青霉液态发酵生产纤维素酶的研究
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摘要
纤维素酶可用于水解纤维素,提高其活性并降低其成本,对纤维素原料的生物转化具有重要意义。本论文以山东大学微生物技术国家重点实验室提供的纤维素酶产生菌—青霉JU-A10作为发酵菌株,主要优化了该菌株产纤维素酶的发酵培养基组成和发酵条件,主要结果如下：
(1)通过考察种子培养时间、接种量、装液量、温度、转速和初始pH对产酶的影响,确定了纤维素酶液态发酵的适宜条件为：种子培养时间32h,接种量10%,装液量100mL,温度30℃,转速200r/min,初始pH5.0。在此条件下滤纸酶活和CMC酶活分别提高61.57%和26.68%。
(2)考察了不同纤维素原料,如木糖渣、汽爆高梁杆,S.F和微晶纤维素等对纤维素酶发酵的影响。结果表明,经过汽爆预处理的高粱秆所产酶活均高于未做任何处理的高粱秆,以3：1的液固比在0.75%的硫酸溶液中浸泡12h后,在3MP的压力下汽爆150s最有利于产酶,滤纸酶活和CMC酶活与未作任何处理的高粱秆相比可分别提高86.86%和111.06%,与以木糖渣为原料相比分别提高了13.46%和22.00%。微晶纤维素和S.F不仅可以作为碳源,同时对纤维酶的合成具有很好的诱导作用。微晶纤维素的效果远优于S.F,当加入1%的微晶纤维素,发酵96h时,滤纸酶活和CMC酶活均最高,与不加微晶纤维素时相比分别提高了45.41%和41.52%。以汽爆高梁杆为原料,论文还考察了无机盐如硫酸镁和碳酸钙对产酶的影响,结果表明其最佳添加量分别为0.05%和0.5%。滤纸酶活可分别提高28.00%和15.60%,CMC酶活可分别提高33.33%和6.56%。
(3)确定了青霉JU-A10的生长期和产酶期分别为36h前和36h后,最佳生长温度和最佳产酶温度分别为32℃和28℃。通过对发酵过程温度分段控制,使滤纸酶活和CMC酶活与30℃恒温发酵相比分别提高了15.1%和14.42%。初步确定了青霉JU-A10的适宜生长pH范围和适宜产酶pH范围分都在5.0左右。通过对pH值的在线控制,发现当发酵过程的pH控制在5.0时,滤纸酶活和CMC酶活与只调初始pH值相比分别提高了35.01%和51.21%。
Cellulase could be used for cellulose hydrolysis.Increasing its activity and lowering its production cost was very important for lignocellulose bioconversion.This paper mainly optimize the fermentation medium and fermentation conditions of the fungi-Penicillium strain JU-A10 provided by State Key Laboratory of Microbial Technology, Shandong University. The main experimental results weresummarized as follows:
(1)Mainly studied the influence of seed cultivation time、inoculum、medium volume、temperature、speed and initial pH on cellulase activity, and the result indicated that the optimization fermentation conditoin composed:seed cultivation time 32h, inoculum 10%, medium volume 100ml, temperature 30℃, speed 200r/min and the initial pH5.0. Under these conditions, the filter paper activity and CMC activity increased by 61.57% and 26.68%.
(2)Mainly studied the influence of different cellulose materials, such as xylose residue、sorghum bar steam explosion S.F and microcrystalline cellulose on cellulase activity. The results showed that after steam explosion pretreatment of sorghum straw produced higher cellulase activity than no treatment of sorghum straw and to a 3:1 ratio of liquid solution at 0.75% sulfuric acid after 12h immersion, under pressure in 3MP steam explosion 150s is best for cellulase activity.In this condition, the filter paper activity and CMC activity increased by 86.86% and 111.06% compared to without any treatment of sorghum straw, and increased by 13.46% and 22.00% compared to xylose residues as raw materials increased. Microcrystalline cellulose and SF not only as a carbon source,but they have a good induction on the synthesis on cellulase. The effect of microcrystalline cellulose is far better than SF.When adding 1% microcrystalline cellulose, fermentation 96h, the filter paper activity and CMC activity were the highest, and were increased by 45.41% and 41.52% compared to without microcrystalline cellulose. To steam explosion sorghum as raw material rod, the paper also studied the influence of inorganic salts such as magnesium sulfate and calcium carbonate on cellulase activity and the results indicated that the optimal dosage was 0.05% and 0.5%. Filter paper activity were increased by 28.00% and 15.60%, CMC activity were increased by 33.33% and 6.56%.
(3)The growing period and cellulase production period of Penicillium JU-A10 are before 36h and after 36h, and the optimal temperature of growth and cellulase production are 32℃and 28℃.By section on temperature control of fermentation process, the filter paper activity and CMC activity increased by 15.1% and 14.42% compared with the 30℃constant temperature fermentation. Initially identified the appropriate pH of the growing period and cellulase production period of Penicillium JU-A10 are both around 5.0. On-line control of pH indicated that when the pH control at 5.0 in whole fermentation process, the filter paper activity and CMC activity increased by 35.01% and 51.21% compared to only adjust the initial pH value and.
