Breeding Of Clostridium Acetobutylicum And High-productivity Butanol Fermentation

Butanol is not only an important chemical material, but also a promising new biofuel. As a fuel additive, butanol is superior to ethanol and has a broad application prospect. The performance of strain is very important and the low solvent production is a critical factor that limits ABE fermentation. Immobilized fermentation can improve solvent productivity, but Clostridium acetobutylicum is easily to lose the capacity of producing solvents and forming spores which typically occurs when Clostridium acetobutylicum is repeatedly subcultured in batch culture or grown in continuous culture. In this thesis, we selected higher butanol-yielding strains by treating the original strain with N+ beam implantation and UV radiation. The metabolic regulation of ABE fermentation and strain degeneration were also studied. Besides, immobilized fermentation was also carried out in a fibrous-bed reactor.A series of efficient screening methods were established. Strains could be obtained through screening plates containing glucose, resazurin, 2-butanol or complex. A strain was screened from parent strain treated with N+ beam implantation and UV radiation and it was designated as Clostridium acetobutylicum NU22. In 7% corn medium, NU22 could produce 14.51 g/L butanol with a butanol ratio of 66.93% and the butanol production was 20.41% higher than that of initial strain YT-37.Effects of different electronic carriers and different weak-acid salts on butanol fermentation by Clostridium acetobutylicum NU22 were investigated. Results showed that benzyl viologen at low concentrations could significantly promote butanol production and reduce acetone production with the butanol ratio increasing from 66.93% to 82.35%. When benzyl viologen was present, Clostridium acetobutylicum NU22 could enter solventogenesis rapidly and the fermentation period was shortened, thus butanol productivity increased markedly. Clostridium acetobutylicum NU22 produced 16.10 g/L butanol with a productivity of 0.37 g/(L·h) when grown in corn medium containing 40 mg/L benzyl viologen. Meanwhile, butanol production and productivity were 10.96% and 60.87% higher than those of control, respectively. Weak-acid salts such as acetate, butyrate and phosphate at low concentrations could also promote butanol production. NU22 could produce 16.27 g/L butanol when grown in corn medium containing 4.00 g/L butyrate.A degenerated strain of Clostridium acetobutylicum NU22 was isolated after repeated subcultures and was designated as DNU83. The butanol production of Clostridium acetobutylicum DNU83 in 7% corn medium drastically decreased to one-six of that of Clostridium acetobutylicum NU22, with 2.33 g/L of butanol, 1.26 g/L of acetone and 0.66 g/L of ethanol. Clostridium acetobutylicum DNU83 could restore solvent production in medium containing weak-acid salts such as acetate, butyrate and phosphate, which could be explained in terms of the increased buffering capacity of the medium. When benzyl viologen was added, the promoting action on butanol production was enhanced. When grown in corn medium containing 30 mg/L benzyl viologen and 4.00 g/L dipotassium hydrogen phosphate, Clostridi- um acetobutylicum DNU83 produced 18.01 g/L of butanol which was 24.09% higher than that of Clostridium acetobutylicum NU22. The addition of sodium acetate to medium was found to stabilize solvent production during continuous subcultures and the action was enhanced by benzyl viologen addition. The butanol production of Clostridium acetobutylicum NU22 could maintain 15.50～17.50 g/L during 20th subcultures.Clostridium acetobutylicum NU22 was immobilized by adsorption onto Aquamats-AO. Immobilized fermentation in a fibrous-bed reactor was carried out. In batch immobilized fermentation, the fermentation period of immobilized cells was shortened from 64 h to 40 h and butanol productivity reached 0.34 g/(L·h) which was 54.54% higher than that of control. During 6th repeated-batch immobilized fermentation in FBB, butanol production could maintain 13.00～14.50 g/L and the average productivity of butanol reached 0.28 g/(L·h) which was 27.27% higher than that of control.