可再生纤维素原料生物转化产乳酸的研究
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摘要
纤维素是地球上最丰富、最廉价的可再生资源。利用生物技术将纤维素转化成乳酸，具有重要的社会及经济意义。本文对纤维素酶的生产、纤维二糖酶的生产及固定化、纤维原料的酶法糖化、固定化乳酸杆菌的发酵、纤维二糖酶和乳酸杆菌的共固定化，以及利用串联式生物反应器协同酶解纤维素发酵乳酸等方面进行了研究。
采用液体深层发酵工艺，对纤维素酶生产过程中的主要工艺参数进行了优化，以廉价的木糖渣和豆饼粉作为里氏木霉(Trichoderma reesei ZU-04)的碳源和氮源，大幅度降低了生产成本，并在30 m~3发酵罐中成功地完成了放大试验，纤维素酶活力高达8.16 FPIU／mL(331.7 FPIU／g纤维素)，为纤维素酶的工业化应用打下了良好的基础。
筛选到一个纤维二糖酶高产菌株(黑曲霉Asperillus niger LR-12)，对其固态发酵条件进行了优化，纤维二糖酶活力达到了国际先进水平(438.3 CBIU／g干曲)。研究发现，该菌的孢子中富含纤维二糖酶，采用海藻酸钙凝胶包埋孢子可将纤维二糖酶有效固定。包埋后的黑曲霉孢子在酶解温度(50℃)下不会萌发，但可以在凝胶珠内缓慢地释放纤维二糖酶，与直接固定酶蛋白的传统方法相比，这种新型的固定化技术具有操作经济简便、对酶活力无破坏、固定化酶的半衰期长等优点。该固定化酶在纤维素酶的协同降解过程中具有明显的促进作用。
对纤维素酶解过程中底物性质、底物浓度、纤维素酶用量及酶系组成等关键因子进行了研究。由于里氏木霉纤维素酶系中纤维二糖酶活力很低(CBA／FPA为0.03)，对经稀酸预处理后的玉米芯纤维底物的酶解得率仅为67.4％。通过添加纤维二糖酶，将CBA／FPA增加到0.42，酶解得率可提高至83.6％。进一步利用里氏木霉纤维素酶和固定化纤维二糖酶的协同作用，纤维原料的酶解得率可高达88.2％。这方面的研究结果对于深入了解纤维素酶的协同降解机制具有重要意义。
采用海藻酸钙凝胶包埋固定德氏乳酸杆菌，固定化细胞与游离细胞相比，发酵时间缩短，乳酸得率提高，并能有效地利用纤维原料水解液进行乳酸发酵，乳酸得率可达90.7％。
将纤维原料的酶解、固定化纤维二糖酶和固定化乳酸杆菌的作用有机耦联，构建成新型的三级串联式生物反应器，该反应器可有效解除纤维二糖和葡萄糖对纤维素酶的反馈抑制作用，促进纤维原料的酶水解，乳酸浓度达55.2g/L，纤维素对乳酸的转化率高达90.6％。该反应器性能稳定，反应效率高，固定化酶和固定化细胞可以重复使用，便于自动化控制，对于优化反应工艺，降低操作成本等具有重要意义。采用分批添料式协同酶解发酵工艺，可提高纤维底物的终浓度和产物乳酸的终浓度(达105.2g／L)，有效提高了酶的利用率和乳酸生产效率。
浙江大学博士学位论文
摘要
定在海藻酸钙凝胶珠中，并将这种共固定化体系与纤维原料的酶解过程相祸合，
构建成新型的串联式生物反应器。在共固定化体系的协同作用下，纤维原料对乳
酸的转化率可达91.5%。本研究工作有利于简化工艺流程，减少设备投资，提高
反应效率，国内外至今尚未见到相同的研究报道。
在纤维素酶基因的克隆与表达方面做了一些探索性的研究工作:将纤维素酶
ES基因在克隆质粒pDS引上酶切分离后，连接到表达质粒pSE420上，进一步将
含有目的基因的表达质粒转入宿主细胞E.coll’JM109中，发酵液中的CMC酶活
力达到了31.6U/mL，成功地实现了基因的表达和胞外分泌。这一工作为进一步
构建高效产酶菌株打下了坚实的基础。
本文在固定化纤维二糖酶的制备、利用固定化纤维二糖酶协同降解纤维原
料、利用三级串联式生物反应器优化纤维原料协同糖化发酵过程、共固定化反应
体系的构建，以及利用共固定化体系组建串联式生物反应器协同降解纤维原料发
酵生产乳酸等方面的研究工作具有明显的特色与创新，有关研究结果不仅有重要
的学术价值，而且在促进可再生纤维素资源的转化利用、推动国民经济的可持续
发展等方面具有深远意义。
Cellulosic material is the most abundant renewable carbon source in the world. Cellulose may be hydrolyzed using cellulase to produce glucose, and glucose can be used for lactic acid production. The utilization of renewable biomass can not only save the foodstuff but also reduce the environmental pollution.
