水稻秸秆同时糖化与乳酸发酵的研究
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摘要
植物生物质作为自然界最主要的碳水化合物储存物，包含纤维素、半纤维素和木质素三部分。因此，水稻秸杆的降解需要纤维素酶各组分与半纤维素酶的协同作用。本研究从自然界分离，并通过紫外线、γ-射线、亚硝基胍进行诱变，筛选到了适于水稻秸秆降解的纤维素酶产生菌T-LSQ-18，进行固体发酵条件的优化，探讨水稻秸秆降解过程中纤维素酶各组分及半纤维酶之间的配比对水稻秸杆水解的效果，当木霉T-LSQ-18与黑曲霉SP-77的固体酶(麥曲)的比例在6：1时，其水解水稻秸秆的效果最好。通过HPLC分析酶解液中的单糖与二糖；通过X-射线衍射分析水稻秸秆处理前后的纤维素结晶度的变化；扫描电子显微镜观测不同酶液对水稻秸秆纤维表面结构的影响，从不同侧面证实了纤维素酶各组分之间的协同关系。
在100ml、150ml的三角瓶与血清瓶中进行水稻秸秆的同时糖化与乳酸发酵条件的优化。采用分批培养，在B10F-2000 10L发酵罐中进行放大培养，通过对温度、pH、搅拌速度、溶解氧等参数的控制，初步优化了水稻秸秆同时糖化与乳酸发酵过程的10L发酵罐的放大培养工艺。通过各种发酵工艺的优化，使水稻秸杆的净减率达35.3％，乳酸生成量达22.3g／L。
Plant carbohydrate, the major reservior of fixed carbon in nature, is made up of cellulose, hemicellulose and lignin. The degradation of rice straw is required the cooperation of cellulase and xylanases. In this study, producing high eco-1, 4- B -D-glucanase , A Trichoderma Reesei-T-LSQ-18 is screened, which is isolated from nature and mutated by UV-ray,Co-ray and NTG. The solid fermentative medium of T-LSQ-18 and SP-77 is opitized. Through experiments of degrading race straw by different ratio fermentative mixture between T-LSQ-18 and SP-77, the effeciency of degrading race straw is the best when the ratio of T-LSQ-18 and SP-77 is six to one. Analysis of mono-sacchrides and di-sacchrides in enzyme degradative liquor by high pressure liquid chromatography,comparison of X-ray diffraction of race straw crystalline intensity before and after incubation with cellulases, scanning electron microscopy(SEM)investigating micrographical changes of rice straw structure after incubation with cellulases,reflect the cooperation of cellulases from different sides.
The conditions of Simultaneous Saccharification and Lactic acid fermentation of rice straw is optimized in 100mL and 150mL trigonal tank. Though batch cultivation, the fermentative condition of Simultaneous Saccharification and Lactic acid fermentation of rice straw in BIOF-2000 10L tank fermentaor is optimized by experients of temperature,pH,stirred rate and dissolved oxgen. At last,the weight of rice straw decreases 35.2 per cent,and the productivity of Lactic acid reach 22.3 g/L.
在100ml、150ml的三角瓶与血清瓶中进行水稻秸秆的同时糖化与乳酸发酵条件的优化。采用分批培养，在B10F-2000 10L发酵罐中进行放大培养，通过对温度、pH、搅拌速度、溶解氧等参数的控制，初步优化了水稻秸秆同时糖化与乳酸发酵过程的10L发酵罐的放大培养工艺。通过各种发酵工艺的优化，使水稻秸杆的净减率达35.3％，乳酸生成量达22.3g／L。
Plant carbohydrate, the major reservior of fixed carbon in nature, is made up of cellulose, hemicellulose and lignin. The degradation of rice straw is required the cooperation of cellulase and xylanases. In this study, producing high eco-1, 4- B -D-glucanase , A Trichoderma Reesei-T-LSQ-18 is screened, which is isolated from nature and mutated by UV-ray,Co-ray and NTG. The solid fermentative medium of T-LSQ-18 and SP-77 is opitized. Through experiments of degrading race straw by different ratio fermentative mixture between T-LSQ-18 and SP-77, the effeciency of degrading race straw is the best when the ratio of T-LSQ-18 and SP-77 is six to one. Analysis of mono-sacchrides and di-sacchrides in enzyme degradative liquor by high pressure liquid chromatography,comparison of X-ray diffraction of race straw crystalline intensity before and after incubation with cellulases, scanning electron microscopy(SEM)investigating micrographical changes of rice straw structure after incubation with cellulases,reflect the cooperation of cellulases from different sides.
The conditions of Simultaneous Saccharification and Lactic acid fermentation of rice straw is optimized in 100mL and 150mL trigonal tank. Though batch cultivation, the fermentative condition of Simultaneous Saccharification and Lactic acid fermentation of rice straw in BIOF-2000 10L tank fermentaor is optimized by experients of temperature,pH,stirred rate and dissolved oxgen. At last,the weight of rice straw decreases 35.2 per cent,and the productivity of Lactic acid reach 22.3 g/L.
