【作者】 李鑫；

【导师】 史仲平；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 博士

【摘要】 丁醇是重要的平台化合物，也是一种高效的清洁液态燃料。利用产溶剂梭菌（Clostridia）和生物质原料发酵生产丁醇受到人们越来越多的关注。在使用梭菌发酵生产溶剂过程中，溶剂产物丁醇、丙酮和乙醇的比例约为6:3:1（w/w）。在保证总溶剂生产效率不变或者提高的前提下，尽量提升主产品—丁醇占总溶剂的比例、或者丁醇/丙酮比，一直是研究者所追求的目标。另外，玉米、小麦等农产品受到种植条件的制约，使用上述生物质生产生物丁醇影响到了人类食物的供给安全。因此，亟需寻找廉价、对种植条件要求低的高产非粮原料作为替代原料生产生物丁醇。针对上述问题，本论文以丙酮丁醇梭菌Clostridium acetobutylicumATCC824为实验菌株，以发酵途径关键酶基因转录水平测定和代谢通量计算为分析手段，以玉米和木薯为原料，在7L厌氧发酵罐下，开展丁醇发酵研究。论文提出了丁醇发酵的有效优化调控策略，并对使用不同原料下的发酵性能进行了比对研究，旨在提高丁醇/丙酮比、实现非粮木薯原料对传统玉米原料的有效替代、改善丁醇发酵的整体性能。主要结果总结如下：（1）研究和比较了以木薯和玉米为原料、传统和原位萃取发酵操作模式下的丁醇发酵性能。结果表明，木薯原料发酵的相转型（产酸相→产溶剂相）严重滞后、丁醇产量和生产效率低下，整体发酵性能无法达到玉米原料发酵的相应水平。通过一系列试行发酵实验最终发现，在以木薯为原料、油醇为原位萃取剂的萃取发酵中，发酵产气显著减弱后适时添加2.5g·L-broth–1酵母浸粉，发酵产气迅速回升、发酵相转型可在数小时内顺利完成，最终丁醇总产量达到35.38g·L-broth–1。（2）适时添加酵母浸粉（2.5g·L-broth–1）的优化调控策略，适用于以木薯为原料的多种丁醇发酵操作模式，可解除发酵相转型延滞、促进相转型的发生和完成。丁醇总产量分别达到13.61g·L–1（传统）、34.37g·L–1（油醇萃取）和18.37g·L–1（生物柴油萃取）。更加重要的是，以木薯为原料、辅以适时酵母浸粉添加的优化调控策略，可以大幅度提高丁醇发酵的丁醇/丙酮比。其中，油醇萃取发酵的丁醇/丙酮比可接近3.0:1，相比玉米原料、相同发酵操作模式下的水平提高了61.5%，整体发酵性能的改善更加明显。（3）分析测定了木薯原料丁醇发酵中，代谢途径的关键酶编码基因转录水平，发酵液中游离氨基酸的浓度及其变化。结果表明：适时添加酵母浸粉后，催化有机酸（乙酸、丁酸）吸收的辅酶A转移酶得到激活，其编码基因ctfAB的转录水平提高了约15倍；同时，菌体开始大量合成有利于丁醇生产的组氨酸族和天冬氨酸族氨基酸。两者共同作用，促进了木薯原料发酵的相转型、极大地提高了丁醇产量和生产效率。（4）在以木薯和玉米为原料进行发酵的条件下，利用代谢通量计算的手段、对碳代谢和还原力再生的最适匹配关系进行了分析，并对代谢途径中关键酶基因转录水平/中间产物进行了测定。结果表明：在木薯原料发酵产溶剂期，丁酸激酶基因buk和辅酶A转移酶基因ctfAB转录水平低、丁酸生成/吸收再利用闭环的代谢强度低；催化丁醇合成最后两步反应的NADH依存型关键酶、丁醛脱氢酶和丁醇脱氢酶的基因adhE和bdhB转录水平也低；但是，NADH再生速率水平高。高NADH再生速率和低丁酸闭环代谢强度共同作用，最终导致木薯丁醇发酵丁醇/丙酮比大幅提高。（5）分别以葡萄糖和酵母浸粉为碳氮源，探讨了不同碳氮配比对丁醇发酵相转型和丁醇/丙酮比的影响。结果发现，产酸期的碳氮比要控制在适中（46.7~93.4mol·mol–1）水平，此时，菌体能够正常生长、有机酸不过量生成，相转型可以顺利完成；产溶剂期的碳氮比保持在较高水平（≥93.4mol·mol–1）时，丁醇/丙酮比可达到3.0:1以上。以木薯为原料、在发酵转型期适时添加酵母浸粉的丁醇发酵优化调控策略的有效性和可靠性得到了进一步的实验验证。（6）依据理论分析结果，弱化丁酸生成/吸收再利用闭环的代谢强度有望提高丁醇发酵的丁醇/丙酮比和总溶剂生产效率。为此，在使用木薯和玉米为原料进行传统丁醇发酵、发酵进入产溶剂期后，脉冲式地添加少量丁酸（总添加量3.0g·L-broth–1）或乙酸（4.0g·L-broth–1），期望达到提高丁醇/丙酮比和总溶剂生产效率的目的。其中，添加少量丁酸的调控策略对玉米原料丁醇发酵最为有效，平均丁醇/丙酮比可以提高23%、从1.92上升到2.36:1；平均总溶剂生产效率也可以提高16%、从0.355g·L–1·h–1上升到0.410g·L–1·h–1。但是，上述调控策略对提高木薯原料丁醇发酵的性能没有明显效果，因为使用木薯原料进行丁醇发酵时，辅酶A转移酶活性低、丁酸闭环代谢强度已经很低，外添丁酸起不到降低丁酸闭环代谢强度的效果。更多还原

【Abstract】 Butanol is an important platform chemical, and clean, high-performance liquid bio-fuelas well. Bio-butanol production by Clostridia spp. with biomass as the substrates has becomemore and more attractive. In butanol fermentation by Clostridia, solvent products of butanol,acetone and ethanol are produced at a ratio of6:3:1(w/w). Continuously increasing butanol tototal solvents ratio or butanol/acetone ratio without sacrificing the total solvents productivityhas been the objective pursued by many researchers. On the other hand, bio-butanolproduction using corn and sugar as the substrates has threatened food supply safety forhuman-beings, as the arable lands for those valuable agricultural products are limited.Therefore, using the cheap and high productive non-grain crops capable of growing atin-arable lands as the substrate