【作者】 谭秀花；

【导师】 余晓斌；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 硕士

【摘要】 随着石化资源日益耗竭,各国开始重视可再生资源的利用。丁醇是重要的有机溶剂和化工原料,作为生物燃料,性能优于乙醇。丁醇发酵中,原料成本占总成本的比例高达60%～70%,并且由于丁醇对菌体的毒害作用,一般总溶剂产量只有18～20g/L,溶剂得率为0.34g/g。溶剂产量低、生产成本高,使发酵法无法与石油化工竞争。本文通过丁醇浸泡及N+束注入诱变筛选丁醇耐受性强、溶剂产量高的菌种,并用小麦淀粉废水与木薯等廉价原料代替玉米,降低生产成本,提高生物丁醇的竞争力。通过丁醇浸泡处理,筛得高耐受性菌株BR30-2,丁醇产量达11.77g/L,比出发菌提高16.65%。再经N+束注入诱变,筛得高产菌BH-9,丁醇与总溶剂产量分别达14.51g/L23.14g/L,较出发菌提高43.81%、38.28%,BH-9稳定性良好。研究了电子转移介体、有机酸盐、pH缓冲剂、还原剂以及震荡和通气对丁醇发酵的影响。刃天青低于0.50%o时可用作筛选高还原力菌株的指示剂。电子转移介体中,中性红显著提高丁醇比例；亚甲基蓝调控作用不明显；甲苯胺蓝抑制作用较强。有机酸盐均可提高溶剂产量,添加0.30%乙酸钠或0.10%丁酸钠时,丁醇产量分别达16.20g/L、16.59g/L。pH缓冲剂中,K2HPO4可以促进溶剂生成,浓度为0.40%时丁醇产量达16g/L；CaCO3、两种缓冲盐(Na2HPO4:KH2PO4=1:1)不利于溶剂生产。还原剂Na2S、L-半胱氨酸盐酸盐、Vc均不利于丁醇发酵。间歇震荡对溶剂产量影响不大；不同发酵时期通气对发酵影响效果不同；相同发酵时期,通气时间越长,氧气对菌体抑制作用越明显。小麦淀粉废水中添加木薯进行丁醇发酵试验,通过单因子试验和正交试验确定的优化培养基配方为：木薯粉5%,蛋白A 3%,丁酸钠0.10%, K2HPO40.10%。最适培养条件为：温度37℃,初始pH7,种龄24h,接种量3%,装液量110mL/250mL,发酵周期80h。丁醇与总溶剂产量达19.32g/L、32.34g/L,溶剂产量与溶剂得率分别比玉米丁醇提高61.70%、21.91%,生产成本大大降低。更多还原

【Abstract】 With the depletion of fossil resources, people paid attention to using renewable resources. Butanol is an important organic solvent and chemical material. As a promising new biofuel, butanol is better than ethanol. However, raw materials price almost accounts for 60%～70% of the total production costs. As butanol is toxic to the bacteria, solvent and productivity are only 18～20g/L and 0.34g/g respectively. When competing with chemical synthesis, fermentation had no advantage because of low solvent yield and high production cost. In this paper, high butanol tolerance and butanol-yielding strains were bred after high concentration of butanol soaking and N+ beam implantation. Using wheat starch wastewater and cassava instead of corn as raw materials for butanol fermentation, production costs were reduced, competitivity was improved accordingly.A high butanol tolerance strain BR30-2 was bred after high concentration butanol soaking, and the output of butanol reached 11.77g/L, increasing by 16.65% compared with the original strain. Using BR30-2 as original strain for N+ beam implantation, a higher butanol-yielding strain BH-9 was bred, of which butanol and total solvent were up to 14.51g/L and 23.14g/L, increasing by 43.81% and 38.28% respectively compared with BR30-2. And BH-9 had a good stability.Effects of electron transfer mediator, acetate, butyrate, pH buffers, reducing agents, agitation and ventilation on butanol fermentation were studied. Resazurin (<0.50%o) could be used as a indicator for breeding high reducing strains. Neutral red increased the ratio of butanol significantly; methylene blue had little effect on solvent production while toluidine blue inhibited growth strongly. Acetate and butyrate increased solvents. When added 0.30% sodium acetate or 0.10% sodium butyrate, butanol reached 16.20g/L and 16.59g/L respectively. K2HPO4 stimulated the formation of solvent, and butanol reached 16g/L when its concentration was 0.40%; CaCO3, two buffer salts (Na2HPO4:KH2PO4=1:1) were not conducive to butanol fermentation. Na2S, Vc and L-cysteine hydrochloride as reducing agents were not good for solvent production either. Intermittent agitation had little effect on solvent; Ventilation had different effects in different fermentation periods. During the same period, the inhibition effect from oxygen was increased while extending ventilation time.Using wheat starch wastewater and cassava as raw materials for butanol fermentation, the optimal fermentation medium and conditions were gotten through uni-factor test and orthogonal design. The optimal medium compositions were:cassava power 5%, protein A 3%, butyric acid sodium 0.10% and K2HPO4 0.10%. The optimal culture conditions were: temperature 37℃, initial pH7, seed age 24h, inoculum 3%, medium volume 110mL/250mL, fermentation time 80h. Under the optimal conditions, butanol and total solvent reached 19.32g/L and 32.34g/L respectively, solvent and productivity increased 61.70% and 21.91% compared with corn, and production costs were decreased remarkably.更多还原