引文
[1] 夏黎明．可再生纤维索资源酶法降解的研究进展．林产化工通讯．1999，(33)：23～27
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[4] 林风．纤维素酶的生物化学和分子生物学研究进展．生命科学，1994，6(1)：19～21
[5] 陈育如，夏黎明．纤维废渣水解及L-乳酸发酵的研究．高校化学工程学报,
2000,14(5):437～447
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[8] Lu ZhaoXin,Minom Kumakura New immobilization method of filaments cells with thin paper carrier surfaces modified by radiation[J]Jmicro Methods, 1994,19(1):29～38
[9] 郭德宪，曹健．利用生物技术降解纤维素的研究进展．郑州工程学院学报 2001．22．3．82～86
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[12] 王丹，林建强．直接生物转化纤维素类资源生产燃料乙醇的研究进展．山东农业大学学报，2002，3(4)：525～529
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[14] Okada H D D Tada K, Molexular characterization and heterologous expression of the gene encoding a low-moleculan-mass endoglucannase from Trichoderma reesei QM9414 Applied and Environmental Microbiolgy 1998 64 (2):555～563
[15] 汪天虹，刘纯强．黄单胞菌β葡萄糖苷酸在大肠杆菌中的克隆和表达．遗传，1991，13(3)：14～18
[16] 林风．纤维素酶的生物化学和分子生物学研究进展．生命科学，1994，6(1)：19～21
[17] Henrissar B Enzymatic hydrolysis of cellulose. Gene, 1989,81(7):83～95
[18] Nideteky B, Steiner W, Enzyme systems for ligo-celluloses degra[19] Harkki A et al, Enzyme Microb. Techool. 1991,13:227
[19] Harkki A et al, Enzyme Microb.. Techool. 1991,13:227
[20] Holtzapple M T el al, Biotexhnol, Bioeng, 1994,44:1122
[21] Hideki Baba. Development of bioconversion of cellulosic wastes [J]. Microb Technol, 1993,(11):18～26
[22] Nidetzky B, Steioer S, Hayn M, Claeyussens M. Biochem. J. 1994,29,705
[23] Enari T Mel al. CRC Critical Reviews in Biotechnology, 1987,5(1):67
[24] Awafo V A el al, Applied Biochemistry and Biotechnology. 1996,57/58:461
[25] Fujii M, Homma T, Ooshima K, and Taniguchi M, Ap-plied Biochemistry and Biotechnology, 1991,28/29;145
[26] Vanhanen S et al, Curr. Genet. 1989,15;181
[27] Barmett C C et al, Bio/Technology, 1991,9:562
[28] Stangl H el al, Curr. Genet, 1993,23:115
[29] Chahal P S, Chahal D S, and Le G B B. Applied Bio-chemistry and Biotechnology 1996,57/58:433
[30] Tao S, Beihui L and Zuohu L J Chem Tech Biotech-nol, 1997,69;429
[31] Simons H A et al, DSS Contract 43SS KN107-14416. 1985:4-5
[32] Beltrame P L et al. Bioresource Technol 1992.39.165
[33] Okada H Sekiya T Yodoyama K, et al efficient secretion of Trichoderma rcesei callobiohydronlase II in schizosaecharonlyces pornbe and characterization of it products. Appl Microbal Biotechnol; 1998,49:301～308
[34] Patterson J A Cellulase enzyme[J] Microb Technol, 1989,(11):187～189
[35] STERNBERG D Fronrier in bioprocessing[J] Microbial, 1987,(23):139～147
[36] 艾高灿．麯霉与木霉纤维素酶基因组的属间遗传表达相容性．微生物学报，1998，38(3)：186～192
[37] Grethieim H E and Saddller J N, Appl Biochem. Biotechnol. 1992,34/35,37
[38] Ramos L P, Breuil C and Saddller J N Appl, Biochem Biotechnol, 1992,34/35,37
[39] Thompson D N Chen H C and Grethlein H E Bioresource Technol, 1991,39;155
[40] Well J Westgate P, Kohlman K and Ladisch M R enzyme Microb Techmol, 1994,16;1002
[41] Himmel M E el al , Handbook on Bioethnol; Production and Utilization (Edited by Wyman C E), Taylor&Fan-cis, 1996,143～161
[42] 王淑军．纤维酶酶解稻壳的条件试验 微生物通报， 1995，6：191～195
[43] 陈育如，夏黎明．植物纤维原料预处理技术的研究进展，化工进展，1999，(4)：24～26
[44] 卢雪梅，马登波，高培基．木素生物降解的分了生物学进展．纤维素科学与技术．1994，21～24
[45] 陈育如，夏黎明．纤维素废渣酶水解及L-乳酸发酵的研究．高校化学工程学报，2000，14(5)：437～441
[46] 郑佐兴，刘祖同，孙之荣．乳酸生产菌的选育及发酵的研究．清华大学学报，1990，30(6)：21～27
[2] 邹仪明．植物纤维素化学(第2版)[M]．北京，中国轻工业出版社，1995，152～154
[3] Gillkes N R Hydrulysis of cellulose[J]. Biores. Technol, 1991,5(3):21-35
