以玉米芯为原料生物转化木糖醇的研究
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摘要
木糖醇被广泛用于食品、医药、化工、轻工及国防等领域。随着社会的进步和经济的发展,木糖醇的需求量将会逐年增加。以木质纤维素类生物质生产木糖醇不仅能够降低木糖醇生产的原料成本,同时在废物处理、环境保护等方面起到一定的作用。木糖是木质纤维素水解物中含量仅次于葡萄糖的一种单糖。木糖的有效利用是木质纤维素生物转化生产木糖醇的关键环节之一。因此,高糖利用率的木糖发酵菌种的选育一直是该领域的热门课题。
本论文以发酵工程的理论为指导,从自然环境中分离筛选出一株发酵木糖产木糖醇的菌株,对该菌株进行了鉴定,同时对其发酵木糖生产木糖醇的工艺进行了参数优化,为工业化生产木糖醇奠定了一定基础。研究内容与结果如下:
1.从玉米芯中筛选出产木糖醇的菌种菌株e,通过形态学鉴定,菌株e有典型的酵母菌落形态,能同化硝酸钾,同化乙醇,不能产生类淀粉化合物。
2.对筛选出的菌株e进行分子生物学鉴定,鉴定结果:菌株e为丝孢酵母属中的Trichosporon coremiiforme。
3.通过单因素试验得出摇瓶上的木糖醇最佳发酵条件为:碳源浓度为40g/l,氮源浓度为6g/l,pH为7,接种量为6%,装液量为60ml,选取种子龄为23h,37℃下发酵4d。
4.在单因素实验的基础上进行了正交试验,正交试验最佳工艺条件为:碳源浓度为40g/l,氮源浓度为6g/l,pH为7,接种量为6%,在此最优条件下木糖醇转化率达到69.1%。通过优化设计比初始木糖醇转化率提高了51.1%。
5.从玉米芯到木糖的生产过程中,预处理对木糖产量有重要影响,细菌B7对玉米芯预处理2天较为合适,预处理过程中最适玉米芯添加量为10%。通过预处理,半纤维素转化率由原来的7.64%上升到18.14%,增长了10.5%。
The biotransformation research used corn cob as raw materials into xylitol
Xylitol as one of intermediate products of glucose metabolism in the human body,is widely used as a functional sweeteners.
In addition,xylitol can improve liver function,therapy acute and chronic hepatitis,acute myocarditis,diabetes,renal failure,etc.It is also used widely in chemical industry,light industry and national defense and other fields.
China is a large agricultural country,which products corncob more than 1000 million tons per year,but most of these resources are not fully utilized,and even resulted in different levels of environmental pollution.So how to use these plant fiber resources effective has become one of the important studies for many countries.Expansion of production of bioethanol utilizing lower-value lignocellulose offers a great potential for not only reducing the production cost but also protecting the environment as it produces far less air pollutants than gasline.D-Xylose is the most abundant monosaccharide in lignocellulose hydrolysate after glucose.Consequently,ethanolic fermentation of xylose is of major concern for the efficient utilization of lignocellulosic hydrolysates to produce fuel ethanol.Therefore,to obtain a bacterial strain that can largely use xylose to produce ethanol is always a hot topic within this area.
This study was based on microbiology theory.According to habitation of xylose zymogenic,a bacterium,which can ferment xylose to product ethanol,was filtered from natural environment.It was identified by analyzing its physiological and biochemical characteristics and applying several molecule biological means.Then the parameters of technique of fermenting xylose to product ethanol were optimized,to lay a foundation of the cellulose to the alcohol production technique.The main content of this research is as follows.
1.After the first riddled of proliferation cultured,separating 36 kinds of bacteria,and then through the resumption of screening and the cell transformation,we found the capacity to product xylitol of the strain e was significantly higher than other strains,whose rate of xylose transformed is 18%,so we select the strain e for further study.Strain e is typical yeast colony morphology,its cell short-oval to spherical,budding reproduction,no ascospores,no ballistospore,formation a large number of pseudohyphal hypha in the coverslip on corn meal agar culture medium.Strain e can assimilate potassium nitrate,ethanol,but cannot produce like-starch.
2.Applying modern molecular biology techniques to classify and identify the screened strain e,the results as follow:strain e is the Trichosporon coremiiforme of trichosporon.The registration number of nucleotide sequence of its D1/D2 of rDNA genes is FJ491404 in GenBank.
