白腐菌连续开放预处理秸秆提高糖化率研究
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摘要
利用秸秆进行燃料乙醇的生产,既可以废物利用,也可以缓解当前化石能源日益紧缺的危机。利用白腐菌预处理秸秆,可以提高秸秆在燃料乙醇生产工艺中的糖化效率,而进行白腐菌预处理,一个关键的问题就是开放模式的解决。
本论文通过对实验室常用菌株的筛选,成功构建出两类开放体系:(1)本身利于在秸秆上开放生长,但糖化效果不佳的Bp2开放体系;(2)糖化效果好,但却不利于在秸秆上开放培养的2538开放体系。在此基础上,成功地实现了两种开放模式的连续化。
培养条件单因子试验结果表明:培养条件中培养时间和接种量对两种开放体系影响效果显著,其中培养天数极其显著;而基质含水量与含水pH值不显著,但两因素对糖化率的影响存在着一个有利范围。通过正交试验优化出Bp2开放体系的最佳培养条件为:培养时间为15天, pH值为7.0,接种量为38.5%,基质含水量为77.8%;2538开放体系的最佳培养条件为:培养天数为15天, pH值为3.0,接种量为42.9%,含水量为71.4%。在最佳条件下,两者糖化率分别为32.3%和44.6%,分别比对照提高了26.2%和69.8%。
通过对Bp2和2538开放体系的连续实验实现了初步的放大,Bp2可以成功实现三批连续开放,使得糖化效果明显提高。2538连续开放虽然随着培养批次的增加糖化效果变差,但可成功实现五批连续开放,且糖化效果比对照明显提高。影响连续开放培养批数的一个关键原因是体系中杂菌的增多,以致影响白腐菌在连续开放培养体系中占据主导生态位。
本论文首次成功构建了秸秆固体发酵两类连续开放预处理模式,探讨了培养条件对开放体系糖化效果的影响规律,优化了两类体系的最佳培养条件,对秸秆转化燃料乙醇研究的放大开放预处理提供了一定的理论和技术支持。
Production of ethanol by using corn straw can not only reduce the straw wastes, but also alleviates the pressure of the lacking of fossil fuel. The pretreatment of corn straw by white-rot fungus can improve the saccharification ratio of the production of fuel ethanol. A key technology of the biopretreatment is non-sterile conditions of the fermentation system.
In this paper, two kinds of non-sterile fermentation systems have been excogitated by screening 8 staple basidiomycetes in the lab :1, The Pleurotus ostreatus system., which has growth superiority in corn straw. But the saccharification efficiency of this system is discontented.2,the Echinodontium taxodii 2538 system, which make against the growth of the fungi. But the saccharification efficiency of this system is the best.
Based on the study of the relationship between fermentation conditions and saccharification efficiency, it is indicated that the influences of different factors differed a lot. Culture time is the most distinctive factor orthogonal experiment, inoculation volume is the second. Water content and pH value is indistinctive. Based on the orthogonal exprements , The optimized fermentation condition of the Pleurotus ostreatus system is: culture time 15 days, inoculation volume 38.5%, Water content 77.8%, pH value 7. The optimized fermentation condition of the Echinodontium taxodii 2538 system is: culture time 15 days, inoculation volume 42.9%, Water content 71.4%, pH value 3. Under the optimized fermentation condition, saccharification efficiency of the Pleurotus ostreatus system is 32.3%, which improved by 26.2%. saccharification efficiency of the Echinodontium taxodii 2538 system is 44.6%, which improved by 69.8%.
Based on the study of the continuous pretreatments by white rot fungi under non-sterile conditions, it is indicated that Pleurotus ostreatus system can continue three times with steady saccharification efficiency, the Echinodontium taxodii 2538 system can continue 5 times at least. The key factor of the continuous pretreatments by white rot fungi under non-sterile conditions is the amount of the infectant microorganism.
The study contrived the continuous pretreatments by white rot fungi under non-sterile conditions for the first time, and found out the relationship between fermentation conditions and saccharification efficiency. This paper provided important theoretic and practical significance for the ethanol production by using corn straw.