substitutes for bio-butanol production has become an urgenttask. Focusing on those problems, in this dissertation, butanol fermentations were conductedin a7L anaerobic fermentor and their performance was compared, using Clostridiumacetobutylicum ATCC824as the protocol strain and corn/cassava as substrates. Optimalbutanol production strategies using different substrates or metabolic regulation modes wereproposed to enhance butanol/acetone ratio and entire fermentation performance, as well as torealize the effective substrate substitution of using non-grain cassava crop to replace the cornmaterial, with the aids of metabolic flux calculations and key enzymes transcriptional levelanalysis. Major contents and results of the dissertation were summarized as follows:(1) Fermentation performance with traditional and in-situ extractive operation modes,using cassava and corn as substrates were investigated. In fermentations on cassava substrate,the severe phase shift delay leads to a very low butanol concentration or productivity, theoverall performance could not reach that of fermentations on corn substrate. Preliminaryanalysis on the apparent fermentation data, components of the medium and substrate itselfspeculated that low nitrogen source content in cassava-based substrate accounts for the poorperformance of fermentations on cassava. Through a serial of trial-and-error experiments, itwas found that in extractive fermentation on cassava with oleyl alcohol as the extractant, byadding2.5g·L-broth–1of yeast extract after the occurrence of significant gas productiondecrease, phase shift smoothly occurred by the indication of a quick gas production recovery.Butanol concentration reached competitively high levels of35.38g·L-broth–1.(2) The optimal regulation strategy of adaptively adding yeast extract (2.5g·L-broth–1) atphase shift stage could be effectively applied for all fermentations on cassava-based substrate,including traditional and extractive operation modes with oleyl alcohol/bio-diesel as theextractants, and final butanol concentration could reach the competitively high levels of13.61g·L-broth–1,34.37g·L-broth–1and18.37g·L-broth–1respectively, as compared with those offermentations on corn. More importantly, the proposed regulation strategy could significantlyincrease butanol/acetone ratio in fermentations on cassava-based substrate. Especially,butanol/acetone ratio in extractive one using oleyl alcohol as extractant achieved to3.0:1withthe strategy, and had a61.5%increment compared to that in corn-based substrate. Overall performance was largely improved.(3) When adopting the proposed regulation strategy for fermentations on cassava-basedsubstrate, variations of the key enzyme genes transcriptional levels and concentrations of freeamino acids released into broth were analyzed. The results showed that after activating thestrategy, CoA-transferase was stimulated and the encode gene ctfAB transcriptional level wasenhanced by15-fold; histidine and aspartic amino acid families, which are beneficial forbutanol synthesis, were also significantly accumulated. Both contributed to the smooth phaseshift, which largely enhanced buatnol concentration and productivity in turn.