| Butanol is an industrial commodity and also considered to be a more promising gasoline substitute compared to ethanol. Renewed attention has been paid to ABE (acetone-butanol-ethanol) fermentation with the renewable and inexpensive substrates, including energy crops, agricultural residues, forestry and food processing wastes, on account of the depletion of oil resources and deteriorating environment.The production process for cellulosic butanol involves cellulase production, lignocellulose hydrolysis, co-fermentation of hexose and pentose to butanol, which is very complicated and makes cellulosic butanol uncompetitive with butanol from fossil. The CBP (consolidated bioprocessing) can offere lower cost and higher efficiency for cellulosic butanol production by combing these procedures above in one pot reaction.To produce cellulosic butanol by CBP, a single microorganism or microbial system must be developed to utilize lignocellulose at a high rate of conversion and produce solvents at high yields and titers. The aim of the work was to construct new highly efficient CBP to produce cellulosic butanol directly with high titer and high productivity by mixed culturing celluloytic anaerobe and solventogenic bacterium, or genetically engineering celluloytic anaerobic clostridia alone.Firstly, three cellulosome-producing anaerobic clostridia including Clostridium thermocellum, Clostridium cellulovorans and Clostridium cellulolyticum were singly cultured with AECC (alkali extracted deshelled corn cobs) as sole carbon source respectively. The comparison of AECC decomposition, fermentable sugars accumulation, metabolite (organic acids and alcohol) production by three bacteria were investigated and evaluated. It was observed that C. thermocellum degraded 44.4 g/L of AECC and accumulated 19.1 g/L of total sugars in 100 h, which represented a good carbon source provider for solventogenic bacterium to manufacture biobutanol. C. cellulovorans did not accumulate as much sugars as C. thermocellum, but produced 6.72 g/L of butyrate in 100 h, which could be reassimilated by solventogenic bacterium and tranformed to butanol. In contrast, slow growth and weak metabolism made C. cellulolyticum not a proper candidate to supply sufficient fermentable sugars or organic acids for solventogenic bacterium in mixed culture.To refrain from producing acids only, a novel strategy for sequential co-culture of C. thermocellum ATCC 27405 and C. beijerinckii NCIMB 8052 was proposed to produce solvents efficiently without feeding butyrate in one pot reaction with AECC as the sole carbon source. In this strategy, soluble sugars accumulation by C. thermocellum hydrolyzing AECC was considered to be paramount for the CBP and was promoted considerably by contrast with previous co-culture studies. To achieve more sugar accumulation, the culture conditions of C. thermocellum were investigated and optimized as follows:the segmental pH control strategy (pH controlled at 7.5 during the first 60 h, and 6.0 afterwards), initial concentrations of 8 g/L for YE and 75 g/L for AECC of 30-40 mesh. Besides, when C. beijerinckii was inoculated at 96 h with an inoculation volume of 1%, ABE fermentation performed best at 33℃ with an initial pH of 6.0. Under the combinatorial optimal culture parameters for sugars accumulation and ABE production, the CBP decomposed 88.9 g/L of AECC and manufactured 19.9 g/L of solvents (acetone 3.96, butanol 10.9 and ethanol 5.04 g/L) in 200 h without feeding butyrate, which was 70.3% and 81.0% higher than those obtained with the initial co-culture conditions.Since the culture temperature of C. thermocellum (60 ℃) doesn’t match that of C. C. beijerinckii (37℃), the CBP has to be carried out in two stages, which affected ABE productivity negatively. Accordingly, a homothermal artificial symbiotic system was constructed by co-culturing mesophile Clostridium cellulovorans 743B and C. beijerinckii NCIMB 8052 to synchronize AECC saccharification and ABE fermentation. In the symbiosis, C. cellulovorans, an anaerobic, celluloytic and butyrate-producing mesophile, was selected to saccharify lignocellulose and produce butyric acid, instead of adding cellulase and butyric acid to the medium, so that the soluble sugars and butyric acid generated can be utilized by C. beijerinckii to produce butanol. Effects of C. beijerinckii inoculation timing, inoculation ratio of two strains and pH control strategy on co-cultures were studied and evaluated. The best cluture conditions were:simultaneous inoculation, inoculation ratio of 2:10 (v/v) between C. beijerinckii and C. cellulovorans, pH control at 7.0 during first 24 hours. Under the optimal conditions, the co-culture decomposed 68.6 g/L of AECC and produced 11.8 g/L of solvents (2.64 g/L of acetone,8.30 g/L of butanol and 0.87 g/L of ethanol) in less than 80 h. Compared with the sequential co-culture of C. thermocellum and C. beijerinckii, the fermentation time of the homothermal CBP was shortened by 120 h, and the butanol productivity was enhanced by 90.6%. Besides, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the dynamics of the abundance of each strain during the co-culturing process, which figured out the roles of each strain at different periods in the symbiosis.Mixed culture process was efficient for cellulosic butanol production but complicated for process regulation. To simplify the CBP, we attempted to develop a gene transfer method for C. cellulovorans, with the goal of producing butanol by C. cellulovorans alone via metabolic engineering. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA (plasmid pXYl) into C. cellulovorans at a low efficiency of 4.6 transformants/μg DNA by utilizing conditions common to Clostridium acetobutylicum electroporations protocol with a little modification. Systematic investigation of various parameters affecting the electrotransformation efficiency was carried out. It was found that when C. cellulovorans was harvested at growth phase of OD600 0.75, washed with buffer ETM and ET with a initial pH 7.0, electrically pulsed with a voltage of 2.0 kv and reincubated for 5 hours prior to selective plating, the electrotransformation efficiency was 76.8 transformants/μg DNA, which was 15.7 times higher than that obtained before optimization.It was found that C. cellulovorans has good butanol tolerance, which suggested that C. cellulovorans is a promising heterologous host for n-butanol production from lignocellulose. So a new CBP for cellulosic butanol production was developed by introducing the butyraldehyde dehydrogenase gene adhE2 (CA-P0035) from C. acetobutylicum into C. cellulovorans, which endued C. cellulovorans the ability to produce butanol from AECC directly. However, the CBP only produced 0.374 g/L butanol, which could be attributed to unsufficient NADH supply and competitive pathways in C. cellulovorans.To improve the symbiosis of C. beijerinckii and C. cellulovorans, the two strains were genetically engineered by over-expressing butyrate kinase and CoA-transferase respectively. The new symbiotic system degraded 54.5 g/L of AECC, produced 9.32 g/L of solvents (1.73 g/L of acetone,6.47 g/L of butanol and 1.12 g/L of ethanol) in 108 h. Compared with the wild strains community, it enhanced by 16.5% and 57.4% respectively in AECC decomposition and butanol production, which implied efficient solvents conversion ratio improvement by metabolic engineering. The fed-batch strategy promoted AECC decomposition and butanol production further.14.1 g/L of solvents including 9.72 g/L of butanol were manufactured from 75.8 g/L of AECC in 108 h, which was 19.5% and 10.5% respectively higher than that obtained by wild strains symbiosis using fed-batch strategy. |
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