面都有較好的生長。從菌相分析中看到加入M. purpuratum使水中的優勢菌門從Proteobacteria變成Actinobacteria,虱目魚腸道中則變成有95%的Pseudomonas,並增加了水和底泥中病原菌的存在量;加入R. sulfidophilum的水中菌相從PCoA分析中看到在養殖期間有很不穩定的變動,虱目魚腸道中也同樣的看到Pseudomonas佔有不小的比例(12%),也增加了弧菌的多樣性;加入混合光合菌的養殖水從PCoA分析中看到菌相變動穩定,而虱目魚腸道的菌相介於對照組和加入R. sulfidophilum的組別之間,暗示腸道菌相對虱目魚的存活與生長有很大的關聯。進行了腸道細菌的變化與虱目魚生長的相關性分析,發現Bacillus的存在量和Lactobacillus的比例對虱目魚的存活率有著正相關(Pearson correlation, r> 0.9),而部分的弧菌序列對魚的長度、重量也有正向的影響(Pearson correlation, r> 0.75)。整體而言,加入任一種光合菌都能夠改善養殖環境、減少抗藥基因發生率,而混合施用相對能夠減少魚的死亡率,更能夠穩定環境和魚腸道菌相,並促進虱目魚的生長。

Without the use of antibiotics, the development and use of probiotics has received increasing attention in order to prevent cultures from disease and increase production value. In this study, we set up four water tanks and cultivated milkfish (Chanos chanos) in these tanks. We supplied Marichromatium purpuratum for one tank, Rhodovulum sulfidophilum for another, mixture of two above bacteria for another, and the last one as control. Investigated the effectiveness of the aquaculture environment and the milkfish with added these phototrophic bacteria. We measured the physicochemical properties of the water and sediments in the tanks, received that phototrophic bacteria can balance the change of total nitrogen in water, M. purpuratum increase sulfur-oxidizing bacteria (SOB) and sulfate, R. sulfidophilum decrease chemical oxygen demand (COD) but increase ammonia nitrogen, admixture of two bacteria can decrease COD and maintain the content of total phosphorus. Furthermore, these experimental groups could decrease the presence of sulfonamide resistance genes (sul1 and sul2).
After eight weeks feeding, there was no deadly fish in control, but experimental groups had raised the mortality rate to 33% (M. purpuratum)、29% (R. sulfidophilum),and 10% (mixture of two bacteria), respectively. However, the participation of R. sulfidophilum and mixture of two bacteria increased the length and weight of the fish. The analysis of bacteria community structure indicated admixture can stable the flora in the aquaculture environment and the fish gut. Also, we found the amount of Bacillus and the percentage of Lactobacillus (Pearson correlation, r> 0.9) in fish gut have the positive correlation to survival rate. In addition, some Vibrio sequences quantity in fish gut had the positive correlation to the length and weight (Pearson correlation, r> 0.75). Overall, mixture of the phototrophic bacteria used in this study could reduce the mortality rate than other experimental groups, more stabilize to the community structure of bacteria, and promote the growth of fish.

摘要 1
Abstract 3
壹、前言 5
一、全球漁業現況 5
二、台灣漁業現況 5
三、虱目魚(Chanos chanos) 6
四、水產益生菌 6
五、氮循環對養殖環境的影響 7
六、硫循環對養殖環境的影響 8
七、磺胺類抗生素的使用對養殖池的影響 8
貳、研究目的 10
參、實驗架構 11
肆、材料方法 12
一、養殖池的設置與養殖時程 12
二、樣本採集和前處理 13
1.樣本採集 13
2.前處理 13
三、底泥pH值測定 14
四、抗生素 14
1.Sulfamethoxazole (SMX) 14
2.Cyclohexmide 14
五、 培養基 15
1.Marine Broth (MB) 15
2.Tryptic Soy Broth (TSB) 15
3.Thiosulfate-Citrate-Bile salts-Sucrose (TCBS) agar 16
六、總活菌數計數 16
七、弧菌計數 17
八、抗磺胺類藥物抗藥菌計數 17
九、總菌DNA萃取 18
1.水樣總菌DNA萃取 18
2.底泥總菌DNA萃取 18
3.魚腸道總菌DNA萃取 18
十、偵測16S rDNA、抗藥基因和弧菌專一性基因 19
1.聚合酶連鎖反應(Polymerase chain reaction, PCR) 19
2.即時聚合酶連鎖反應(Real-Time Quantitative PCR, qPCR) 20
十一、菌群結構分析 20
1.總菌相分析 20
2.弧菌相分析 21
十二、相關性分析 21
伍、實驗結果 22
一、水和底泥的物化性質檢測 22
1.水質檢測 22
2.底泥檢測 23
二、環境樣本中的微生物數量 24
1.總活菌數 24
2.水中的總弧菌數 25
3.虱目魚腸道的弧菌比例 26
三、菌相結構分析 26
1.定序結果概覽 26
2.菌相組成 28
3.參與氮循環的菌屬推測和分析 31
4.參與硫循環的菌屬推測和分析 33
5.潛在病原菌發生率 33
6.虱目魚腸道中益生菌的發生率 34
四、虱目魚生長情形 35
五、弧菌結構分析 36
1.定序結果概覽 36
2.弧菌組成 37
六、磺胺類抗藥菌比例以及基因檢測 38
1.水中磺胺類抗藥基因的檢測 38
2.虱目魚腸道中磺胺類抗藥菌的比例 39
3.虱目魚腸道磺胺類抗藥基因的檢測 39
七、弧菌檢測 40
1.水中弧菌的檢測 40
2.虱目魚腸道中弧菌的檢測 40
八、相關性分析結果 41
陸、討論 42
一、光合菌對養殖環境物化性質的影響 43
二、光合菌對養殖環境菌群的影響 44
三、光合菌對養殖環境弧菌的影響 45
四、光合菌對養殖環境中潛在病原菌的影響 46
五、光合菌對養殖環境中磺胺類抗藥基因發生率的影響 46
六、腸道菌相對虱目魚的影響 47
七、腸道弧菌對虱目魚的影響 48
八、腸道益生菌對虱目魚的影響 49
九、虱目魚腸道中的抗藥基因發生率 50
柒、參考文獻 53
捌、結果圖表 60
玖、附錄 102
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