| 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. |
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