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研究生:郭榮哲
研究生(外文):Jung-CheKuo
論文名稱:微藻Picochlorum sp. S1b 抑制弧菌生長活性的研究
論文名稱(外文):Study on vibrio static activity of Picochlorum sp. strain S1b
指導教授:陳逸民陳逸民引用關係
指導教授(外文):Yi-Min Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:89
中文關鍵詞:綠水系統Picochlorum sp.紅細菌科哈維氏弧菌弧菌
外文關鍵詞:green water systemPicochlorum sp.RhodobacteraceaeVibrio harveyivibrio
相關次數:
  • 被引用被引用:1
  • 點閱點閱:425
  • 評分評分:
  • 下載下載:5
  • 收藏至我的研究室書目清單書目收藏:0
實驗室前人的研究指出,以微藻Picochlorum sp. S1b作為綠水系統的材料,添加在青斑魚吋苗養殖池的作法,可以明顯降低池水中弧菌的數量。弧菌數量的降低,推測和S1b產生抑菌物質、與弧菌競爭特定營養,抑或與一些具有抑制弧菌能力的細菌共存有關。為進一步的了解何者為真,我們選取兩株弧菌,Vibrio harveyi strain BCRC12907與V. campbellii strain BCRC12909,利用孔洞擴散法 (well diffusion method),分析S1b粗萃物 (45 mg/mL) 的抑菌能力,同時將弧菌分別接種到無菌與有菌 (開放式培養) 的S1b培養液 (弧菌及藻細胞的密度分別為3.35 x 103 CFU/mL 以及 2.00 x 106 ind/mL) ,共同培養48小時後,觀察弧菌數量的變化。結果顯示,S1b的粗萃物並不具有明顯的抑菌活性。弧菌在與無菌和有菌的S1b藻液共培養後,數量會出現明顯的變化,其中與前者共培養的弧菌數會增加到1.47 x106 CFU/mL,為初始接種量的500倍,然而與後者共培養的弧菌數則低至無法檢出 (〈 15 CFU/mL)。由此推測S1b抑制弧菌的活性來自其共存細菌,而非藻細胞的抗菌物質。
為了進一步的分析S1b藻液內可能含有哪些能抑制弧菌的共存細菌,我們將上述實驗中的有菌S1b藻液,利用marine agar進行菌株的培養與分離,隨後利用孔洞擴散法,分析活菌的抑制弧菌活性,以及利用16S rDNA的分析與NCBI資料庫的比對法鑑定菌種。由此,我們一共分離出80個菌株,有14株及11株具有明顯抑制V. harveyi strain BCRC12907及V. campbellii strain BCRC12909 (抑菌圈〉 2.0 mm) 的活性,其中有3株具有抑制兩種弧菌的能力。進一步利用與S1b藻液共培養後,添加弧菌的方法,確認這些菌株的抑制弧菌活性之後,發現strain 7、8、46、48、50及61等品系確實能快速降低弧菌的數量。具有抑制弧菌生長能力的菌種以紅細菌科 (Rhodobacteraceae)、黃桿菌科 (Flavobacteriaceae) 的菌為主,其抑制弧菌的機制有待進一步的探究。

In the laboratory previously research, we have found that applied a marine microalga Picochlorum sp. S1b in tanks of grouper (Epinephelus coioides) larvae which significantly reduce the number of vibrio density in the water. We speculated that S1b produce vibrio static ingredients, compete-specific nutrition or some bacteria symbiosis with S1b inhibit vibrio growth. We selected two species of vibrio, Vibrio harveyi strain BCRC12907 and V. campbellii strain BCRC12909 and evaluated the vibrio static activity of S1b crude extract (45 mg/mL), according to the inhibition zone formed on agar plates through well diffusion method. Then spiked vibrio (3.35 x 103 CFU/mL) into the axenic and non-axenic (opened culture) S1b, measurement the viability of vibrio, to understand whether the vibrio static activity from its symbiotic bacteria.
Results show that the S1b crude extract does not have significant inhibitory activity, then the vibrio viability in axenic and non-axenic S1b were significant differences for co-culture 48 hours, the former vibrio density increased to 1.47 x106 CFU/mL, 500 times of initial inoculum; however, the vibrio density of the latter, remained non-detectable (〈 15 CFU/mL). Thus the S1b inhibit the vibrio through its symbiotic bacteria, rather than microalga itself.
In order to further study the non-axenic S1b containing bacteria which can inhibit the vibrio, we isolated bacteria randomly from the non-axenic medium spread marine agar plate, followed by well diffusion method, analysis which strain has the ability to inhibit vibrio, as well as cloning the 16S rDNA sequence and alignment with database to identify the strain. As a result, a total 80 strains, respectively, 14 and 11 can inhibit the V. harveyi BCRC12907 and V. campbellii BCRC12909 growth (inhibition zone 〉 2.0mm), while 3 bacteria can inhibit both two species of vibrio. Further co-cultured with the axenic S1b, and spike the vibrio, confirm which strain process vibrio static activity. We found that indeed some strains of isolate reduce the density of vibrio (strains 7,8,46,48,50 and 61). Through the 16S rDNA alignment, the bacteria process vibrio static activity is roughly Rhodobacteraceae, Flavobacteriaceae, further we will search for related research and speculated the proper vibrio static activity mechanism of these lsolated.

中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
前言 1
1-1 弧菌的特性與分類地位 1
1-2 弧菌的重要性 2
1-3 養殖環境弧菌的控制 4
1-4 Picochlorum sp. strain S1b具有抑制弧菌的能力 11
1-5 研究策略與目的 12
材料與方法 13
2-1 藥品與儀器 13
2-2 藻株來源與保存 15
2-3 S1b細胞內含物的抑菌活性分析 15
2-4 S1b藻液的抑菌活性分析 17
2-5 S1b藻液菌相組成分析 21
2-6 菌種單離 22
2-7 來自S1b藻液菌株的弧菌抑制活性分析 23
2-8 菌種鑑定 24
實驗結果 26
3-1 S1b細胞內含物的抑菌活性分析 26
3-2 S1b藻液的抑菌活性分析 26
3-3 S1b藻液菌相組成分析 27
3-4 菌種單離與弧菌抑制活性分析 28
3-5 菌種鑑定 28
討論 31
總結 37
參考文獻 67
附錄 82
附錄一、弧菌生化特性檢索表 82
附錄二、培養基與試劑配方 83
附錄三、儀器與藥品 85
附錄四、自動迴旋接種設備計數方法 87
附錄五、PCR反應條件與引子組成 88
附錄六、已知具有抑制弧菌活性的菌種名稱與分類地位 89

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