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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生命科學系所 > 依資料類型分類 > 碩博士論文 >  黑翅土白蟻腸道之微生物菌相解析及其固氮菌與木質纖維素分解菌之功能性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152373

標題: 黑翅土白蟻腸道之微生物菌相解析及其固氮菌與木質纖維素分解菌之功能性分析
Bacterial community analysis and isolation of nitrogen-fixing and lignocellulolytic bacteria from the termite gut of Odontotermes formosanus
作者: 楊佳慈
Yang, Jia Cih
Contributors: 黃介辰
Chieh-Chen Huang
生命科學系所
關鍵字: 生質燃料;黑翅土白蟻;微生物族群;木質纖維素分解菌固氮;共代謝
biofuel;lignocellulose;Odontotermes formosanus;microbial communities;lignocellulolytic bacteria;nitrogen fixation bacteria;cometabolism
日期: 2013
Issue Date: 2013-11-07 13:23:40 (UTC+8)
Publisher: 生命科學系所
摘要: 人類大量煉製石油作為能源替社會帶來快速的繁榮發展與便利,但燃燒石油的同時也增加大氣中二氧化碳等溫室氣體含量上升,造成地球暖化問題等環境問題已不容忽視,因此各國致力發展生質燃料(biofuel)以代替石油使用。由於第一代生質燃料存在與糧食作物重疊的缺點,第二代生質燃料則發展用稻草、蔗渣、莖桿、木材等廢棄物之木質纖維素(lignocellulose)作為煉製醇類來源,成為未來綠色能源的重要目標。白蟻(termite)能利用木頭、枯枝、落葉等木質纖維素作為營養來源,是地球碳循環中重要的分解者,因此是研究第二代生質燃料的良好生物模型。本研究選擇台灣地區常見之黑翅土白蟻(Odontotermes formosanus)工蟻作為研究材料,探討黑翅土白蟻的腸道微生物在分解植物木質纖維素以及缺乏氮源的營養條件下,如何提供白蟻生長所需要之碳源及氮源。實驗第一部份先分析黑翅土白蟻腸道之微生物菌相與優勢菌種的功能,使用非培養法(culture- independent methods),把擴增的黑翅土白蟻腸道微生物之16S rDNA,利用變性梯度膠體電泳(DGGE)技術將不同微生物的序列分離進行比對鑑定。依照微生物序列的相同度(identify)可將黑翅土白蟻的腸道微生物歸納成四個目(oder)分別為:Enterobacteriales, Bacteroidales, Clostridiales, Spirochaetales。為更一步探討腸道微生物的功能,因此實驗第二部分以培養法(culture-dependent methods)進行菌種篩選。分別設計天然性基質(芒果樹粉、狼尾草)以及人工性基質(carboxymethyl cellulose, xylan, pectin)四種添加不同碳源並且無氮源的選擇性培養基,注入黑翅土白蟻工蟻的腸道微生物進行培養分離。從不同培養基篩選之分離菌株鑑定結果有Enterobacteriales、Lactobacillales、Bacillales、Actinomycetales四種目之微生物。實驗第三部分則測試分離菌株分解木質纖維素的醣類水解酶活性,以及固氮酶基因nifH及氨氮(NH3+)釋出之能力的能力。結果顯示分離菌株Paenibacillus wclg4可分泌纖維素酶及半纖維素酶水解纖維素與半纖維素,而Klebsiella wccg12菌種在缺乏氮源的培養基中固定氮氣生長產生約41mg/L氨氮。非培養法與培養法的結果均可鑑定到腸桿菌目(Enterobacteriale)之Klebsiella sp.,推測白蟻在攝取木質纖維素缺乏氮源的營養條件下,Klebsiella sp.有固定氮源及發酵醣類產生醇類的功能,並且釋出氮源及醇類給予分解菌及其他微生物利用。而木質纖維素分解菌參與分解木質纖維素的過程,可釋出的醣類給予固氮菌等微生物利用。因此實驗第四部分以共培養分離菌株Klebsiella wccg12與Paenibacillus wclg4在添加人工纖維素且無氮源的培養基中測試兩者是否有共代謝(cometabolism)的合作關係。雖然實際微生物族群與環境及代謝作用複雜許多,從本研究共培養白蟻腸道微生物的方法,讓我們能探討微生物在白蟻的消化系統運中扮演的功能,未來或許可借鏡白蟻分解植物纖維的方式,發展更簡便有效率的生物反應器生產生質能源。
Fungus-growing termites (Order Isoptera, Family Termitidae, subfamily Macrotermitinae) have over 330 species belonging to 12 genera and have agricultural symbiosis with fungi Termitomyces in their subterranean nests. They are a group of higher termites, distributed throughout the Asian and African continents. Several hypotheses concerning the role of fungi Termitomyces has been proposed to have the ability to degrade lignin and thus providing the termite with nitrogen-rich food; but little is known about the lignocellulosic degradation potential of those fungus-growing termites’s gut microflora. Furthermore, the mutualistic relationship of termite gut bacteria, the host macrotermitine termite and Termitomyces have not yet been adequately addressed in the degradation of lignocellulose and their roles in the carbon and nitrogen metabolism. The aim of this study was to investigate the link between the functionality and the diversity of microbial communities of the termite gut by culture dependent and independent methods. The symbiotic bacteria in termite gut were detected by denaturing gradient gel electrophoresis (DGGE) in culture independent method. The representative phylotypes were affiliated to four orders, Enterobacteriales, Bacteroidales, Clostridiales, and Spirochaetales. And we isolated bacteria by repeated batch culture using the gut microflora in different carbon substrates (natural and artificial) and also nitrogen deficient medium. Majority of the bacterial community profiles using the batch cultures were monitored by molecular gene-targeted 16S rRNA analysis. From the results, clones from four orders belonging to Enterobacteriales, Lactobacillales, Bacillales, Actinomycetales were obtained. And a phylogenetic tree was constructed using the neighbor-joining method to study the distance between the different species. The Genus Klebsiella was found to be the dominant population by the culture dependent method (batch culturing) and culture independent method (DGGE). The role of the isolate Paenibacillus wclg4 was detected secrete cellulase and hemicellulase. From our study isolate Klebsiella wccg12 was detected to help nitrogen fixation, as the termite’s diet was nitrogen deficient and this isolate produced significant amounts of ammonia when grown in nitrogen deficient medium. Therefore, it was concluded that the macrotermitine termite gut microbiota played an important function in lignocellulosic degradation and nitrogen fixation.
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