(1)通过考察种子培养时间、接种量、装液量、温度、转速和初始pH对产酶的影响,确定了纤维素酶液态发酵的适宜条件为：种子培养时间32h,接种量10%,装液量100mL,温度30℃,转速200r/min,初始pH5.0。在此条件下滤纸酶活和CMC酶活分别提高61.57%和26.68%。
(2)考察了不同纤维素原料,如木糖渣、汽爆高梁杆,S.F和微晶纤维素等对纤维素酶发酵的影响。结果表明,经过汽爆预处理的高粱秆所产酶活均高于未做任何处理的高粱秆,以3：1的液固比在0.75%的硫酸溶液中浸泡12h后,在3MP的压力下汽爆150s最有利于产酶,滤纸酶活和CMC酶活与未作任何处理的高粱秆相比可分别提高86.86%和111.06%,与以木糖渣为原料相比分别提高了13.46%和22.00%。微晶纤维素和S.F不仅可以作为碳源,同时对纤维酶的合成具有很好的诱导作用。微晶纤维素的效果远优于S.F,当加入1%的微晶纤维素,发酵96h时,滤纸酶活和CMC酶活均最高,与不加微晶纤维素时相比分别提高了45.41%和41.52%。以汽爆高梁杆为原料,论文还考察了无机盐如硫酸镁和碳酸钙对产酶的影响,结果表明其最佳添加量分别为0.05%和0.5%。滤纸酶活可分别提高28.00%和15.60%,CMC酶活可分别提高33.33%和6.56%。
(3)确定了青霉JU-A10的生长期和产酶期分别为36h前和36h后,最佳生长温度和最佳产酶温度分别为32℃和28℃。通过对发酵过程温度分段控制,使滤纸酶活和CMC酶活与30℃恒温发酵相比分别提高了15.1%和14.42%。初步确定了青霉JU-A10的适宜生长pH范围和适宜产酶pH范围分都在5.0左右。通过对pH值的在线控制,发现当发酵过程的pH控制在5.0时,滤纸酶活和CMC酶活与只调初始pH值相比分别提高了35.01%和51.21%。
Cellulase could be used for cellulose hydrolysis.Increasing its activity and lowering its production cost was very important for lignocellulose bioconversion.This paper mainly optimize the fermentation medium and fermentation conditions of the fungi-Penicillium strain JU-A10 provided by State Key Laboratory of Microbial Technology, Shandong University. The main experimental results weresummarized as follows:
(1)Mainly studied the influence of seed cultivation time、inoculum、medium volume、temperature、speed and initial pH on cellulase activity, and the result indicated that the optimization fermentation conditoin composed:seed cultivation time 32h, inoculum 10%, medium volume 100ml, temperature 30℃, speed 200r/min and the initial pH5.0. Under these conditions, the filter paper activity and CMC activity increased by 61.57% and 26.68%.
(2)Mainly studied the influence of different cellulose materials, such as xylose residue、sorghum bar steam explosion S.F and microcrystalline cellulose on cellulase activity. The results showed that after steam explosion pretreatment of sorghum straw produced higher cellulase activity than no treatment of sorghum straw and to a 3:1 ratio of liquid solution at 0.75% sulfuric acid after 12h immersion, under pressure in 3MP steam explosion 150s is best for cellulase activity.In this condition, the filter paper activity and CMC activity increased by 86.86% and 111.06% compared to without any treatment of sorghum straw, and increased by 13.46% and 22.00% compared to xylose residues as raw materials increased. Microcrystalline cellulose and SF not only as a carbon source,but they have a good induction on the synthesis on cellulase. The effect of microcrystalline cellulose is far better than SF.When adding 1% microcrystalline cellulose, fermentation 96h, the filter paper activity and CMC activity were the highest, and were increased by 45.41% and 41.52% compared to without microcrystalline cellulose. To steam explosion sorghum as raw material rod, the paper also studied the influence of inorganic salts such as magnesium sulfate and calcium carbonate on cellulase activity and the results indicated that the optimal dosage was 0.05% and 0.5%. Filter paper activity were increased by 28.00% and 15.60%, CMC activity were increased by 33.33% and 6.56%.
(3)The growing period and cellulase production period of Penicillium JU-A10 are before 36h and after 36h, and the optimal temperature of growth and cellulase production are 32℃and 28℃.By section on temperature control of fermentation process, the filter paper activity and CMC activity increased by 15.1% and 14.42% compared with the 30℃constant temperature fermentation. Initially identified the appropriate pH of the growing period and cellulase production period of Penicillium JU-A10 are both around 5.0. On-line control of pH indicated that when the pH control at 5.0 in whole fermentation process, the filter paper activity and CMC activity increased by 35.01% and 51.21% compared to only adjust the initial pH value and.
引文
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