| With exhaustion of fossil fuels, global pollution and climate change, it is urgent to seek renewable, low cost, clean and high efficiency energy alternatives. Cellulosic ethanol has attracted greater attention over the past decade. Consolidated bioprocessing (CBP) is a breakthrough for bioethanol production due to its energy saving strategy. The key issue of CBP is to obtain or engineer a suitable microorganism converting lignocellulose to ethanol in the presence of variety inhibitors. Clostridium thermocellum and other thermophilic anaerobic bacteria have been used and demonstrated to be potential candidates due to their simultaneous saccharification and fermentation merits in CBP. However, these bacteria still need to be genetically engineered due to their low ethanol yield and poor tolerance of chemical compounds generated in the fermentation system. Thus, the scientific aims of this study are:(1) Optimize the fermentation strategy in order to maximize the activity of cellulosome (from Clostridium thermocellum); (2) Develop a genetic tool for the transformation of Clostridium thermocellum LQR1 and for subsequent genetic engineering. This tool will be further applied and validated in the mesophilic anaerobic bacterium Clostridium cellulolyticum H10; (3) Adapt the Clostridium thermocellum so that it can tolerates 5% (v/v) ethanol by mutagenesis screening. The activity of cellulosome was tested under varying concentrations of chemical compounds derived from lignocellulose pretreatment and fermentation. We found that, firstly, the cellulolytic activity of cellulosome was actually promoted by formate, acetate and lactate; secondly, cellulosome was tolerant up to 5 mM furfural, 50 mM p-hydroxybenzoic acid and 1 mM catechol. Furthermore, the cellulosome exhibited higher ethanol tolerance and thermostability than commercialized fungal (Trichoderma reesei) cellulase. To probe the implication of these unique enzyme-features, Clostridium thermocellum JYT01 was cultured under conditions optimal for cellulosome activity. This CBP system yielded 491 mM ethanol, the highest level reported thus far for C. thermocellum monocultures.In order to increase transformation efficiency of Clostridium thermocellum LQR1, the competent cells were prepared from the isoniazid induced spheroplast cells. The result showed that Texas red-conjugated dextran was successfully delivered into Clostridium thermocellum LQR1 under the field strength between 15-18kV/cm, which was generated by a BCM electroporator. In addition, approximately 102 pfu/μg DNA transformation efficiency was achieved in Clostridium cellulolyticum H10 when the pIKM1 plasmid was employed as control. The transformation strategy developed in this study also could be applied in other thermophilic bacteria.The wild type Clostridium thermocellum LQR1 only tolerates less than 1% (v/v) ethanol, which is a big gap towards 5% (v/v), the industrial requirement for large scale bioethanol production. To this end, we adapted three Clostridium thermocellum ethanol tolerant strains by EMS mutagenesis. All 3 strains exhibited up to 6% (v/v) ethanol tolerance. Two of them named Clostridium thermocellum M1560 and M1530 showed the same ethanol production as wt in cellulose fermentation.In conclusion, the optimized cellulosic ethanol fermentation strategy was developed, by which 491 mM ethanol was generated, the highest yield reported so far. Furthermore, a genetic electro-transformation strategy was developed in Clostridium thermocellum LQR1 and Clostridium cellulolyticum H10. These findings pave a new way for design, selection and optimization of the cellulosome-ethanologen partnership. Our work is also a milestone in lighte of genetic engineering of thermophilic anaerobic bacteria to advance cellulosic ethanol production. |
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