Construction And Performance Of A Staged Butyrate-butanol Fermentation Process With Rice Straw As Substrate

Bio-butanol is known as an outstanding alternative liquid fuel which has drawn extensive attention towards the research.However,the bio-butanol application has been hampered by the high dosage of enzymes involved in lignocellulose hydrolysis,the low consumption rate of xylose(hydrolysate of hemicellulose),the high yield of by-products and the low productivity of butanol production.In the present research,a staged butyrate-butanol fermentation process using the lignocellulosic material as substrate was proposed.Construction and process optimization of butyrate-producing culture,the study of the influence of components of rice straw hydrolysate and butyrate fermentation broth on butanol fermentation,construction and performance of staged butyrate-butanol fermentation process were performed.This provides a theoretical and technical basis for the development of economical and efficient biobutanol process.To enhance the degradation and conversion rate of rice straw,based on the mutualism relationship between different microbes in the mixed culture,a mesophilic bioprocess using bioaugmentated culture and a thermophilic bioprocess using the constructed co-culture was developed to ferment rice straw to butyric acid.The produced butyric acid was used as co-substrate in subsequent butanol fermentation.Bioaugmentation with Clostridium tyrobutyricum ATCC 25755 has obviously enriched Clostridium Ⅳ.Genes involved in energy production and conversion and carbohydrate transport and metabolism were enriched resulting in the increment of butyric acid production from 5.53 to 18.05 g/L.The co-culture of C.thermocellum ATCC 27405 and C.thermobutyricum ATCC 49875 produced 33.14 g/L of butyric acid from pretreated rice straw in fed-batch fermentation.In the co-culture,C.thermocellum ATCC 27405 had provided substrates to C.thermobutyricum ATCC 49875.In return,C.thermobutyricum ATCC 49875 had improved the metabolism of C.thermocellum ATCC 27405 by relieving feedback inhibition of sugars produced.Secondary metabolism of by-products,acetic and lactic acid,to butyric acid by C.thermobutyricum ATCC 49875 also contributed to the hyper-production of butyric acid.To evaluate the feasibility of the staged butyrate-butanol fermentation processes using rice straw as a substrate,butanol fermentation with rice straw hydrolysate(xylose,glucose,and cellobiose)and fermentative product(VFAs)was performed with the help of C.beijerinckii NCIMB 8052.The strain C.beijerinckii is capable of converting xylose,glucose,and cellobiose to butanol,but the butanol production was low,especially while using xylose as substrate.The consumption of xylose,glucose,and cellobiose was obviously promoted by supplementing 3 g/L of butyric acid as cosubstrate that results in the increment of the butanol production by 1.52-9.91 times.With 5 g/L butyric acid as a co-substrate and inoculation of 0.59 g-dry cell/L in the culture,a butanol production of about 11.89 g/L was obtained.Supplement of 0-1.5 g/L acetic acid influenced the assimilation rate of butyric acid during butanol fermentation but it does not affect the yield of butanol.The cell growth and butanol production would be inhibited by a high density of external VFAs but a reasonable VFAs concentration would not influence the butanol production and reached 12.14 g/L.Expressions of genes revealed that the butyrate was re-assimilated through the reversed butyrate formation pathway.Furthermore,in the butanol fermentation with reasonable concentration of VFAs,adh E gene was up-regulated,which suggested that the staged butyrate-butanol fermentation processes using rice straw as substrate was feasible and effective.Two staged butyrate-butanol fermentation processeses were constructed,i.e.butanol fermentation with rice straw hydrolysate and mesophilic butyrate fermentation broth as co-substrate(FMP)and butanol fermentation with rice straw hydrolysate and thermophilic butyrate fermentation broth as co-substrate(FTP),respectively.The overall performances of the two processes were evaluated.The butanol production reached 12.85 g/L and 15.86 g/L in FMP and FTP,respectively,which was much higher than 6.27 g/L of the butanol fermentation with rice straw hydrolysate as substrate(FSP).The translational analysis indicated that the buk and adh E genes were up-regulated in FMP and FTP which illustrates the mechanism of staged butyrate-butanol fermentation processes.The results suggest that FMP and FTP have obvious advantages in butanol production and energy yield.Enhanced production of 108.5 and 149.4 g butanol was obtained from 1 kg of rice straw by using FMP and FTP,respectively and the energy yield reached 4418.3 and 6043.7 k J/kg.The total consumption of cellulase enzyme was reduced by 3 fold in the new processes.The staged butyrate-butanol fermentation process is a promising and economical strategy for butanol production from renewable biomass.