3.Obtained fermentation xylitol best conditions through single-factor test on the shake-flask are as follows:carbon concentration of 40g / 1,nitrogen concentration of 6 g / 1, pH 7,inoculation size 6%,and liquid 60ml,and selected seed age is 23 hours,37℃fermentation for 4 days.
4.Based on the single factor experiment,we do orthogonal test,which optimum conditions are carbon source concentration,40g/l;nitrogen source concentration,6g/l;initial pH,7.0;inoculum size,6%,the same to the single factor experiment,under this optimum condition,the conversion rate of xylitol reaches 69.1%.By optimizing the design,the conversion rate of the xylitol advanced 51.1%than initial.The conversion rate of Trichosporon coremiiforme is no significant differences compare to the yeast strains used for xylitol fermentation at domestic and abroad.However,in low-carbon source concentration, Trichosporon coremiiforme has particularly high conversion rate,but the corn cob,straw and other crops after the initial degradation has the low concentrations of monosaccharide,so Trichosporon coremiiforme is more suitable for the biological transformation of the initial degradation products of the corn cob,straw and other crop,which is important in comprehensive utilization of waste and environmental protection.
5.In the production process from corn cob to xylose,pretreatment have an important impact on the production of xylose,and bacterial B7 pre-treats corncob for 2 days more appropriate,which process of the optimum content of corn cob is 10%.Through the pretreatment,hemicellulose conversion rate increased from 7.64%to 18.14%,rised 10.5%. Although hemicellulose is not very high conversion rate,only 18.14%,and xylose output is only 6.92g / 1,but the double strains step-by-step approach has a great development prospects, because the method uses two types of bacteria,bacteria B7 and Aspergillus niger,with the advantages of simple raw materials and low cost compare to the traditional enzyme approach and high-temperature cooking method and has the environment protection superiority.Besides this,the double strains step-by-step approach utilizes the scrap corn cob,reached the goal of waste utilization.We believe that after further study,double strains step-by-step approach will attain the satisfactory results in all respects.
In this paper,we screen out the strain using xylose produce xylitol,confirm the appropriate conditions for xylitol production,and through corncob xylose conversion to a preliminary study,create the double strains step-by-step approach,based for the multi-strains treatment method.Applying the double strains step-by-step approach not only solve the com cob environment pollution problem,but also obtain high output of xylitol,so it has practical application of high significance.
本论文以发酵工程的理论为指导,从自然环境中分离筛选出一株发酵木糖产木糖醇的菌株,对该菌株进行了鉴定,同时对其发酵木糖生产木糖醇的工艺进行了参数优化,为工业化生产木糖醇奠定了一定基础。研究内容与结果如下:
1.从玉米芯中筛选出产木糖醇的菌种菌株e,通过形态学鉴定,菌株e有典型的酵母菌落形态,能同化硝酸钾,同化乙醇,不能产生类淀粉化合物。
2.对筛选出的菌株e进行分子生物学鉴定,鉴定结果:菌株e为丝孢酵母属中的Trichosporon coremiiforme。
3.通过单因素试验得出摇瓶上的木糖醇最佳发酵条件为:碳源浓度为40g/l,氮源浓度为6g/l,pH为7,接种量为6%,装液量为60ml,选取种子龄为23h,37℃下发酵4d。
4.在单因素实验的基础上进行了正交试验,正交试验最佳工艺条件为:碳源浓度为40g/l,氮源浓度为6g/l,pH为7,接种量为6%,在此最优条件下木糖醇转化率达到69.1%。通过优化设计比初始木糖醇转化率提高了51.1%。
5.从玉米芯到木糖的生产过程中,预处理对木糖产量有重要影响,细菌B7对玉米芯预处理2天较为合适,预处理过程中最适玉米芯添加量为10%。通过预处理,半纤维素转化率由原来的7.64%上升到18.14%,增长了10.5%。
The biotransformation research used corn cob as raw materials into xylitol
Xylitol as one of intermediate products of glucose metabolism in the human body,is widely used as a functional sweeteners.
In addition,xylitol can improve liver function,therapy acute and chronic hepatitis,acute myocarditis,diabetes,renal failure,etc.It is also used widely in chemical industry,light industry and national defense and other fields.
