纤维质酒精发酵的菌种选育及发酵条件的研究
摘要
本文研究了酿酒酵母(Saccharomyces cercvisiaeYT318)发酵纤维素物质(以玉米秸秆为原料)转化为燃料酒精的工艺过程，通过紫外线诱变法选育高产菌株YT318。YT318菌株与出发菌株YT18相比，产酒精能力提高了15％，发酵周期缩短了12小时。利用蒸汽爆破法对纤维质原料预处理其优化条件为：压力为1.2MPa，持压时间5min，总转化率为67.72％。从而建立了一种预处理原料对后期发酵作用效应的快速分析方法。
本文还对后期酒精发酵过程及营养因子进行了系统研究发现YT318对木糖、葡萄糖有较强的酒精发酵力。过高的糖浓度，有毒的物质，温度，酸度等都能够成为抑制发酵的因素。这些因素有些是在发酵前期起作用，如底物抑制作用和过高的酸度，有些则在发酵后期影响发酵的进行，如酒精和二氧化碳，及有机酸的抑制作用。值得注意的是本文讨论的各个因素的协同作用对发酵的抑制作用更强。
在发酵初期高于10％的总糖会产生抑制作用。因此发酵醪中低水平的底物浓度有利于酒精产量提高。采用分批补料的供糖方式的发酵可将原料利用率从82.62％提高到88.51％。因此在发酵初期底物抑制作用比发酵后期酒精的毒性作用要大。氮源的添加有利于酒精的生产。同样发酵条件下，添加氮源可以将发酵时间大幅度缩短。尿素是最佳的无机氮源，由于酵母细胞对氮源吸收利用特点，在发酵初期氮源需要保持较高水平。氧气有利于细胞生长和存活因子的生产，因此在发酵过程中保持一定量的分子氧对发酵有促进作用。
YT318菌株作为最佳工艺发酵菌对经预处理的玉米秸杆，最适发酵工艺条件：温度35℃，最佳摇床转速，100r/min，最适pH为5.5，经过60小时发酵，酒精浓度可达5.5V/V，残总糖在0.7％以下。表明该菌种对于实现纤维质原料酒精发酵是可行的。
This paper studied the technological process of fermenting cellulose to fuel ethanol by saccharomyces cercvisiae YT318 which was obtained through UV mutation. Comparing to original Saccharotnycete YT18, YT318 increased in ethanol production capacity by 15% and decreased in fermentation with 12h. The optimized conditions of preparing cellulose with vapour-exploding was: pressure at 1.2 Mpa, time 5 min, productivity 67.72%.
Systematic research on later part of alcohol fermentation and nutrient factors was conducted. It found that YT318 had great capability of alcohol-production on glucose and xylose. Excessive glucose density, toxious material, temperature, acid and so on could inhibit fermentation. Some factors took effect in earlier part of fermentation, while others in latter part. But the synergetic effects of these factors inhibited fermentation even more.
Earlier part of fermentation were inhibited at 10% total sugar concentration. Low substrate density was beneficial to improvement of ethanol output. Utilization ratio on raw material could increase from 82.62% to 88.51% in fed-batch fermentation. So it showed substrate inhibition in earlier part of fermentation took more effect that alcohol toxicity in latter part of fermentation.
Adding of nitrogen source helped produce alcohol and would shorten fermentation period. Area was the best inorganic nitrogen source which should maintain at high concentration at earlier part of fermentation because of saccharomycete's feature.
Oxygen was beneficial to cell growth and living factors production. It would enhance the fermentation if fixed amount of oxygen was maintained.
As the best saccharomycete to prepared corn stalk, YT318 had a suitable culture
conditions: temperature 35℃, flaker speed l00r/min, pH 5.5. Ethanol concentration reached 5.5 V/V after 60 h's fermentation and residual sugar was under 0.7%. This demonstrated that it was feasible to produce alcohol in fermentation on cellulose with YT318.
本文还对后期酒精发酵过程及营养因子进行了系统研究发现YT318对木糖、葡萄糖有较强的酒精发酵力。过高的糖浓度，有毒的物质，温度，酸度等都能够成为抑制发酵的因素。这些因素有些是在发酵前期起作用，如底物抑制作用和过高的酸度，有些则在发酵后期影响发酵的进行，如酒精和二氧化碳，及有机酸的抑制作用。值得注意的是本文讨论的各个因素的协同作用对发酵的抑制作用更强。
在发酵初期高于10％的总糖会产生抑制作用。因此发酵醪中低水平的底物浓度有利于酒精产量提高。采用分批补料的供糖方式的发酵可将原料利用率从82.62％提高到88.51％。因此在发酵初期底物抑制作用比发酵后期酒精的毒性作用要大。氮源的添加有利于酒精的生产。同样发酵条件下，添加氮源可以将发酵时间大幅度缩短。尿素是最佳的无机氮源，由于酵母细胞对氮源吸收利用特点，在发酵初期氮源需要保持较高水平。氧气有利于细胞生长和存活因子的生产，因此在发酵过程中保持一定量的分子氧对发酵有促进作用。
YT318菌株作为最佳工艺发酵菌对经预处理的玉米秸杆，最适发酵工艺条件：温度35℃，最佳摇床转速，100r/min，最适pH为5.5，经过60小时发酵，酒精浓度可达5.5V/V，残总糖在0.7％以下。表明该菌种对于实现纤维质原料酒精发酵是可行的。
This paper studied the technological process of fermenting cellulose to fuel ethanol by saccharomyces cercvisiae YT318 which was obtained through UV mutation. Comparing to original Saccharotnycete YT18, YT318 increased in ethanol production capacity by 15% and decreased in fermentation with 12h. The optimized conditions of preparing cellulose with vapour-exploding was: pressure at 1.2 Mpa, time 5 min, productivity 67.72%.
