| Plant fiber resource is the most potential raw material for ethanol production,and it is also a rich biomass resource.At present,however,a significant portion is not fully utilized except for a small amount used in textile,paper and construction industries,or as fuelwood(thermal efficiency is only about 10%)and roughage.The hydrolysates of plant fibers are mostly in the form of xylooligosaccharide or xylose,which are easy to be used by microorganisms and produced metabolites by microbial fermentation,and can be converted into ethanol or other high value-added,diversified products.It is reported that there are a few kinds of yeast in nature that ferment xylose to ethanol,but the ethanol yield of wild yeast is low.In recent years,the mutation breeding technology of ARTP,which is developed at room temperature and atmospheric pressure,has shown great potential in biological breeding due to the combination of many possible biological mutagenic factors such as electron,ion,photon,excited neutral particle,free radical,ultraviolet radiation and so on.On the basis of natural isolation and screening of yeast strains,this study focuses on the application of new ARTP mutation breeding technology to select a good yeast strain that uses xylose and ferments high-yield ethanol.As a production strain,xylose extract is used as raw material for ethanol fermentation test,and its application effect is verified.In order to obtain good strains of xylose efficient use,improve the utilization of agricultural and sideline products and added value,and further develop new products.The main results are as follows:(1)Isolation,screening and identification of ethanol producing yeast using xylose 126strains of yeast producing ethanol from xylose were isolated,screened and identified from a variety of samples.Five wild yeast A25,A42,A64,A70 and A95 with relatively high ethanol production were obtained by comparing their ethanol production capacity in various ways.The highest ethanol yield was A42,28?,160 r/min,and the ethanol content was 13.93 g/L after60 h fermentation.According to the morphological and molecular biological identification of the five strains,they are respectively Meyerozyma guilliermondii A25,Wickerhamomyces anomalus A42,Wickerhamomyces anomalus A64,Wickerhamomyces anomalus A64Wickerhamomyces anomalus A70 and Wickerhamomyces anomalus A95.The characteristics of five strains were compared and A25 and A42 were selected for follow-up test.(2)ARTP mutation breeding of strains A25 and A42 Domestication and cultivation of Meyerozyma guilliermondii A25 and Wickerhamomyces anomalus A42 were carried out.The ethanol content of the domesticated strains A25 and A42 increased by 15.06%and 17.58%respectively.Through the study of the mutation conditions of ARTP,it was determined that the best mutation conditions were the concentration of the suspension 10~8 each m L and the mutation treatment time 120 s.under this condition,the two yeast strains A25 and A42 were mutated,and the lethal rates were 95.00%and 97.53%respectively.Then,two rounds of screening were carried out for 1000 mutants by the method of tubule fermentation and TTC colorimetry.57 mutants were fermented in liquid state.The ethanol content of metabolites was determined by gas chromatography and compared.Finally,the strains A25-55 and A42-338with the highest ethanol yield were screened,and their ethanol yields were 16.58±0.38 g/L and 20.65±0.15 g/L,increased by 50.27%and 41.75%respectively.Finally,the genetic stability test was carried out with the Wickerhamomyces anomalus A42-338 as the target strain selected in this study,and the change rate of ethanol production in each generation of eight generations was less than 2%,which had good genetic stability.(3)Study on the application of Wickerhamomyces anomalus A42-338 Firstly,response surface method was used to optimize the xylose extraction process by enzymatic treatment of Camellia oleifera seed shell.The final optimized conditions were xylanase addition 5%,enzymolysis time 10 h,enzymolysis temperature 49?,substrate concentration20 g/L.Under the optimized conditions,xylose concentration was 11.63 g/L and glucose concentration was 3.32 g/L.Through the optimization experiment of the ratio of xylose extract and glucose,the xylose extract was compounded with glucose into xylose:glucose1:3 g/g fermentation medium,inoculated with Wickerhamomyces anomalus A42-338,the ethanol yield was the highest at 28?,160 r/min for 60h,the ethanol content was 19.88 g/L,the reducing sugar content was 9.06 g/L,and the xylose residual sugar content was 1.79 g/L The utilization rate of xylose was 84.61%,the utilization rate of glucose was 81.05%,and the conversion rate of sugar was 0.3976 g ethanol/g sugar.Wickerhamomyces anomalus A42-338 can be used in the production of oil tea seed xylose extract and glucose mixed sugar ethanol fermentation. |
PDF Full Text Request