Ethanol Fermentation From Corncob Hydrolysate Using A Self-Screened Pichia Guilliermondii

Lignocellulosic raw material is an abundant renewable resource. Development of an efficient lignocellulosic-based bio-production platform could be beneficial for the reduction of fossil oil consumption and carbon-dioxide emission. Here, a strain with tolerance to lignocellulosic hydrolysate inhibitors is important to the cost-effective fermentation using lignocellulosic hydrolysate. In this thesis work, microbial screening, adaptation, process optimization and genetic engineering were conducted to produce ethanol and other useful chemicals from lignocellulosic hydrolysate without either detoxification or external nutrient supplementation.Firstly, an inhibitor-tolerant strain was obtained by screening from various soil samples. The strain could ferment glucose and xylose in the minimum medium containing a mixture of inhibitors including furfural, HMF and acetate. From 18s rRNA sequence and alignment analysis, the screened strain was identified as Pichia guilliermondii. It could utilize the non-detoxified lignocellulosic hydrolysate to produce value-added chemicals such as xylitol. Interestingly, it could degrade 6 g L-1 HMF within 20 hours, much faster than other reported yeasts. Further experimental results indicated that besides its NADPH dependent aldehyde dehydrogenase activity, this P. guilliermondii strain also had a PMS/DCPIP (phenazine methosulphate/2,6-dichlorophenol-indophenol) dependent HMF oxidase activity.The P. guilliermondii strain could also produce ethanol from the non-detoxified corncob residue (CCR) hydrolysate. However, the accumulation of reactive oxygen species (ROS) during fermentation led to the loss of cell viability and productivity, especially after 100 hours of fermentation. To solve this critical problem, after rational analysis, a biotin-addition strategy was designed to improve the cellular anti-oxidation ability during lignocellulosic-ethanol fermentation. By increasing the biotin concentration in seed culture medium, in the latter fermentation process the intracellular biotin pool (IBP) was successfully elevated, which facilitated the synthesis of porphyrin, a critical structure in catalase. As a result, the cellular catalase activity was increased by 1.8 folds and the ROS level was reduced by 46% in the high-IBP cells compared to the control. The viability of high-IBP cells was also higher compared to the control at the 190th hour, and finally the ethanol titer was enhanced by 28% in batch fermentation. The high-IBP cells were also applied to repeated-batch fermentation with the non-detoxified CCR lignocellulosic hydrolysate, and the titer and average productivity of ethanol reached 55.5 to 59.2 g L-1 and 0.9 g L-1 h-1. The results are considered to be favorable to future industrial application of this lignocellulosic bioethanol process.As we understand, this wild-type P. guilliermondii is a Crabtree-negative yeast, in which glucose is mostly used for cell mass formation rather than ethanol production under aerobic condition. In contrast, in Crabtree-positive organisms such as Saccharomyces cerevisiae, the glucose can be fermented into ethanol even under aerobic condition when glucose is excessive. Here, whether the Crabtree-negative P. guilliermondii strain could be genetically engineered to exhibit different metabolism in aerobic fermentation is an interesting issue for the cellular physiology/metabolism study as well as future application. As the genetic modification platform for P. guilliermondii was lacking, a uracil auxotrophic P. guilliermondii (WT) was at first screened out; then, the CAT8 gene, which encodes a putative global transcriptional activator in glucose signaling cascade, was disrupted by homologous recombination. The aerobic fermentation performance of the mutant (ΔCAT8) in both synthetic medium and CCR hydrolysate was investigated, and its specific oxygen uptake rate was found to decrease to 2/3 of the WT strain while the ethanol accumulation increased significantly compared to the WT. Transcriptional analysis suggested a comprehensive regulatory function of CAT8 in the central metabolic pathway. The facts imply that the CAT8 gene may play an important role in regulating the glucose metabolism of P guilliermondii.In conclusion, we successfully obtained a strain with excellent tolerance to lignocellulosic hydrolysate inhibitors through screening and adaptation. After rational bioprocessing manipulation, a high ethanol titer was achieved in cyclic CCR hydrolysate fermentation. In addition, the genetic manipulation platform was established and the CAT8 role was revealed. The information on cellular inhibitor degradation and anti-oxidation ability enhancement was also obtained. This work is considered helpful to related studies on other Crabtree-negative yeasts.