Systematic research on later part of alcohol fermentation and nutrient factors was conducted. It found that YT318 had great capability of alcohol-production on glucose and xylose. Excessive glucose density, toxious material, temperature, acid and so on could inhibit fermentation. Some factors took effect in earlier part of fermentation, while others in latter part. But the synergetic effects of these factors inhibited fermentation even more.
Earlier part of fermentation were inhibited at 10% total sugar concentration. Low substrate density was beneficial to improvement of ethanol output. Utilization ratio on raw material could increase from 82.62% to 88.51% in fed-batch fermentation. So it showed substrate inhibition in earlier part of fermentation took more effect that alcohol toxicity in latter part of fermentation.
Adding of nitrogen source helped produce alcohol and would shorten fermentation period. Area was the best inorganic nitrogen source which should maintain at high concentration at earlier part of fermentation because of saccharomycete's feature.
Oxygen was beneficial to cell growth and living factors production. It would enhance the fermentation if fixed amount of oxygen was maintained.
As the best saccharomycete to prepared corn stalk, YT318 had a suitable culture
conditions: temperature 35℃, flaker speed l00r/min, pH 5.5. Ethanol concentration reached 5.5 V/V after 60 h's fermentation and residual sugar was under 0.7%. This demonstrated that it was feasible to produce alcohol in fermentation on cellulose with YT318.
引文
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46. Jones, M., and Pierce, J. S. Adsorption of amino acids from wort by yeast.J. Inst. Brewing. 1964, 70:307-315.
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48．刘天福，赵华龄．农产品加工技术经济手册．上海：上海科学技术文献出版社，1987．199-201．
49. Thomas, K. C. & Ingledew, W. M., Fuel alcohol production: effects of free amino nitrogen of fermentation of very high gravity wheat mashes. Appl.Environ. Microbiol., 1990, 56: 2046-50.
50．任立伟，侯霖等．‘97中国吉林玉米深加工国际研讨会论文集，426-438
51．刘天福，赵华龄．工业微生物学．上海：上海科学技术文献出版社，1987．199-201．
52．杜连祥．工业微生物试验技术．北京：轻工出版社，2000．199-201．
53．贺延龄．厌氧纤维素分解菌的分离鉴定与草浆非纤维细胞的乙酸发酵，博士学位论文，天津工业学院，1988
54．赵昕，高培基．半纤维素的酒精发酵．工业微生物，1993，23(2)：25-27．
55．宋书玉，郭军英，张亚坤等．酸性蛋白酶用与酒精浓醪发酵的研究．酿酒科技，1997，
95(5)：55-56．
56．夏淑兰．现代微生物实验技术．北京：轻工业出版社．1988：36～48
57．黄宇彤，杜连祥．玉米原料高浓度酒精发酵中试的研究．天津轻工业学院学报，2002，1：56-58．
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2. Lynch J.M., Jappl. Bacterial. Symp. Suppl., 1987,71-83,