本论文通过对实验室常用菌株的筛选,成功构建出两类开放体系:(1)本身利于在秸秆上开放生长,但糖化效果不佳的Bp2开放体系;(2)糖化效果好,但却不利于在秸秆上开放培养的2538开放体系。在此基础上,成功地实现了两种开放模式的连续化。
培养条件单因子试验结果表明:培养条件中培养时间和接种量对两种开放体系影响效果显著,其中培养天数极其显著;而基质含水量与含水pH值不显著,但两因素对糖化率的影响存在着一个有利范围。通过正交试验优化出Bp2开放体系的最佳培养条件为:培养时间为15天, pH值为7.0,接种量为38.5%,基质含水量为77.8%;2538开放体系的最佳培养条件为:培养天数为15天, pH值为3.0,接种量为42.9%,含水量为71.4%。在最佳条件下,两者糖化率分别为32.3%和44.6%,分别比对照提高了26.2%和69.8%。
通过对Bp2和2538开放体系的连续实验实现了初步的放大,Bp2可以成功实现三批连续开放,使得糖化效果明显提高。2538连续开放虽然随着培养批次的增加糖化效果变差,但可成功实现五批连续开放,且糖化效果比对照明显提高。影响连续开放培养批数的一个关键原因是体系中杂菌的增多,以致影响白腐菌在连续开放培养体系中占据主导生态位。
本论文首次成功构建了秸秆固体发酵两类连续开放预处理模式,探讨了培养条件对开放体系糖化效果的影响规律,优化了两类体系的最佳培养条件,对秸秆转化燃料乙醇研究的放大开放预处理提供了一定的理论和技术支持。
Production of ethanol by using corn straw can not only reduce the straw wastes, but also alleviates the pressure of the lacking of fossil fuel. The pretreatment of corn straw by white-rot fungus can improve the saccharification ratio of the production of fuel ethanol. A key technology of the biopretreatment is non-sterile conditions of the fermentation system.
In this paper, two kinds of non-sterile fermentation systems have been excogitated by screening 8 staple basidiomycetes in the lab :1, The Pleurotus ostreatus system., which has growth superiority in corn straw. But the saccharification efficiency of this system is discontented.2,the Echinodontium taxodii 2538 system, which make against the growth of the fungi. But the saccharification efficiency of this system is the best.
Based on the study of the relationship between fermentation conditions and saccharification efficiency, it is indicated that the influences of different factors differed a lot. Culture time is the most distinctive factor orthogonal experiment, inoculation volume is the second. Water content and pH value is indistinctive. Based on the orthogonal exprements , The optimized fermentation condition of the Pleurotus ostreatus system is: culture time 15 days, inoculation volume 38.5%, Water content 77.8%, pH value 7. The optimized fermentation condition of the Echinodontium taxodii 2538 system is: culture time 15 days, inoculation volume 42.9%, Water content 71.4%, pH value 3. Under the optimized fermentation condition, saccharification efficiency of the Pleurotus ostreatus system is 32.3%, which improved by 26.2%. saccharification efficiency of the Echinodontium taxodii 2538 system is 44.6%, which improved by 69.8%.
Based on the study of the continuous pretreatments by white rot fungi under non-sterile conditions, it is indicated that Pleurotus ostreatus system can continue three times with steady saccharification efficiency, the Echinodontium taxodii 2538 system can continue 5 times at least. The key factor of the continuous pretreatments by white rot fungi under non-sterile conditions is the amount of the infectant microorganism.
The study contrived the continuous pretreatments by white rot fungi under non-sterile conditions for the first time, and found out the relationship between fermentation conditions and saccharification efficiency. This paper provided important theoretic and practical significance for the ethanol production by using corn straw.
