微生物处理玉米秸秆生产蛋白饲料的研究
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摘要
从牛瘤胃中分离高产纤维素酶细菌，经纤维素刚果红平板初筛、摇瓶培养、固态发酵复筛、得到一株芽孢杆菌X4，该菌不仅可降解纤维素，且自身具固氮能力。最适生长PH为7左右，最适生长温度为37℃。在发酵过程中与其他菌株能很好共生。同时从牛瘤胃中分离、筛选得酵母Y2，实验证明该菌株与其他发酵菌株具良好配伍性，能利用混菌发酵产生的还原糖迅速生长，达到较高的生物量。
利用氨法和白腐真菌L：对玉米秸秆进行前期处理，依据多种微生物共生及代谢的特性，采用多菌种混合共发酵方式，将康宁木霉和选育出的高活性黑曲霉S",瘤胃细菌X4，酵母Y：，接种于前处理过的玉米秸秆上共发酵，利用正交试验，得出：在发酵温度30℃，PH5.0发酵9天后，粗蛋白含量达24.61％，纤维素降解率为48.37％。
对酵母Y：原生质体的制备、再生条件进行探索：采用对数生长前期(约24h)的菌体细胞，用1.5％蜗牛酶，0.1％p—巯基乙醇，处理60min，原生质体形成率可达95％，用0.6ml／l蔗糖做渗透稳定剂，原生质体再生效果最佳，再生率为6.32％。
采用He-Ne激光诱变酵母Y：原生质体，获得一株是出发菌株产生物量1.31倍的酵母Y：。经酯酶同工酶谱分析，其酶带条纹数，迁移率等均发生了明显变化，利用Y：和混菌共发酵实验结果表明，产物中粗蛋白可提高到28.10％，此时纤维素降解率为51.12％。
High-yield cellulase bacteria were isolated from rumen. After the primary screening test by cellulose-congo red agar medium, the second one by flask cultivation and then solid fermentation, A Bacillus X4 was obtained. This strain not only was capable of degrading cellulose, but also possessed the ability to fix Nitrogen. The optimum PH of it was at 7.0, The suitable temperature for the growth of )Q was about 37癈. The strain X4 was able to symbiosis well with other selected strains in the co-fermentation. The yeast called Y2 was separated from rumen too. The experiments showed that yeast Y2 matched better with mixed strains. And it grew quickly with reducing sugar which was produced by other selected strains in the co-fermentation resulting in higher biomass.
According to characteristics of the multi-microorganisms symbiosis and their metabolism, the corn straws were pretreated with the ammonia and white-rot fungi Lx, Trichoderma koningii Oud C413, high activity Aspergillus niger Sy, rumen bacterium X4 and rumen yeast Y2 were carefully selected and were inoculated on pretreated corn straws powder for multi-strains co-fermentation. The result of orthogonal experiment showed: with the fermentation temperature 30癈, PH 5.0, After 9 days, the contents of raw proteins reached 24.61% While the degradation ratio of cellulose was 48.37%.
The suitable condition of protoplast formation and regeneration of yeast Y2 were studied. The result of experiment showed that: the early exponential phase cells (about 24h) were easy to form protoplast, under the action of 1.5% snail enzyme and 0.1% (3 -mercaptoethanol, the ratio of protoplast formation was about 95% in 60 minutes. On the condition
that 0.6mol/l sucrose was used as osmotic stabilizer, the ratio of protoplast regeneration was the highest. The ratio of its regeneration was 6.32%.
A mutagenic strain called Y2' was achieved using irradiating protoplasts by He-Ne laser ( A =632.8nm). Its biomass yield increased as much as 1.31 times than that of starting strain. And it match excellently with other mixed strains. The esterase(EST) isoenzyme electropheresis bands of the mutagenic strain Y2' compared with that of starting strain changed dramatically. The result of co-fermentation utilizing 2' indicated the content of crude protein in production reached 28.10% while at the same time the degradation rate of cellulose came to 51.12%.