China is a large agricultural country,which products corncob more than 1000 million tons per year,but most of these resources are not fully utilized,and even resulted in different levels of environmental pollution.So how to use these plant fiber resources effective has become one of the important studies for many countries.Expansion of production of bioethanol utilizing lower-value lignocellulose offers a great potential for not only reducing the production cost but also protecting the environment as it produces far less air pollutants than gasline.D-Xylose is the most abundant monosaccharide in lignocellulose hydrolysate after glucose.Consequently,ethanolic fermentation of xylose is of major concern for the efficient utilization of lignocellulosic hydrolysates to produce fuel ethanol.Therefore,to obtain a bacterial strain that can largely use xylose to produce ethanol is always a hot topic within this area.
This study was based on microbiology theory.According to habitation of xylose zymogenic,a bacterium,which can ferment xylose to product ethanol,was filtered from natural environment.It was identified by analyzing its physiological and biochemical characteristics and applying several molecule biological means.Then the parameters of technique of fermenting xylose to product ethanol were optimized,to lay a foundation of the cellulose to the alcohol production technique.The main content of this research is as follows.
1.After the first riddled of proliferation cultured,separating 36 kinds of bacteria,and then through the resumption of screening and the cell transformation,we found the capacity to product xylitol of the strain e was significantly higher than other strains,whose rate of xylose transformed is 18%,so we select the strain e for further study.Strain e is typical yeast colony morphology,its cell short-oval to spherical,budding reproduction,no ascospores,no ballistospore,formation a large number of pseudohyphal hypha in the coverslip on corn meal agar culture medium.Strain e can assimilate potassium nitrate,ethanol,but cannot produce like-starch.
2.Applying modern molecular biology techniques to classify and identify the screened strain e,the results as follow:strain e is the Trichosporon coremiiforme of trichosporon.The registration number of nucleotide sequence of its D1/D2 of rDNA genes is FJ491404 in GenBank.
3.Obtained fermentation xylitol best conditions through single-factor test on the shake-flask are as follows:carbon concentration of 40g / 1,nitrogen concentration of 6 g / 1, pH 7,inoculation size 6%,and liquid 60ml,and selected seed age is 23 hours,37℃fermentation for 4 days.
4.Based on the single factor experiment,we do orthogonal test,which optimum conditions are carbon source concentration,40g/l;nitrogen source concentration,6g/l;initial pH,7.0;inoculum size,6%,the same to the single factor experiment,under this optimum condition,the conversion rate of xylitol reaches 69.1%.By optimizing the design,the conversion rate of the xylitol advanced 51.1%than initial.The conversion rate of Trichosporon coremiiforme is no significant differences compare to the yeast strains used for xylitol fermentation at domestic and abroad.However,in low-carbon source concentration, Trichosporon coremiiforme has particularly high conversion rate,but the corn cob,straw and other crops after the initial degradation has the low concentrations of monosaccharide,so Trichosporon coremiiforme is more suitable for the biological transformation of the initial degradation products of the corn cob,straw and other crop,which is important in comprehensive utilization of waste and environmental protection.
5.In the production process from corn cob to xylose,pretreatment have an important impact on the production of xylose,and bacterial B7 pre-treats corncob for 2 days more appropriate,which process of the optimum content of corn cob is 10%.Through the pretreatment,hemicellulose conversion rate increased from 7.64%to 18.14%,rised 10.5%. Although hemicellulose is not very high conversion rate,only 18.14%,and xylose output is only 6.92g / 1,but the double strains step-by-step approach has a great development prospects, because the method uses two types of bacteria,bacteria B7 and Aspergillus niger,with the advantages of simple raw materials and low cost compare to the traditional enzyme approach and high-temperature cooking method and has the environment protection superiority.Besides this,the double strains step-by-step approach utilizes the scrap corn cob,reached the goal of waste utilization.We believe that after further study,double strains step-by-step approach will attain the satisfactory results in all respects.
In this paper,we screen out the strain using xylose produce xylitol,confirm the appropriate conditions for xylitol production,and through corncob xylose conversion to a preliminary study,create the double strains step-by-step approach,based for the multi-strains treatment method.Applying the double strains step-by-step approach not only solve the com cob environment pollution problem,but also obtain high output of xylitol,so it has practical application of high significance.
引文
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