引文
[1]钱伯章.世界能源的消费现状和可再生能源发展趋势(上).节能与环保, 2005, 3: 8-11
[2]徐绍峰.中国能源状况与经济社会可持续发展分析.经济论坛, 2005, 7: 8-12
[3]王新新.中国的能源安全与能源战略选择.中国科技论坛, 2007, 1: 17-22
[4]李俊峰,时璟丽,王仲颖.大力推进可再生能源的发展.可再生能源, 2006, 12: 23-27
[5]王小孟,谭江林,陈金珠.我国生物质能源开发利用的现状.江西林业科技, 2006, 5: 45-49
[6]孙振钧.巾国生物质产业及发展取向.农业下程学报, 2004, 20(5): 1-5
[7]杜风光,冯文生.燃料乙醇发展现状和前景展望.现代化工, 2006, 1: 6-10
[8]马永红,魏祯,陈清江等.基于随机前沿面的全球石化行业技术效率相关研究.现代化工, 2005, 25(3): 62-66
[9]丁喆.太阳赐予的礼物——生物质能.科技智囊, 2006, 10: 8-22
[10]黄忠水,纪威,鄂卓茂.我国开发燃料酒精的综合效益分析.节能, 2001, 12: 3-7
[11]张昆,吴桢,梅广.纤维素发酵生产燃料酒精研究粮食与油脂, 2007, 2: 10-13
[12]黄爱玲,周美华.玉米秸杆水解的酶法与稀酸法比较.东华大学学报(自然科学版), 2005, 5: 110-114
[13]张德强,黄镇亚,张志毅.木质纤维生物量一步法(SSF)转化成乙醇的研究进展.北京林业大学学报, 2001, TQ35113
[14]张继泉,孙玉英,关凤梅等.玉米秸秆稀硫酸预处理条件的初步研究..纤维素科学与技术, 2002, 2: 32-36
[15]庞晓华.纤维素酶成本大幅降低,生物基乙醇燃料推广将不日而期.化工生产与技术, 2005: 23
[16]张继泉,王瑞明,孙玉英.利用木质纤维素生产燃料酒精的研究进展.酿酒科技, 2003, 1: 39-42
[17] J.韦伯斯特(著),张素轩(译).第一版..真菌导论[M].中国林业出版社, 1982, 11
[18] Kirk T. K., Farrell R. L. Enzymatic“combustion”: the microbial degradation oflinin. Annual Review of Microbiology, 1987, 41: 465-505
[19] Eriksson K. E. L., Blanchette R. A, Ander. P. Microbial and Enzymatic Degradation of wood and wood components. Berlin Heidelberg: Springer Verlag Comparative Biochemistry and Physiology Part A: Physiology, 1991, 4: 691
[20] Andre F., Jaime R., Juanita F., et al. World Journal of Microbiology & Biotechnology, 2001, 17: 31-34
[21] David M. C., Andrew F. S., Taylor, et al. New Phytologist, 2001, 152(1): 151-155
[22] Pavel K., Alena K., Jaroslav V., et al. World Journal of Microbiology & Biotechnology, 1999, 15: 269-276
[23]李慧蓉.环境科学进展, 1996, 4(6): 69-77
[24] Argyropoulos, D. S., Menachem S. B. Lignin In: Eriksson K. E. L. Advances in Biochemical Engineering Biotechnology, 1997, 5(7): 127-158
[25] Hiromichi I., Masanori W., Yoichi H et al. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. Journal of Biotechnology, 2003, 8(5): 273-280
[26] Leidig E, Prusse U, Vorlop K. D., et al. Biotransformation of Poly R-478 by continuous cultures of PVAL-encapsulated Trametes versicolor under non-sterile conditions. Bioprocess Eng., 1999, 21: 5-12
[27] Libra J A., Borchert M., Banit S. Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions. Biotechnol. Bioeng., 2003, 82(6): 736-744
[28] Gao D., Wen X., Qian Y. Decolorization of reactive brilliant red K-2BP with the white rot fungi under non-sterile conditions. Chinese Science Bulletin, 2004, 49(9): 981-982
[29]李稳宏,吴大雄,高新等.麦秸纤维素酶解法产糖预处理工艺条件研究.西北大学学报(自然科学版), 1997, 27(3): 227-230
[30] Goering H. K, Van P. S. Forage Fibre Analysis USDA-ARS Agric. Handbook. Washington: Gov. Print, 1971: 357-598
[31]荚荣,汤必奎,张晓宾.藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用.生物工程学报, 2004, 20(2): 302-305
