https://scholars.lib.ntu.edu.tw/handle/123456789/78237
Title: | 高纖維飲食大鼠盲腸中纖維素分解菌之培養 Enrichment of cellulose-saccharifying bacteria communities from rat fed with high-fiber diet |
Authors: | 陳紀臻 Chen, Chi-Chen |
Keywords: | 纖維素產糖;優化培養;次世代定序;定量即時聚合?鏈鎖反應;腸道菌;cellulose;cellulosic saccharification;cecum;enrichment;real-time PCR;Next-generation sequencing | Issue Date: | 2016 | Abstract: | 近年來,纖維素被認為有潛力成為穩定的生質能源來源。科學家先利用酸或酵素水解纖維素成還原糖類,微生物再利用還原糖產生生質能源,如乙醇與氫氣。與酸水解纖維素相比,酵素水解雖效率較差,但所需要的環境較為溫和,運作成本也較低。為了增加生質能源產量,科學家積極尋找能高效率分解纖維素產糖的微生物。此篇論文的目的為從高纖維飼料餵養之大鼠盲腸中尋找高效率產糖的纖維素分解菌。大鼠盲腸內的微生物被優化培養在含有水溶性纖維素的培養基中,並以剛果紅染色法選擇能代謝水溶性纖維素的菌落,將其養成菌液後以二硝基水楊酸試劑測定菌落的產糖能力。其中單一菌落RCF (rat cecum colony F)的水溶性纖維素產糖能力最佳。全基因定序(whole-genome sequencing)的結果顯示此單一菌落內共含有九種菌株,主要為四株梭狀芽胞桿菌、大腸桿菌、瘤胃脫硫腸狀菌及產氣腸桿菌。且根據前人的研究,梭狀芽胞桿菌被推測為此菌落內主要的纖維素產糖者,而大腸桿菌、瘤胃脫硫腸狀菌及產氣腸桿菌則被視為只有耗糖的能力。同時,我們發現RCF分解晶體纖維素產糖的能力不佳。因此,為了增加RCF的晶體纖維素產糖效率,本研究針對RCF中的菌種特性採用了抗生素處理法、酒精處理法及高溫處理法,期待能增加菌液中梭狀芽胞桿菌的比例。革蘭氏陰性菌抗生素(萘啶酸)被用來抑制大腸桿菌、瘤胃脫硫腸狀菌及產氣腸桿菌等耗糖菌,而乙醇及高溫則被用來去除菌液中的非產孢子菌(梭狀芽胞桿菌及瘤胃脫硫腸狀菌之外的所有菌種)的比例,提高族群的纖維素產糖量。結果顯示抗生素能夠有效增加RCF的固態纖維素產糖能力。然而,次世代定序與定量即時聚合酶鏈鎖反應(real-time PCR)的結果卻顯示此抗生素減少了梭狀芽胞桿菌在菌液中的數量及比例,推翻了我們預計產糖量會與梭狀芽胞桿菌比例同時增加的假設。因此我們推測有三個可能:1. 萘啶酸改變了菌液中梭狀芽胞桿菌族群的組成比例,增加高產糖的菌株的比例,減少了高耗糖的菌株。2. RCF內有其他能分解纖維素產糖的菌種,而抗生素抑制了梭狀芽胞桿菌,減少了纖維素的競爭,使得其他纖維素分解菌增加。3. 原本預計未有纖維素產糖能力的大腸桿菌其實能夠分解纖維素,因此產糖量與大腸桿菌的比例同時增加。 Cellulose is one of the highly abundant organic compounds on earth, which may be one of the alternative energy resources. Cellulose can be chemically or biologically degraded into simple sugars, which can be used to generate bioenergy such as ethanol and hydrogen. Chemical hydrolysis of cellulose requires high energy input. Therefore, bio-degradation may be a feasible process. The objective of the study is to isolate and identify cellulose-saccharifying microorganisms from rat cecum fed with high-fiber diets. A Sprague Dawley (SD) rat was fed with high fiber diets for 6 months. Mandel-Reese (MR) media with sodium carboxymethyl cellulose (CMC) as the sole carbon source, was used to enrich cellulolytic microbes. Congo red overlay method was used to select the cellulose-degrading colonies on the isolation plates. The congo red-positive colonies were inoculated into MR media with CMC, and the reducing sugar were measured by 3,5-dinitrosalicylic acid (DNS) colorimetric method. Enrichment culture RCF (rat cecum colony F), the one with highest CMC-saccharifying rate, was selected for crystalline cellulose-saccharifying tests and further studies. The whole-genome sequencing result from the 30-day-incubation RCF culture showed a community of bacteria consisted with four Clostridium spp., Desulfotomaculum ruminis (D. ruminis), Escherichia coli (E. coli), Enterobacter aerogenes, Pseudoflavonifractor capillosus (P. capillosus) and Cellulomonas cellasea. In addition, time-coursed reducing sugar concentrations from α-cellulose indicated that microbial community RCF consumed more reducing sugars than it produced within 8 days of incubation. According to previous studies, Clostridium spp. were most likely to be cellulolytic and D. ruminis, E. coli and Enterobacter aerogenes were speculated to be non-cellulolytic in this microbial community. In order to enhance the sugar yields from crystalline cellulose, α-cellulose, three treatments based on the characteristics of community were conducted: antibiotic, heat and alcohol treatments. Nalidixic acid, an antibiotic against gram-negative bacteria, was used to inhibit D. ruminis, E. coli and Enterobacter aerogenes. Heat and alcohol were applied to favor spore formers, Clostridium spp. and D. ruminis. Species- distribution with time was analyzed using real-time PCR and 16S ribosome RNA gene amplicon sequencing. Amongst these three treatments, antibiotic enhanced the sugar yield successfully. However, real-time PCR and 16S rRNA gene amplicon sequencing results showed that both the microbial populations and fractions of Clostridum spp. were reduced substantially by antibiotic treatment. Three possible reasons for the sugar yield enhancement after antibiotic treatment were: 1. Antibiotic changed the Clostridium population structure into more cellulose-saccharifying community. 2. Antibiotic treatment provided the competitive advantage of minor microorganism having high cellulose-saccharifying ability. 3. The predicted non-cellulolytic E. coli could perform cellulosic saccharification. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/277240 | DOI: | 10.6342/NTU201600223 | Rights: | 論文公開時間: 2018/6/11 論文使用權限: 同意有償授權(權利金給回饋學校) |
Appears in Collections: | 環境工程學研究所 |
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ntu-105-R02541127-1.pdf | 23.54 kB | Adobe PDF | View/Open |
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