[4] 林风．纤维素酶的生物化学和分子生物学研究进展．生命科学，1994，6(1)：19～21
[5] 陈育如，夏黎明．纤维废渣水解及L-乳酸发酵的研究．高校化学工程学报,
2000,14(5):437～447
[6] Esterbauer H et al, Bioresources Technology. 1991,36～51
[7] Kluosov A A Symposium on enzymatic hydrolysis of cellulose[J]. Biochem,1990(29):10577-10585
[8] Lu ZhaoXin,Minom Kumakura New immobilization method of filaments cells with thin paper carrier surfaces modified by radiation[J]Jmicro Methods, 1994,19(1):29～38
[9] 郭德宪，曹健．利用生物技术降解纤维素的研究进展．郑州工程学院学报 2001．22．3．82～86
[10] Stockton B C et al, Biotechnol. Lett. 1991,13,57
[11] Duff S J B.Cooper D G, and Fuller O M, Enzyme Mi-crob. Technol. 1987,9;47
[12] 王丹，林建强．直接生物转化纤维素类资源生产燃料乙醇的研究进展．山东农业大学学报，2002，3(4)：525～529
[13] Grajek W. Enzyme Microb. Technol. 1987,9:744
[14] Okada H D D Tada K, Molexular characterization and heterologous expression of the gene encoding a low-moleculan-mass endoglucannase from Trichoderma reesei QM9414 Applied and Environmental Microbiolgy 1998 64 (2):555～563
[15] 汪天虹，刘纯强．黄单胞菌β葡萄糖苷酸在大肠杆菌中的克隆和表达．遗传，1991，13(3)：14～18
[16] 林风．纤维素酶的生物化学和分子生物学研究进展．生命科学，1994，6(1)：19～21
[17] Henrissar B Enzymatic hydrolysis of cellulose. Gene, 1989,81(7):83～95
[18] Nideteky B, Steiner W, Enzyme systems for ligo-celluloses degra[19] Harkki A et al, Enzyme Microb. Techool. 1991,13:227
[19] Harkki A et al, Enzyme Microb.. Techool. 1991,13:227
[20] Holtzapple M T el al, Biotexhnol, Bioeng, 1994,44:1122
[21] Hideki Baba. Development of bioconversion of cellulosic wastes [J]. Microb Technol, 1993,(11):18～26
[22] Nidetzky B, Steioer S, Hayn M, Claeyussens M. Biochem. J. 1994,29,705
[23] Enari T Mel al. CRC Critical Reviews in Biotechnology, 1987,5(1):67
[24] Awafo V A el al, Applied Biochemistry and Biotechnology. 1996,57/58:461
[25] Fujii M, Homma T, Ooshima K, and Taniguchi M, Ap-plied Biochemistry and Biotechnology, 1991,28/29;145
[26] Vanhanen S et al, Curr. Genet. 1989,15;181
[27] Barmett C C et al, Bio/Technology, 1991,9:562
[28] Stangl H el al, Curr. Genet, 1993,23:115
[29] Chahal P S, Chahal D S, and Le G B B. Applied Bio-chemistry and Biotechnology 1996,57/58:433
[30] Tao S, Beihui L and Zuohu L J Chem Tech Biotech-nol, 1997,69;429
[31] Simons H A et al, DSS Contract 43SS KN107-14416. 1985:4-5
[32] Beltrame P L et al. Bioresource Technol 1992.39.165
[33] Okada H Sekiya T Yodoyama K, et al efficient secretion of Trichoderma rcesei callobiohydronlase II in schizosaecharonlyces pornbe and characterization of it products. Appl Microbal Biotechnol; 1998,49:301～308
[34] Patterson J A Cellulase enzyme[J] Microb Technol, 1989,(11):187～189
[35] STERNBERG D Fronrier in bioprocessing[J] Microbial, 1987,(23):139～147
[36] 艾高灿．麯霉与木霉纤维素酶基因组的属间遗传表达相容性．微生物学报，1998，38(3)：186～192
[37] Grethieim H E and Saddller J N, Appl Biochem. Biotechnol. 1992,34/35,37
[38] Ramos L P, Breuil C and Saddller J N Appl, Biochem Biotechnol, 1992,34/35,37
[39] Thompson D N Chen H C and Grethlein H E Bioresource Technol, 1991,39;155
[40] Well J Westgate P, Kohlman K and Ladisch M R enzyme Microb Techmol, 1994,16;1002
[41] Himmel M E el al , Handbook on Bioethnol; Production and Utilization (Edited by Wyman C E), Taylor&Fan-cis, 1996,143～161
[42] 王淑军．纤维酶酶解稻壳的条件试验 微生物通报， 1995，6：191～195
[43] 陈育如，夏黎明．植物纤维原料预处理技术的研究进展，化工进展，1999，(4)：24～26
[44] 卢雪梅，马登波，高培基．木素生物降解的分了生物学进展．纤维素科学与技术．1994，21～24
[45] 陈育如，夏黎明．纤维素废渣酶水解及L-乳酸发酵的研究．高校化学工程学报，2000，14(5)：437～441
[46] 郑佐兴，刘祖同，孙之荣．乳酸生产菌的选育及发酵的研究．清华大学学报，1990，30(6)：21～27