[32]吴坤,张世敏,朱显峰.木质素生物降解研究进展.河南农业大学学报, 2000, 34(4): 349-354
[33]林鹿,庞春生,杨柳.白腐菌对甘蔗渣木质纤维的降解及其影响因子(Ⅲ)——愈创木酚对木质纤维不同组分降解的影响.中国造纸学报, 2004, 19(1): 183-185
[34]林鹿,杨柳,庞春生.白腐菌对甘蔗渣木质纤维的降解及其影响因子(Ⅱ)——黎芦醇对木质纤维不同组分降解的影响.中国造纸学报, 2004, 19(1): 180-182
[35] Wang Y., Zhu T., Deng Z. Study on Activities of the Laccase and Polyphenol Oxidase of Corilus versicolor. Chinese Journal of Ecology, 1999, 18(6): 62-64
[36]丁少军,王传槐, Buswell J A.不同培养条件对云芝(Coriolus versicolor)木质素降解酶产酶影响的研究.纤维素科学与技术, 1994, 2(2): 36-46
[37] Glenn J. K., Gold M. J. Purification and characterization of an extracellular Mn(Ⅱ) dependent peroxidase from the lignin degrading basidiomycete Phanerochaete chrysosporium. Archives of Biochemistry and Biophysics, 1985, 242: 329-341
[38] Glenn J. K., Morgan M. A., Mayfield M. B. et al. An extracellular H2O2 requiring enzyme preparation involved in lignin biodegradation in white-rot basidiomycete Phanerochaete chrysosporium. Biochemical and biophysical Research Communications, 1983, 114: 1077-1083
[39] Hatakka A., Uusi-Rauva A. K. Degradation of 14C labeled poplar wood lignin by selected white-rot fungi. European Journal of Applied Microbiology and Biotechnology, 1983, 17: 235-242
[40] Kirk T. K., Farrell R. L. Enzymatic“combustion”: the microbial degradation of linin, Annual Review of Microbiology, 1987, 41: 465-505c
[2]徐绍峰.中国能源状况与经济社会可持续发展分析.经济论坛, 2005, 7: 8-12
[3]王新新.中国的能源安全与能源战略选择.中国科技论坛, 2007, 1: 17-22
[4]李俊峰,时璟丽,王仲颖.大力推进可再生能源的发展.可再生能源, 2006, 12: 23-27
[5]王小孟,谭江林,陈金珠.我国生物质能源开发利用的现状.江西林业科技, 2006, 5: 45-49
[6]孙振钧.巾国生物质产业及发展取向.农业下程学报, 2004, 20(5): 1-5
[7]杜风光,冯文生.燃料乙醇发展现状和前景展望.现代化工, 2006, 1: 6-10
[8]马永红,魏祯,陈清江等.基于随机前沿面的全球石化行业技术效率相关研究.现代化工, 2005, 25(3): 62-66
[9]丁喆.太阳赐予的礼物——生物质能.科技智囊, 2006, 10: 8-22
[10]黄忠水,纪威,鄂卓茂.我国开发燃料酒精的综合效益分析.节能, 2001, 12: 3-7
[11]张昆,吴桢,梅广.纤维素发酵生产燃料酒精研究粮食与油脂, 2007, 2: 10-13
[12]黄爱玲,周美华.玉米秸杆水解的酶法与稀酸法比较.东华大学学报(自然科学版), 2005, 5: 110-114
[13]张德强,黄镇亚,张志毅.木质纤维生物量一步法(SSF)转化成乙醇的研究进展.北京林业大学学报, 2001, TQ35113
[14]张继泉,孙玉英,关凤梅等.玉米秸秆稀硫酸预处理条件的初步研究..纤维素科学与技术, 2002, 2: 32-36
[15]庞晓华.纤维素酶成本大幅降低,生物基乙醇燃料推广将不日而期.化工生产与技术, 2005: 23
[16]张继泉,王瑞明,孙玉英.利用木质纤维素生产燃料酒精的研究进展.酿酒科技, 2003, 1: 39-42
[17] J.韦伯斯特(著),张素轩(译).第一版..真菌导论[M].中国林业出版社, 1982, 11
[18] Kirk T. K., Farrell R. L. Enzymatic“combustion”: the microbial degradation oflinin. Annual Review of Microbiology, 1987, 41: 465-505
[19] Eriksson K. E. L., Blanchette R. A, Ander. P. Microbial and Enzymatic Degradation of wood and wood components. Berlin Heidelberg: Springer Verlag Comparative Biochemistry and Physiology Part A: Physiology, 1991, 4: 691
[20] Andre F., Jaime R., Juanita F., et al. World Journal of Microbiology & Biotechnology, 2001, 17: 31-34