3．陈滔声．化学通报，1983，2：1-6
4．刘天福，赵华龄．农产品加工技术经济手册．上海：上海科学技术文献出版社，1987．199-201．
5．高寿清．我国酒精工业现状与玉米生产酒精展望，中国发酵工业协会理事会论文集．无锡：轻工业出版社，2000．93-97．
6. Carroll R.Keim et al, Biotech,1989,7,22-29
7. Fein, J. E. Biotech. Lett. 1984, 6:123-127.
8. Chung, I. S. and Lee, Y. Y. Enzyem Microb. Tech.1985, 7:217-21.
9. Ju. N. H. Biotech. Lett. 1984, 12:837-46.
10.Canlis Coelho de Lerralho,Neto. Int. Ind.,Biotech, 1987,6(6),232-235
11．志水一允．燃料化学原料与开发，1981，273
12．熊谷知荣子．发酵工学杂志，1982，60：77-84．
13．文钱桥等．中国酿造．1990，(7)：25～30
14．陈国符主编．植物纤维化学，轻工业出版社，1980
15. Tsao G.T.et al, Proc. Biochem, 1982,17,34-38
16. Kosaric, N., Ong, S. L. and Duvnjak, Z. Biotech. Bioeng. 1982, 24:691-70.
17. Cromie, S. and Daelle, H. W. Europe. J. Appl. Microb. Biotech.1981,11:116-121.
18．陈嘉翔编著．制浆化学，轻工业出版社，1990
19．陈嘉翔主编．制浆原理与工程，轻工业出版社，1990
20. Matsumoto, N., Fukuski, O., Miyanaga, M, etc. Agr. Biol. Chem.1982,46:1549-54.
21．邬义明主编．植物纤维化学(第二版)，轻工业出版社，1990
22. Rhee, S. K., Pagan, R. J., Wong, L. J. Ferment. Technol. 1984, 62:297-82.
23．马瑞德等．工业微生物，1980，1，23-25
24. Wayman et al. Biotech. Lett., 1996,80(10),742-752
25. Mandels,M. et al., Ferment Technol.,1976,54,267
26. Mandels,M. et al., Biotech. Bioeng.,1974,16,1471
27．志水等．木材学会志，1977，23，562
28．东顺一．高分子加工，1983，32(6)，30-35
29. Kumakra etal., Bioresour.Technol.,1991,32,189-195
30. Fan, L.T., Adv. Biochem. Eng/Biotechnol., 1982,23,158-187
31. Daelle, H. W. J. Appl. Microbiol. Biotech. 1982,16:136-42.
32. Galani, I., Drainas, C. and Typas, M. A. Biotech. Left. 1985, 7:673-79.
33. Koll et al, Chem. Int., Ed. Eng.,1988,34,754
34．杨斌．食品与发酵工业，1995，6，61-65
35. Goldstein I.S.et al,Biotech,1993,18(19),65
36. Lantero, O. J. and Fish, J. J., Process for producing ethanol. US Patent 5,231,017.27 July 1993.
37．许开天编著．酒精蒸馏技术，轻工业出版社，1990
38. Lee, J. S. the Marget Jones memorial Lecture: amino acids in malting and brewing. J. Inst. Brewing. 1982, 88:228-233.
39．江西食品发酵工业科学研究所．酶制剂生产和在食品工业中的应用，轻工业出版社，1977
40．李洪刚，段堂荣．纤维素酶在酒精生产中的应用，酿酒科技，1996，总75(3)：44-45．
41. Chen, L. F. and Gong, C. S. Appl. Microbiol. Biotech. 1986, 25:208-212.
42. Thomas, K. C. & Ingledew, W. M., Fuel alcohol production: effects of free amino nitrogen of fermentation of very high gravity wheat mashes. Appl.Environ. Microbiol., 1990, 56: 2046-50.
43. Jones, A. M., Thomas, K. C. & Ingledew, W. M., Ethanolic fermentation of blackstrap molasses and sugar cane juice using very high gravity technology.J. Agric. Food Chem., 1994, 42:1242-6.
44. Thomas, K. C. & Ingledew, W. M., production of 21% (v/v)ethanot by fermentation of very high gravity (VHG) wheat mashes. J. Ind. Microbiol., 1992, 10:61-8.
45. Ingledew, W. M., Jones, A. M., Bhattry, R. S. & Rossnagel, B. G., Fuel alcohol production from hullless barley. Cereal Chem., 1995, 72: 147-50.
46. Jones, M., and Pierce, J. S. Adsorption of amino acids from wort by yeast.J. Inst. Brewing. 1964, 70:307-315.
47. Leao, C. and Van Uden, N. Effects of ethanol and other alkanols on passive proton influx in the yeast Saccharomyces cerevisiae. Biochem. Biophys. Acta.1984, 774:43-48.
48．刘天福，赵华龄．农产品加工技术经济手册．上海：上海科学技术文献出版社，1987．199-201．
49. Thomas, K. C. & Ingledew, W. M., Fuel alcohol production: effects of free amino nitrogen of fermentation of very high gravity wheat mashes. Appl.Environ. Microbiol., 1990, 56: 2046-50.
50．任立伟，侯霖等．‘97中国吉林玉米深加工国际研讨会论文集，426-438
51．刘天福，赵华龄．工业微生物学．上海：上海科学技术文献出版社，1987．199-201．
52．杜连祥．工业微生物试验技术．北京：轻工出版社，2000．199-201．
53．贺延龄．厌氧纤维素分解菌的分离鉴定与草浆非纤维细胞的乙酸发酵，博士学位论文，天津工业学院，1988
54．赵昕，高培基．半纤维素的酒精发酵．工业微生物，1993，23(2)：25-27．
55．宋书玉，郭军英，张亚坤等．酸性蛋白酶用与酒精浓醪发酵的研究．酿酒科技，1997，
95(5)：55-56．
56．夏淑兰．现代微生物实验技术．北京：轻工业出版社．1988：36～48
57．黄宇彤，杜连祥．玉米原料高浓度酒精发酵中试的研究．天津轻工业学院学报，2002，1：56-58．
58. Kunkee, R. E. and Bisson, L. F., Wine making yeasts. In The Yeasts, London:Academic Press. 1993, 2nd, Vol.5, 69-127.
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