The Process And Molecular Mechanisms Of Acetone-butanol-ethanol Production By Lignocellulose Fermentation

The increasing oil price and the unsustainable stock of fossil fuels on earth prompted the demand for the production of chemicals and fuels from renewable resources. Butanol is compatible with gasoline, and also has a higher energy density comparing with ethanol. It is therefore expected that butanol plays a major role in next generation of renewable biofuels. Research done in this thesis aims at improving biobutanol production by Clostridium species. Following is a summary of the results of these investigations.1. Corncob pretreatment using a new pretreatment technology—Wet disk millingCorncob was pretreated using WDM (wet disk milling) methods. The smooth and compact surface of ground corncob is disrupted in this pretreatment. The surface of particles turned rough and irregular after10milling cycles, suggesting the disruption of the highly ordered structure composed of cellulose, hemicellulose, and lignin, resulting in the increase of the material’s accessibility to cellulase.Sugar yields of71.3%for glucose and39.1%for xylose from pretreated corncob were observed after enzymatic hydrolysis, a2.24-fold improvement over unpretreated corncob. The relationship between sugar yields and particle size of the pretreated corncob was investigated, suggesting a smaller particle size benefits enzymatic hydrolysis with the WDM pretreatment approach. Analysis of the correlation between parameters representing particle size and efficiency of enzymatic hydrolysis predicted that frequency90%is the best parameter representing particle size for the indication of the readiness of the material for enzymatic hydrolysis. Furfural and hydroxymethyl furfural that were reported to affect fermentation were analyzed. Neither of them was detected in the WDM pretreated corncob.2. ABE production using Clostridium acetobutylicum SE-1from corncob hydrolysateAcetone-butanol-ethanol (ABE) production from corncob was achieved using an integrated process combining wet disk milling (WDM) pretreatment with enzymatic hydrolysis and fermentation by C. acetobutylicum SE-1.ABE production from corncob was carried out with both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes using C. acetobutylicum SE-1. Interestingly, when considering the time for fermentation as the time for ABE production, a comparable rate of ABE productivity and yield in the SHF process (0.20g/L·h and0.36g/g) could be observed when glucose (0.17g/L·h and0.34g/g) or a mixture of glucose and xylose (0.22g/L·h and0.35g/g) mimicking the corncob hydrolysate were used as the substrate for fermentation. This result suggested that the WDM is a suitable pretreatment method for ABE production from corncob owe to the mild conditions. A higher ABE production rate could be observed with the SSF process (0.15g/L· h) comparing with SHF process (0.12g/L·h) when combine the time for saccharification and fermentation and consider it as the time for ABE production. This is possibly a result of low sustained sugar level during fermentation. The mass and energy balance of ABE production for both SSF and SHF approaches was evaluated. An energy efficiency (net energy input/total energy output) of112%was achieved for SHF process and142%was achieved for SSF process. These investigations lead to the suggestion that this new WDM pretreatment method has the potentials to be exploited for efficient ABE production from corncob.3. Simultaneous utilization of glucose and xylose for butanol production using C.beijerinckii SE-2Most butanol-producing strains prefer glucose over xylose, leading to a slower butanol production from lignocellulose hydrolysate. It’s therefore beneficial to find and use a strain that can simultaneously use both glucose and xylose. In this study, we found that a C.beijerinckii SE-2strain assimilated glucose and xylose simultaneously and produced ABE. In addition, a classic diauxic growth behavior was not seen. A similar rate of initial sugar consumption (0.8g/L·h for glucose and1.0g/L·h for xylose) could be observed in the two cultures, suggesting this strain could use either glucose or xylose as the substrate and it has a similar capability to degrade these two sugars.With different initial glucose:xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. ABE production profiles were similar on different substrates.4. Optimization of butanol production from corncob hydrolysate by fermentation with C. beijerinckii SE-2WDM pretreated corncob hydrolysate were used for butanol fermentation with C. beijerinckii SE-2. Optimization of media employing central composite design (CCD) and response surface methodology (RSM) were performed to evaluate effects of6factors including contents of CSL, CH3COONH4, K2HPO4-KH2PO4, MnSO4·H2O, MgSO4·7H2O and FeSO4·7H2O. The optimum recipe contains3.6g/L CH3COONH4,2.4g/L CSL,1g/L K2HPO4-KH2PO4,0.01g/L MnSO4·H2O,0.2g/L MgSO4·7H20and0.01g/L FeSO4·7H2O. The concentration of butanol could reach11.76g/L with this recipe,42%higher than the regular recipe. This study indicated that RSM is a useful approach for optimizing operational conditions for butanol production.5. Transcriptional response of glucose and xylose supplementation to C. beijerinckii SE-2cultureIn C. acetobutylicum, it was shown that xylose-related genes are repressed in the presence of glucose, the favorable substrate for ABE production. Previous reports indicated that the xylose metabolism of C. beijerinckii is free from carbon catabolite repression. Indeed, the relief from carbon catabolite repression will dramatically improve xylose metabolism when glucose is present, and is possibly the reason for the simultaneous glucose and xylose consumption shown above. The removal of glucose inhibition on xylose metabolism, were it to be true, would be a major advantage for C. beijerinckii SE-2. Contrary to previous suggestions, three genes(xylA-Ⅱ, xylB and xylT) which were suggested to be involved in the xylose utilization showed strong inhibition when glucose is supplemented to the media and induction when xylose is supplemented to the media. These observations suggest the carbon catabolite repression is still present in C. beijerinckii SE-2and the simultaneous xylose/glucose consumption has to be due to other reasons. It is therefore most likely that the rate limiting step of the degradation of glucose and xylose is in the production of solvent from pyruvate.