[21] David M. C., Andrew F. S., Taylor, et al. New Phytologist, 2001, 152(1): 151-155
[22] Pavel K., Alena K., Jaroslav V., et al. World Journal of Microbiology & Biotechnology, 1999, 15: 269-276
[23]李慧蓉.环境科学进展, 1996, 4(6): 69-77
[24] Argyropoulos, D. S., Menachem S. B. Lignin In: Eriksson K. E. L. Advances in Biochemical Engineering Biotechnology, 1997, 5(7): 127-158
[25] Hiromichi I., Masanori W., Yoichi H et al. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. Journal of Biotechnology, 2003, 8(5): 273-280
[26] Leidig E, Prusse U, Vorlop K. D., et al. Biotransformation of Poly R-478 by continuous cultures of PVAL-encapsulated Trametes versicolor under non-sterile conditions. Bioprocess Eng., 1999, 21: 5-12
[27] Libra J A., Borchert M., Banit S. Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions. Biotechnol. Bioeng., 2003, 82(6): 736-744
[28] Gao D., Wen X., Qian Y. Decolorization of reactive brilliant red K-2BP with the white rot fungi under non-sterile conditions. Chinese Science Bulletin, 2004, 49(9): 981-982
[29]李稳宏,吴大雄,高新等.麦秸纤维素酶解法产糖预处理工艺条件研究.西北大学学报(自然科学版), 1997, 27(3): 227-230
[30] Goering H. K, Van P. S. Forage Fibre Analysis USDA-ARS Agric. Handbook. Washington: Gov. Print, 1971: 357-598
[31]荚荣,汤必奎,张晓宾.藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用.生物工程学报, 2004, 20(2): 302-305
[32]吴坤,张世敏,朱显峰.木质素生物降解研究进展.河南农业大学学报, 2000, 34(4): 349-354
[33]林鹿,庞春生,杨柳.白腐菌对甘蔗渣木质纤维的降解及其影响因子(Ⅲ)——愈创木酚对木质纤维不同组分降解的影响.中国造纸学报, 2004, 19(1): 183-185
[34]林鹿,杨柳,庞春生.白腐菌对甘蔗渣木质纤维的降解及其影响因子(Ⅱ)——黎芦醇对木质纤维不同组分降解的影响.中国造纸学报, 2004, 19(1): 180-182
[35] Wang Y., Zhu T., Deng Z. Study on Activities of the Laccase and Polyphenol Oxidase of Corilus versicolor. Chinese Journal of Ecology, 1999, 18(6): 62-64
[36]丁少军,王传槐, Buswell J A.不同培养条件对云芝(Coriolus versicolor)木质素降解酶产酶影响的研究.纤维素科学与技术, 1994, 2(2): 36-46
[37] Glenn J. K., Gold M. J. Purification and characterization of an extracellular Mn(Ⅱ) dependent peroxidase from the lignin degrading basidiomycete Phanerochaete chrysosporium. Archives of Biochemistry and Biophysics, 1985, 242: 329-341
[38] Glenn J. K., Morgan M. A., Mayfield M. B. et al. An extracellular H2O2 requiring enzyme preparation involved in lignin biodegradation in white-rot basidiomycete Phanerochaete chrysosporium. Biochemical and biophysical Research Communications, 1983, 114: 1077-1083
[39] Hatakka A., Uusi-Rauva A. K. Degradation of 14C labeled poplar wood lignin by selected white-rot fungi. European Journal of Applied Microbiology and Biotechnology, 1983, 17: 235-242
[40] Kirk T. K., Farrell R. L. Enzymatic“combustion”: the microbial degradation of linin, Annual Review of Microbiology, 1987, 41: 465-505c