【摘要】：氫能是一種理想的替代能源,具有安全、低耗、廉價(jià)、綠色等優(yōu)點(diǎn)。木質(zhì)纖維素是生物煉制過(guò)程中重要的原料,其結(jié)構(gòu)穩(wěn)定,難以被有效轉(zhuǎn)化。木糖作為木質(zhì)纖維素水解后的重要成分,只有轉(zhuǎn)化為木酮糖后才能被微生物利用,而許多微生物不具有相關(guān)的酶類,使得木糖的轉(zhuǎn)化效率低。目前絕大多數(shù)已知的利用木糖發(fā)酵產(chǎn)氫細(xì)菌中,產(chǎn)氫能力不能達(dá)到生產(chǎn)需求。本研究針對(duì)以上問(wèn)題,以實(shí)驗(yàn)室富集的厭氧降解纖維素產(chǎn)甲烷菌系FSC為材料,通過(guò)厭氧亨蓋特技術(shù)分離純化了一株厭氧發(fā)酵木糖產(chǎn)氫細(xì)菌FSC-15,對(duì)其系統(tǒng)發(fā)育學(xué)地位、生理生化特征、產(chǎn)氫能力進(jìn)行了分析。通過(guò)末端代謝產(chǎn)物抑制實(shí)驗(yàn),研究了菌株對(duì)末端產(chǎn)物的耐受性;通過(guò)單因素實(shí)驗(yàn)和正交實(shí)驗(yàn)對(duì)其發(fā)酵木糖的最佳產(chǎn)氫條件進(jìn)行了優(yōu)化;解析了戊糖、己糖及混合糖對(duì)菌株生長(zhǎng)和產(chǎn)氫代謝的影響;評(píng)價(jià)了菌株FSC-15對(duì)纖維素厭氧降解產(chǎn)甲烷菌系FSC水解濾紙和水稻秸稈產(chǎn)甲烷的影響。研究結(jié)果如下:(1)菌株FSC-15的16S rRNA序列與Clostridium dioils DSM 5431T(AJ458418)和Clostridium beijerinckii NCIMB 8052T(CP000721)相似性都為99.6%,但菌株FSC-15的生理特性與Clostridium beijerinckii NCIMB 8052T(CP000721)更接近。最佳生長(zhǎng)溫度為35℃-40℃,最適生長(zhǎng)p H 7.5。菌株可以耐受初始濃度為1500 mg/L的乙酸,2000 mg/L的丁酸;當(dāng)培養(yǎng)基中乙醇初始濃度為400mg/L時(shí),菌體的生長(zhǎng)和產(chǎn)氫分別降低了57.4%和61.2%;頂空氫分壓為134.2 k Pa時(shí),菌體的生長(zhǎng)被抑制了19.8%。(2)單因素實(shí)驗(yàn)和正交實(shí)驗(yàn)結(jié)果表明,菌株FSC-15的最佳產(chǎn)氫條件為:YE 0.1 g/L,BBL 0.1g/L,NH4Cl 1g/L,Na HCO3 0.8 g/L,Fe3+75μmol/L,Ni2+5μmol/L,培養(yǎng)基初始p H 7.5,培養(yǎng)溫度40℃。(3)混合糖發(fā)酵體系中,不僅菌株的生長(zhǎng)延滯期顯著縮短,生長(zhǎng)速率加快,生物量顯著提高,并且氫氣產(chǎn)量更高。菌株FSC-15分別以木糖和葡萄糖作為底物時(shí),生長(zhǎng)速率沒(méi)有明顯差異,但是隨著發(fā)酵時(shí)間繼續(xù)延長(zhǎng),以木糖為底物的發(fā)酵體系中氫氣產(chǎn)量比其他發(fā)酵體系中高。菌株利用木糖發(fā)酵的轉(zhuǎn)化率為1.845 mol H2/mol木糖;以葡萄糖為底物進(jìn)行發(fā)酵菌株的產(chǎn)氫效率為1.179mol H2/mol葡萄糖。(4)添加2%的菌株FSC-15后,厭氧降解纖維素產(chǎn)甲烷菌系FSC產(chǎn)氫速度明顯加快,氫氣產(chǎn)量提高41.8%。以秸稈為發(fā)酵底物時(shí),加入菌株FSC-15的厭氧降解纖維素產(chǎn)甲烷菌系FSC產(chǎn)甲烷的速率顯著提高,甲烷產(chǎn)量提高1倍。FSC和添加菌株FSC-15后的共培養(yǎng)體系相比,濾紙和秸稈的總降解率沒(méi)有顯著差異。添加菌株FSC-15后,菌系FSC中細(xì)菌和古菌的種類和豐度都有一定的變化。
[Abstract]:Hydrogen energy is an ideal alternative energy, with the advantages of safety, low consumption, cheap, green and so on. Lignocellulose is an important raw material in the process of biological refining, and its structure is stable and difficult to be effectively transformed. Xylose, as an important component of lignocellulose hydrolysis, can only be used by microorganisms after conversion to xylose. However, many microbes do not have related enzymes, which makes the conversion efficiency of xylose low. At present, most of the known hydrogen producing bacteria using xylose fermenting can not meet the production demand. In order to solve the above problems, a strain of anaerobic fermentative xylose hydrogen producing bacteria (FSC-15,) was isolated and purified from methanogenic cellulose-producing bacteria (FSC), which was enriched in laboratory, and its phylogenetic status was studied by anaerobic Hangate technique. Physiological and biochemical characteristics and hydrogen production capacity were analyzed. The tolerance of the strain to the end product was studied by the end metabolite inhibition test, and the optimum hydrogen production conditions of xylose fermentation were optimized by single factor experiment and orthogonal experiment. The effects of pentose, hexose and mixed sugar on the growth and hydrogen production of the strain were analyzed, and the effects of strain FSC-15 on cellulose anaerobic degradation methanogenic strain FSC hydrolysate filter paper and rice straw methanogenesis were evaluated. The results were as follows: (1) the similarity of 16s rRNA sequence of strain FSC-15 with Clostridium dioils DSM 5431T (AJ458418) and Clostridium beijerinckii NCIMB 8052T (CP000721) was 99.6T, but the physiological characteristics of FSC-15 were closer to Clostridium beijerinckii NCIMB 8052T (CP000721). The optimum growth temperature is 35 鈩，

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