利用氨法和白腐真菌L：对玉米秸秆进行前期处理，依据多种微生物共生及代谢的特性，采用多菌种混合共发酵方式，将康宁木霉和选育出的高活性黑曲霉S",瘤胃细菌X4，酵母Y：，接种于前处理过的玉米秸秆上共发酵，利用正交试验，得出：在发酵温度30℃，PH5.0发酵9天后，粗蛋白含量达24.61％，纤维素降解率为48.37％。
对酵母Y：原生质体的制备、再生条件进行探索：采用对数生长前期(约24h)的菌体细胞，用1.5％蜗牛酶，0.1％p—巯基乙醇，处理60min，原生质体形成率可达95％，用0.6ml／l蔗糖做渗透稳定剂，原生质体再生效果最佳，再生率为6.32％。
采用He-Ne激光诱变酵母Y：原生质体，获得一株是出发菌株产生物量1.31倍的酵母Y：。经酯酶同工酶谱分析，其酶带条纹数，迁移率等均发生了明显变化，利用Y：和混菌共发酵实验结果表明，产物中粗蛋白可提高到28.10％，此时纤维素降解率为51.12％。
High-yield cellulase bacteria were isolated from rumen. After the primary screening test by cellulose-congo red agar medium, the second one by flask cultivation and then solid fermentation, A Bacillus X4 was obtained. This strain not only was capable of degrading cellulose, but also possessed the ability to fix Nitrogen. The optimum PH of it was at 7.0, The suitable temperature for the growth of )Q was about 37癈. The strain X4 was able to symbiosis well with other selected strains in the co-fermentation. The yeast called Y2 was separated from rumen too. The experiments showed that yeast Y2 matched better with mixed strains. And it grew quickly with reducing sugar which was produced by other selected strains in the co-fermentation resulting in higher biomass.
According to characteristics of the multi-microorganisms symbiosis and their metabolism, the corn straws were pretreated with the ammonia and white-rot fungi Lx, Trichoderma koningii Oud C413, high activity Aspergillus niger Sy, rumen bacterium X4 and rumen yeast Y2 were carefully selected and were inoculated on pretreated corn straws powder for multi-strains co-fermentation. The result of orthogonal experiment showed: with the fermentation temperature 30癈, PH 5.0, After 9 days, the contents of raw proteins reached 24.61% While the degradation ratio of cellulose was 48.37%.
The suitable condition of protoplast formation and regeneration of yeast Y2 were studied. The result of experiment showed that: the early exponential phase cells (about 24h) were easy to form protoplast, under the action of 1.5% snail enzyme and 0.1% (3 -mercaptoethanol, the ratio of protoplast formation was about 95% in 60 minutes. On the condition
that 0.6mol/l sucrose was used as osmotic stabilizer, the ratio of protoplast regeneration was the highest. The ratio of its regeneration was 6.32%.
A mutagenic strain called Y2' was achieved using irradiating protoplasts by He-Ne laser ( A =632.8nm). Its biomass yield increased as much as 1.31 times than that of starting strain. And it match excellently with other mixed strains. The esterase(EST) isoenzyme electropheresis bands of the mutagenic strain Y2' compared with that of starting strain changed dramatically. The result of co-fermentation utilizing 2' indicated the content of crude protein in production reached 28.10% while at the same time the degradation rate of cellulose came to 51.12%.
引文
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[7] 王仁振，秸秆类粗纤维饲料生化加工技术的研究，饲料工业，1999，20(3)：8～12
[8] T. Kent Kirt. Enzymatic "Combustion": The Microbial Degradation of lignin. Ann Rev. Microbiol. 1987. 41:465～505
[9] 卢雪梅，刘紫鹃，高培基，木质素生物降解的化学反应机制，林产化学与工业，1996，16(2)：75～82
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[11] 雷天富．秸秆饲料化加工技术，陕西省人民教育出版杜，1998：2～3
[12] 贾万福．农作物秸秆饲料的加工利用，中国畜牧杂志，1996，32(1)：43～44
[13] 卢庆萍，王加启．秸秆处理技术研究进展，中国饲料 1999(2)：7～9
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