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研究生:郭東霖
研究生(外文):KUO, DONG-LIN
論文名稱:磺胺類抗生素在水產養殖場環境中之 分解與其相關菌相分析
論文名稱(外文):Biodegradation of Sulfonamides in Aquaculture Environmental and It’s Related Metagenomic Analysis
指導教授:楊鉅文
指導教授(外文):Yang, CHU-WEN
口試委員:楊鉅文張碧芬劉秀美
口試委員(外文):Yang, CHU-WENCHANG, BEA-VENLIU, SHIU-MEI
口試日期:2017-07-14
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:2017
畢業學年度:105
語文別:中文
論文頁數:104
中文關鍵詞:磺胺類抗生素養殖池微生物降解生物反應器Illumina定序菌相分析
外文關鍵詞:Sulfonamidesaquaculture pondsmicrobial degradationbioreactorsIllumina sequencingbacterial community analysis
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台灣養殖漁業因採用密集式養殖法,易造成疾病散播而產生損失,磺胺類藥物常用來抑制魚病發生。磺胺類藥物容易殘留在水體與底泥中。傳統汙水處理難以清除,或是魚民未經處理直接排入水體,造成環境汙染。利用微生物方法將磺胺類抗生素去除為可行方式之一。另外,魚池中有機物質的累積容易造成養殖魚類致病菌的孳生。台南水產試驗所開發多營養階利用養殖系統,除了利用貝類、藻類兩淨化池,還添加光合菌的方式代謝池水中過多的有機物質,抑制魚病之發生。本研究目的探討台南水試所養殖池底泥對磺胺類藥物之降解,建立最佳條件。進行不同濃度、單獨與混合的磺胺甲噁唑(sulfamethoxazole,SMX)、磺胺二甲氧嘧啶(sulfadimethoxine,SDM)、磺胺二甲嘧啶(sulfamethazine,SMZ)降解,分離具降解能力之純菌、加回底泥進行生物反應器實驗。另外以Illumina定序分析多營階皆利用養殖魚池的菌相變化。
結果發現不同時期生態池底泥之批次實驗,為105年4月之生態池降解效果最佳,在好氧與厭氧的狀態下降解SMX。以105年4月之生態池底泥進行不同濃度單獨/混合磺胺批次實驗,結果發現SMX降解效果最好,且在濃度低時較容易降解完。其次為SDM,殘留率還有60%。SMZ最難降解,其濃度幾乎保持在90%以上。從分解SMX的底泥樣本菌相分析中可看到,可能為主要降解SMX之菌屬已知細菌菌屬為Methylophaga、Sulfurovum、Labrenzia。古菌菌屬為Methanococcoides、Methanolobus、Methanosarcina。
將底泥樣本連續稀釋塗抹至含有SMX之平板上進行篩菌,分離到具有降解能力的細菌Vibrio azureus、Vibrio alginolyticus、Marinobacter sp. GX5A52、Pseudomonas sp. AFJ01P。在尋找最佳條件測試後,發現好氧部分Vibrio alginolyticus的效果最好,厭氧部分則是Pseudomonas sp. AFJ01P生長速度較快,且降解速率與Marinobacter sp. GX5A52差不多,選用這兩株菌進行加回底泥的生物反應器實驗。生物反應器實驗共添加四次SMX,前2次添加的降解速率大致與前述純菌加回底泥之降解速率相符。之後隨著添加次數的增加,降解能力也會跟著提升。另外,好氧與厭氧控制組(不額外添加降解菌)皆有有促進降解的趨勢,反應器中SMX濃度快速下降,未來也會對控制組的反應器進行菌相分析,比對可能為具有降解能力之菌屬。
台南水試所試採樣本與民間養殖池部分,入水口水樣比例最高的已知菌綱前五名是Alphaproteobacteria 19.6%、Gammaproteobacteria 12.2%、Actinobacteria 8.9%、Cyanobacteria 5.3%及Flavobacteriia 4.8%。石斑養殖池水樣比例最高的已知菌綱前五名是Alphaproteobacteria 13.9%、Gammaproteobacteria 12.6%、Cyanobacteria 5.6%、Epsilonproteobacteria 3.5%及Flavobacteriia 3.2%。虱目魚養殖池水樣比例最高的已知菌綱前五名是Cyanobacteria 14.5%、Gammaproteobacteria 9.5%、Alphaproteobacteria 9.1%、Actinobacteria 6.8%及Spartobacteria 3.7%。整體大致分石斑養殖池水樣本為一大群,虱目魚養殖池水樣本為另一大群。其中藍綠菌屬GpIIa在水樣中的比例最高。虱目魚養殖池藍綠菌比例比石斑養殖池藍綠菌比例高。藻類以Bacillariophyta及Chlorophyta的比例較高。石斑養殖池中藻類比例比虱目魚養殖池藻類比例高很多。光合菌比例在兩種養殖池都很低。
分析與魚病相關的菌屬比例,在水樣本或底泥樣本,比例最高的菌屬都是Candidatus Pelagibacter及Vibrio。三個石斑養殖池魚病相關的菌屬比例較高(約7%-8%),虱目魚養殖池魚病相關的菌屬比例則多在2%。
本研究中,不同時期的樣本及降解方式,造成樣本間不同的降解結果。希望能提供養殖魚池被磺胺類汙染時之參考與評估。另外不同時期底泥之菌相變化,希望有助於養殖魚池之水質參考。

