| Since the 1970's, renewable fuels and materials have attracted significant interest due to high petroleum prices and awareness of the depletion of fossil fuel reserves. Bioethanol production is considered to be a milestone for sustainable development. Hydrolyzate of biomas includes a number of pentoses and hexoses. There is no single organism yet shown to efficiently convert all these sugars to ethanol. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. E. coli is naturally able to use a wide spectrum of sugars. One of the research work has concentrated on engineering E. coli to selectively produce ethanol.Hemicellulose is a by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer, which can be hydrolyzed into monosaccharides by a dilute acid process. In addition to aromatics, acetic acid, formic acid and methanol released from woody biomass during the acid process and the hydrolysis of carbohydrates also generates toxic compounds. During dilute acid hydrolysis of biomass, monosaccharided are dehydrated and various toxic compounds are formed that can inhibit microorganism growth and ethanol fermentation, such as furfural and 5-hydroxymethylfurfural (HMF). Lignin byproducts during biomass hydrolysis are also inhibitory to microorganisms. Na2SO4 and CaSO4 formed during neutralization of the hydrolyzate contribute to a high osmotic pressure. This mixture of inhibitors will likely be a great challenge to those fermenting strains.In this study, the basic culture conditions of E. coli FBR5 were investigated. The optimal culture conditions are as following:12 mL phosphate buffer,35℃, inoculum size of 5% by volume,12～14 hour-old inoculum and 180 r/min of shake speed.Inhibitors were added into the pure xylose media to investigate the tolerance of E. coli FBR5 to those inhibitors. It's found that FBR5 was inhibited in the present of 5～10 g/L Na2SO4,10 g/L CaCl2,0.50 g/L FeSO4,2.0 g/L furfural and 0.2～0.4 g/L phenol, respectively. The results indicate that the salts in hot-water wood extract hydrolyzate can be inhibitory to FBR5.Sugar Maple hot-water extract hemicellulosic hydrolyzate was concentrated and fractionated by a Nano-filtration membrane process in our lab. In this study, E. coli FBR5 was challenged by concentrated hot-water wood extract hemicellulosic dilute sulfuric acid hydrolyzate. After repeated strain adaptation, a new improved strain:E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of dilute sulfuric acid hydrolyzate in the fermentation media, and xylose was completely utilized by the strain to produce ethanol. Dilute sulfuric acid hydrolyzate was detoxified by ion-exchange, and FBHW can be grown rapidly in the desalted hydrolyzate with a yield of 22.6 g/L ethanol. To avoid the increase of production cost by ion-exchange, mixed-acid hydrolysis was carried out. Then the mixed-acid hydrolyzate was neutralized by mixed-base. Due to the balance of ions in the hydrolyzate, its inhibition to microorganism decreased. FBHW was grown in the concentrated mixed-acid hydrolyzate without any treatment and yielded 36.9 g/L ethanol.High concentration of metal ions could be inhibitory to microorganisms. On the other hand, some ions are essential to growth of microorganism. In this study, ICP method was used to analyze elements content of LB broth media, cells of E. coli FBHW and hydrolyzate. Except for Cu,K,Na and P, all the other metal ions are excess for FBHW to grow, especially for trace elements such as, Fe, Ni, Zn and Mn. To reduce the cost of medium, NH3·H2O was used as the only nitrogen source to replace LB in the hydrolyzate to grow E. coli FBHW. FBHW can be grown in the simplified medium with a low ethanol yield.
|
PDF Full Text Request