Study On The Appliaction Of Glycerol-pretreatment Coupled Enzyme In Preparation Of Bio-ethanol And Extraction Of DNJ

Cellulosic biomass, the most abundant resource on the earth, is potential, renewableand green energy. If this cellulosic biomass could be converted to chemical products, it willnot only alleviate food and energy crises in current human society, but also play animportant role in solving problems of environment pollution caused by crop resources,and has significance for the sustainable development of human society.Firstly, through compositional analysis of natural bagasse, the results showed that thepercentage content of cellulose,hemicellulose and lignin were36%,38%and15%respectively. H2SO4, NaOH and glycerol were selected to pretreat bagasse, resulting in58.49%,64.81%and54.9%of cellulose being obtained in bagasse solide. Therefore,compositional analysis of natural and pretreated bagasse, could provide data for subsequentcellulase production, enzymatic saccharification and mixed fermentation of bagasse.Secondly, cellulase production by the fermentation of Trichoderma koningii （CICC40359）was optimized, and optimum cellulase production conditions were obtained asfollows: the urea concentration was2g/L, soybean meal concentration was12g/L, andinitial pH value was4.0in the initial fermentation medium. According to this optimumconditions, glycerol-pretreated bagasse was used as carbon source to induce cellulaseproduction by the fermentation of Trichoderma koningii. Period of cellulase production was7days which were3days shorter than the untreated bagasse. And the cellulase activity inthe fermentation broth increased up to1.31IU/mL, which was16.96%higher than theuntreated bagasse.Thirdly, using cellulase hydrolyzed bagasse was pretreated by H2SO4, NaOH andglycerol respectively,obtaining the result that glycerol pretreatment made enzymatichydrolysis of bagasse to the maximum. Effects of time and temperature for pretreatingbagasse by glycerol on enzymatic hydrolysis of bagasse were investigated,obtaining theoptimum pretreatment conditions: the pretreatment temperature was210℃,andpretreatment time was3h. According the optimum preatment conditions, time course ofenzymatic hydrolysis of glycerol-pretreated bagasse was studied. When the hydrolysis timewas72h, glucose was released to the maximum value, and the concentration andsaccharification rate were2.57g/L and41.6%respectively, and both of them were betterthan non-pretreated bagasse. Therefore, it was proved that the pretreatment of bagasse byglycerol made saccharification rate of cellulose increase.Fourthly, inoculation volume ratio, total inoculation size between Aspergillus niger and Candida shehatae, and inoculation delay time of Candida shehatae was used as influencingfactors in research on ethanol production through mixed fermentation.And the optimumconditions for ethanol production were determined as follow:inoculation volume ratiobetween Aspergillus niger and Candida shehatae was1:1; total inoculation size of both was10%; and inoculation delay time of Candida shehatae was12h. According to the optimalinoculation conditions,1.33gram ethanol per1litre fermentation broth and86.21%ofethanol yield was obtained when using glycerol-pretreated bagasse as substrate duringmixed fermentation, and it was higher than non-pretreated bagasse(0.63g/L and75.26%).Finally, mulberry leaf was pretreated respectively by H2SO4.HCl and NaOH solution atthe concentration of0.05mol/L. HCl solution was found to be the best pretreatmentreagents for extracting1-Deoxynojirimycin(DNJ) from the mulberry leaf. Thenfermentation broth of4cellulase-producing strains was used to pretreat mulberry leaf, andsubquently pretreated by0.05mol/L HCl solution, and the result showed that Trichodermareesei was the best pretreatment strain for extracting DNJ. When the time was12h forpretreating mulberry leaf by the culture of Trichoderma reesei,1.995mg DNJ per1grammulberry leaf was obtained. So, using cellulase-producing strains to pretreat mulberry leafcould promote release of DNJ,resulting in higher DNJ extraction.In summary, after bagasse pretreated by glycerol, saccharification rate and ethanolyeild during mixed fermentation were higher than non-pretreated bagasse. And mulberryleaf was pretreated by cellulase-producing strain, resulting in higher extraction of DNJthan control. Thus process engineering and data for high value of cellulosic materials wereobtained.