Aquaculture in Taiwan are using intensive farming.When diseases outbreak, it would result in direct economic losses. Therefore, sulfonamides are often used to avoid farmed aquatic animals from getting infected.Sulfonamides remain in water and sediment, and it is difficult to be removed by using traditional sewage treatment.If untreated water was discharged into river or ocean,it would cause environmental pollution.Bioremediation is an effective way to scavenge sulfonamides.In addition, the accumulation of organic matter in the fish pond is likely to lead to increasing of fish-borne pathogens.So Fisheries Research Institute,Council of Agriculture make a development of multi-nutrient comsuming of farming systems.In addition to two of purifying pond, they add photosynthetic bacteria in the fish pond to digest the organic matter. The purpose of this study is to investigate aerobic and anaerobic biodegradation of sulfonamides in different period of sediments from milkfish pond, as well as to establishing an optimum system. Degraded the different concentration of sulfamethoxazole(SMX) and different kinds of sulfonamides, sulfadimethoxine(SDM) and sulfamethazine(SMZ) alone or mixed.Isolate the bacteria which are able to degrade SMX.Second,mix bacterial suspension with sediment, then carried out the bioreactor experiment.Also, we attempted to use Illumina technology for profiling microbial communities in the multi-nutrient comsuming of farming systems.
The results showed that sediment from milkfish pond collect at 2016.4.13 has the best effect to degrade SMX at aerobic and anaerobic situation.Then we use this sediment to degrade the different concentration of sulfonamides alone or mixed. The results showed that alone and low concerntration SMX could be removed fast in the experiments, the remaining of SDM and SMZ were up to 60 % and 90%, respectively. In the microbial community analysis, the top three of bacteria that would be able to degrade sulfonamides are Methylophaga、Sulfurovum、Labrenzia.Top three of archaea are Methanococcoides、Methanolobus、Methanosarcina.Next, we do a series of dilution of sediment, and streak plates which contain SMX.We isolate Vibrio azureus、Vibrio alginolyticus、Marinobacter sp. GX5A52、Pseudomonas sp. AFJ01P that can degrade SMX.Then we mix bacterial suspension with sediment to find an optimum condition.Result showed that Vibrio alginolyticus is the most effective in aerobic and Pseudomonas sp. AFJ01P is the most effective in anaerobic,so we choose these bacteria to carry out bioreactor experiment. In experiments of bioreactor,there are four times of additions.Rate of degradation in first two additions are approximately as the same as before.With the increasing in the number of times addition, the ability of degradation was trend to enhanced. In the future, control group of the reactors will also conduct the microbial community analysis.
Compare with sample from Fisheries Research Institute,Council of Agriculture and folk,Microbial community analysis showed that top five of the highest proportion of known bacterial classes at the water intake are Alphaproteobacteria 19.6%、Gammaproteobacteria 12.2%、Actinobacteria 8.9%、Cyanobacteria 5.3%及Flavobacteriia 4.8%. Top five of the highest proportion of known bacterial classes at grouper fish pond are Alphaproteobacteria 13.9%、Gammaproteobacteria 12.6%、Cyanobacteria 5.6%、Epsilonproteobacteria 3.5%及Flavobacteriia 3.2%. Top five of the highest proportion of known bacterial classes at milkfish pond are Cyanobacteria 14.5%、Gammaproteobacteria 9.5%、Alphaproteobacteria 9.1%、Actinobacteria 6.8%及Spartobacteria 3.7%.Overall, we divid bacteria from grouper fish pond into a large group, and bacteria from milkfish pond into another one.GpIIa has the highest proportion of water samples among these ponds. The proportion of cyanobacteria in the milkfish pond is higher than the grouper pond. The proportion of algae to Bacillariophyta and Chlorophyta are higher. The proportion of algae in the grouacly pond is much higher than that in the milkfish. The proportion of photosynthetic bacteria is low in both ponds. Analysis of the proportion of bacteria associated with fish disease, in water samples or sediment samples, the highest proportion of bacteria are Candidatus Pelagibacter and Vibrio. Disease-associated bacterial in three of the grouper fish pond are higher than the others (About 7% -8%), and the proportion of disease-associated bacteria with the milkfish pond fish is 2%.
In this study, samples of different periods and degradation methods resulted in different degradation pattern between samples.It is hope that this research can provide reference and assessment when when water is contaminated with sulfonamides.Beside, bacterial community changes in different period of fish pond , hoping to contribute to the culture of fish.

目錄

目錄 3
中文摘要 7
英文摘要.......................................................................................................10
第一章 前言 ...................13
第一節 研究緣起 .....13
一、 養殖漁業...............................................................................13
二、 多營養階利用養殖系統.......................................................13
三、 病害防治...............................................................................15
四、 磺胺類抗生素.......................................................................15
第二節 研究目的…………………………………………….....…….16
第二章 材料與方法.....................................................................................17
第一節 實驗材料………………………………………….....….……17
第二節 儀器使用……………………………………….....….………20
第三節 實驗方法..................................................................................21
一、磺胺類抗生素實驗品管 21
二、實驗方法與步驟 22
第四節 分析方法.........………………………….……….....………...24
一、 磺胺類抗生素的分析………………..……….....…………24
二、 分子生物學分析方法…………………………….....…..…..25
第三章 結果……………………………………………….....…………..…28
第一節 養殖池菌相分析……………………………………........……28
第二節 實驗品管………………………................……….....…..…….33
第三節 樣本基本特性分析....…………………………….….........…...34
第四節 批次降解實驗…………………......................................…..….35
第五節 好氧純菌篩選、降解能力與鑑定……………..…..….............36
第六節 生物反應器試驗……………………..……....……….....….….38
第四章 討論..................................................39
第五章 結論..................................................42
第六章 參考文獻..............................................46
圖表.........................................................58

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