【作者】 何景昌；

【导师】 裘娟萍；

【作者基本信息】 浙江工业大学 ， 微生物学， 2009， 硕士

【摘要】 本文改进获得了一种快速有效测定丙酮、丁醇和乙醇产量的方法,其测定条件为:采用AT-FFAP毛细管柱（30 m×0.25 mm×0.33μm）;载气流速:N₂ 35 mL/min,H₂ 35 mL/min,空气350 mL/min;柱温:180℃;进样口温度:180℃;检测器温度:200℃,进样量:1μL。在此条件下,每个样品测定时间5 min。通过多次对丙酮丁醇梭菌微波、离子束和紫外诱变,选育出一株丙酮丁醇高产菌株,命名为B513,该菌株ABE总溶剂能够达到18.27g/L,其中丁醇产量为11.43g╱L,溶剂产量比出发菌株提高了100.77%。本试验从渗透压稳定剂、蔗糖浓度、酶浓度、酶解时间等方面研究了丙酮丁醇梭菌原生质体制备和再生条件。原生质体制备和再生的最适宜条件为:0.3 M蔗糖作为稳定剂,菌体培养16-18 h,溶菌酶浓度1 mg/mL,酶解温度37℃,酶解40 min。在此条件下,原生质体的制备率和再生率分别达到70%和99%。采用紫外线灭活高产菌株B513、热灭活发酵时间短的菌株D01后,双亲融合获得80株融合子,但融合子ABE产量均较低,最高溶剂产量10.31 g/L,发酵时间也没有缩短。另外,研究了发酵温度、热处理时间、接种量、发酵液pH、振荡、发酵时间对丙酮丁醇发酵的影响,确定了最佳发酵条件:发酵温度38℃,菌悬液沸水中热处理1.5 min,接种量3%,玉米醪保持原始pH,发酵液出现醪盖时,每隔10 h摇匀,发酵70 h。在此条件下,经多次发酵,ABE的产量能够达到18 g/L。通过正交试验对细胞固定化条件研究,得出最佳固定化条件为:PVA,SA,氯化钙浓度分别为9%,1.1%,4%,固定化4 h,固定化细胞发酵溶剂产量达到10.82 g/L。固定化细胞耦合抽提发酵研究表明补料分批发酵首次加料最适宜时间范围为70-80 h。作者试图通过采用ptb基因敲除的方法提高发酵主要产物丁醇的产量,但试验结果表明PCR扩增的含Kan抗性基因片段不能成功转化丙酮丁醇梭菌。不同pH对丙酮丁醇代谢途径中酶的活性影响结果表明,较低pH并不能完全诱导丁醇合成酶的产生,因此认为采用基因敲除技术切断丙酮丁醇梭菌代谢途径中的丁酸分支代谢途径,不利于丁醇的产生,导致不能产生溶剂。更多还原

【Abstract】 A rapid and efficient method of detecting acetone,butanol and ethanol was acquired in this paper.Its detecting conditions were showed as below: AT-FFAP capillary column（30 m×0.25 mm×0.33μm）;carrier gas flow rate: N₂ 35 mL/min,H₂ 35 mL/min,air 350 mL/min;column temperature:180℃;injection temperature:180℃;detector temperature:200℃,injection volume:1μL.Under these conditions,detecting time needs 5 min.A high acetone and butanol producing strains,named B513,was obtained through repeated mutation of microwave,UV and ion beam. ABE yield reached 18.27 g/L,in which butanol concentration arrived 11.43 g/L.The production of ABE was increased 100.77%in comparison with that of the parent strain.Protoplast preparation and regeneration conditions were studied on the osmotic pressure stabilizer,sucrose concentration,enzyme concentration, enzyme hydrolysis time.The most appropriate conditions were as follow: sucrose,as a stabilizer,concentration was 0.3 M;cell culture time was 16-18 h;lysozyme concentration was 1 mg/mL;enzymatic hydrolysis temperature was 37℃;enzymatic hydrolysis time was 40 min.Under these conditions,the rates of protoplast preparation and regeneration were 70% and 99%respectively.Strains B513,D01 were inactivated by UV and heat treatment separately.80 fusants were gained by biparental inactivation. ABE yield was lower and maximum solvent production was 10.31 g/L. There was also not shortening on fermentation time.In addition,ABE fermentation conditions were studied on fermentation temperature,heat treatment time,inoculation amount, fermentation broth pH,oscillations,and fermentation time.The optimum fermentation conditions were as follow:fermentation temperature was 38℃;heat treatment time was 1.5 min in boiling water;inoculation amount reached 3%;corn mash maintained the original pH;every 10 h shaked once when mash cap appeared;fermentation time was 70 h.Under these conditions,ABE yield can reach 18 g/L.Research on cell immobilization using orthogonal test has obtained the best immobilization conditions.The concentrations of PVA,SA,CaCl₂ were 9%,1.1%,4%,individually;immobilized time was 4 h;solvent yield reached 10.82 g/L.Study on immobilized cell fermentation coupled extraction shows that 70-80 h was the first suitable feed time in fed-batch fermentation.Author tried to enhance butanol production through knocking-out ptb gene,but results showed that the PCR amplified fragment containing Kan gene couldn’t be successfully transformed into Clostridium acetobutylicum. The result of enzyme activity in ABE metabolic pathway influenced by different pH showed that butanol synthesis enzymes can not be completely induced by low pH.So cutting off butyrate branch metabolic pathway of ABE fermentation through gene knockout technology is unfavorable to butanol production,which could not produce solvent.更多还原