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研究生:許銘元
研究生(外文):Hsu, Ming-Yuan
論文名稱:好氧異營的硝化與脫氮細菌之特性研究
論文名稱(外文):Characterization of aerobic heterotrophic nitrification and denitrification bacteria
指導教授:許世宜
指導教授(外文):Sheu, Shih-Yi
口試委員:許世宜、陳文明、張淑貞
口試委員(外文):Sheu, Shih-Yi、Chen, Wen-Ming、Chang, Shu-Chen
口試日期:2017-07-26
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:海洋生物技術研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:111
中文關鍵詞:硝化菌脫氮菌
外文關鍵詞:nitrification bacteriadenitrification bacteria
相關次數:
  • 被引用被引用:2
  • 點閱點閱:513
  • 評分評分:
  • 下載下載:22
  • 收藏至我的研究室書目清單書目收藏:0
隨著全球人口的增長,糧食的需求量也隨之增加,使得養殖業走向高密度養殖方式。然而高密度的養殖方式容易因魚塭中含有殘餘飼料、生物屍體及排泄物,而形成銨氮、亞硝酸鹽氮及其他有機污染物。因此養殖池中的含氮廢物上升,導致養殖生物大量死亡;所以希望能以微生物法進行硝化-脫氮作用以改善水質達到永續利用。
本實驗的菌株來自於台灣水域環境中的樣品,初步利用BTB快速篩選法與API 20NE快速篩選法,得到推測具有硝酸還原能力的菌株73株。進而以硝酸還原能力試管法測定確認其功能,以獲得具有將硝酸還原成氮氣的菌株,篩選出分別為Vibrio alginolyticus、Marinobacter hydrocarbonoclasticus、Pseudomonas balearica、Paracoccus mangrovi、Rhizobium ipomoeae、Rhizobium selenitireducens、Piscinibacterium candidicorallinum、Shewanella algae、Massilia flava、Gemmobacter lanyuensis、Vogesella amnigena等24株脫氮細菌。
進一步再利用硝化作用能力檢測法,從中挑選出同時擁有硝化-脫氮能力強的菌株,包含海水菌Marinobacter hydrocarbonoclasticus YWPs-2、Marinobacter hydrocarbonoclasticus YWPs-6、Pseudomonas balearica YWPs-11、Pseudomonas balearica YWPs-18和淡水菌Massilia flava YBJ-26及Vogesella amnigena Npb-02,以利於提高水質修復應用效率。
此外,經由菌株shin9-1、DQS-4、R1之基因體研究,探討全基因序列的硝化-脫氮作用基因圖譜與其代謝途徑的關聯性,以提供水質修復應用的理論基礎;期待未來能開發有效配合水質修復工程的應用策略。
With the growth in the global population and the demand for food will increase in the world, the high-density farming of aquaculture industry is obliged to perform. However, high-density farming method results in the accumulation of ammonia nitrogen, nitrite nitrogen and other organic pollutants by the residual feed, corpses and excreta of culture pond. A high level accumulation of nitrogen-containing waste in culture pond results in a large number of Aquatic organisms dead. The hope is the microbial utilization method such as nitrification-denitrification to improve water quality to achieve sustainable use.
In this study, bacterial strains are isolated and purified from water sample in the Taiwan waters. Seventy-three strains with the potential of denitrification are obtained by the rapid screening methods including the BTB and API 20NE screening methods. These strains are further confirmed by determination of nitrate reduction tube test to obtain bacteria with the ability to reduce of nitrate to nitrogen. There are 24 strains possess the ability and they are considered as Vibrio alginolyticus, Marinobacter hydrocarbonoclasticus, Pseudomonas balearica, Paracoccus mangrovi, Rhizobium ipomoeae, Rhizobium selenitireducens, Piscinibacterium candidicorallinum, Shewanella algae, Massilia flava, Gemmobacter lanyuensis and Vogesella amnigena etc. Furthermore, in order to improve efficiency of water remediation, these 24 strains are subjected to screening of simultaneous possessing nitrification-denitrification ability by examination of nitrification ability. Among them, seawater bacteria such as Marinobacter hydrocarbonoclasticus YWPs-2, Marinobacter hydrocarbonoclasticus YWPs-6, Pseudomonas balearica YWPs-11, Pseudomonas balearica YWPs-18 and freshwater bacteria such as Massilia flava YBJ-26 and Vogesella amnigena Npb-02 are able to simultaneously perform high efficiency of nitrification and denitrification.
In addition, investigation on the relationship between the nitrification-denitrification gene map of the whole genome and its metabolic pathway through genomic research of the strains shin9-1, DQS-4 and R1 may provide the theoretical basis for application of water remediation. It is expected that the strategy of effective application in water remediation engineering will be developed in the future.
致謝
摘要 I
Abstract III
目錄 V
表目錄 VII
圖目錄 IX
第壹章、緒論 1
一、前言 1
二、研究目的 4
第貳章、文獻回顧 5
一、含氮汙染來源 5
二、氮的危害 6
三、生物脫氮法-硝化脫氮作用機制 7
四、硝化細菌與脫氮細菌的研究發展 8
五、參與脫氮作用之相關酵素 9
六、篩選脫氮菌方法 12
七、硝酸還原測試 16
八、DNA定序與基因體研究應用 96
第參章、材料與方法 18
一、實驗架構與流程 18
二、樣品採集及來源 19
三、培養基 20
四、BTB快速篩選法之菌株分離 22
五、菌株保存 23
六、硝酸還原能力試管法測定 25
七、16S RNA基因序列分析與比對 26
八、演化樹建構 30
九、API 20NE套組篩選法 30
十、硝化作用能力測試 31
十一、菌株之全基因定序分析 39
第肆章、結果與討論 42
第一部分 脫氮能力菌株篩選 42
I. BTB快速篩選法 42
II. API 20NE快速篩選法 52
第二部分 硝化-脫氮能力菌株篩選 58
第三部分 菌株shin9-1、DQS-4及R1全基因定序分析 67
第伍章、結論 79
第陸章、參考文獻 80
附錄 86
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