(4) The optimal balances in between carbon metabolic and reductive power flows wereanalyzed by metabolic flux calculation, and gene transcriptional levels of the key enzymes inmain metabolic routes were also measured, when fermenting on corn-and cassava-basedsubstrates. The results indicated that during solventogenic phase when fermenting oncassava-based substrate, transcriptional levels of genes ctfAB and buk encodingCoA-transferase and butyrate kinase were low, resulting in a weak metabolic strength inbutyrate formation/re-assimilation closed loop; on the other hand, transcriptional levels ofgenes adhE and bdhB, encoding the two NADH-dependent enzymes catalyzing the final tworeactions for butanol synthesis were also low; however, NADH regeneration rate was muchhigher. The weakened metabolic strength in butyrate closed loop and higher NADHregeneration rate lead to a higher butanol/acetone ratio in cassava-based butanol fermentation.(5) The effect of carbon/nitrogen sources (C/N) ratio on phase shift and butanol/acetoneratio was investigated using glucose/yeast extract as the carbon/nitrogen sources. If C/N ratiowas controlled in a moderate range (46.7～93.4mol·mol–1) during acidogenic phase, cellscould grow normally without severe organic acids accumulation so that phase shift couldsmoothly occurred. On the other hand, during solventogenic phase, a higher C/N ratio (≥93.4mol·mol–1) could increase butanol/acetone ratio above3.0:1. The effectiveness and reliabilityof the optimal regulation strategy of adaptively adding yeast extract for enhancingcassava-based butanol fermentation performance were further experimentally verified.(6) Theoretically, weakening metabolic strength of the butyrate closed loop wouldenhance buatnol/acetone ratio. Consecutively feeding a small amount of butyrate/acetateduring solventogenic phase to weaken the metabolic strengths in butyrate/acetate closed-loopswas attempted to increase butanol/acetone ratio and total solvent productivity in butanolfermentations with corn-and cassava-based media. Consecutively feeding a small amount ofbutyrate (a total of3.0g L-broth–1) is most effective in improving performance of corn-basedbutanol fermentation, as it simultaneously increased average butanol/acetone ratio by23%(1.92to2.36:1) and total solvent productivity by16%(0.355to0.410g·L–1·h–1) as comparedwith those of control. However, the butyrate feeding strategy could not improvebutanol/acetone ratio and total solvent productivity in cassava-based butanol fermentations,where the metabolic strength of butyrate closed-loop had already been very low.更多还原