Since the production of cellulase was the major contribution to the bioconversion process, the submerged fermentation by Trichoderma reesei ZU~04 was studied. Using corn cob residue from xylose factory as the carbon source, the obtained cellulase activity was comparable with that using purified cellulose on the same cellulose basis. The bean cake was used as nitrogen source instead of peptone or yeast extract with the C/N ratio 10.0. The fermentation was scaled up in a 30 m3 stirred fermenter, and the activity of cellulase reached 8.16 FPIU/ml (331.7 FPIU/g cellulose) after 4 days. This research work greatly reduced the production cost, and laid a good foundation for industrial application.
A high yield cellobiase producing strain (Aspergillus niger LR-12) was screened. Using solid-state fermentation, the activity of cellobiase could reach 438.3 CBIU/g dry koji under optimized conditions, and this result was very attractive compared with those reported in recent literature. It was found that the spores of this strain were rich in cellobiase. By entrapping the spores into calcium alginate gels, the cellobiase was immobilized efficiently. The entrapped spores would not germinate at hydrolysis temperature (50 C), but could release cellobiase slowly into the gel beads. Comparing with the traditional immobilization of pure enzyme protein, this new method was more convenient and economical. The activity of enzyme was not destroyed, and the immobilized cellobiase were quite stable with a long half-life. The immobilized cellobiase could accelerate the synergetic hydrolysis process of cellulosic biamass.
During the saccharification of cellulosic material, the main influence factors including character and concentration of substrate, enzyme dosage, and composition of cellulase system were studied. Since the cellulase system from T. reesei was poor in cellobiase (CBA / FPA was 0.03), the hydrolysis yield of corn cob residue was only 67.4%. When cellobiase was added into the system to increase CBA/FPA to 0.42, the hydrolysis yield could be improved to 83.6%. Under the synergetic reaction of T. reesei cellulase and immobilized cellobiase, the yield of hydrolysis was raised to 88.2%.
Lactic acid is an important organic acid and applied in broad fields. Using cellulose instead of starch for lactic acid production was significant for development of national economy. In this work, Lactobacillus delbrueckii was immobilized by
entrapping the cells into calcium alginate gels. Comparing with free cells, the fermentation term of immobilized cells was shortened, and the yield of lactic acid was improved. The immobilized cells could utilize cellulosic hydrolysate to produce lactic acid efficiently, the yield of lactic acid reached 90.7%.
A three-step coupling bioreactor was set up by coupling the cellulose hydrolysis, the immobilized cellobiase and the immobilized L. delbrueckii cells together. In this coupling bioreactor, the feedback inhibition to cellulase reaction caused by the accumulation of cellobiose and glucose was eliminated, the hydrolysis of cellulosic material was promoted, and the yield of lactic acid from cellulose reached 90.6%. The new reactor was stable and efficient, and the immobilized cellobiase and cells could be repeatedly used for a long time. Under fed-batch process, the final concentration of cellulosic substrate and lactic acid were increased to 200 g/L and 105.2 g/1, respectively. The utilization of cellulase and the productive efficiency of lactic acid were both unproved.
Further, the spores of A. niger containing cellobiase and the cells of L. delbrueckii were entrapped together into calcium alginate gels to form a coimmobilized system. The coupling bioreactor with coimmobilized system showed a good p
采用液体深层发酵工艺，对纤维素酶生产过程中的主要工艺参数进行了优化，以廉价的木糖渣和豆饼粉作为里氏木霉(Trichoderma reesei ZU-04)的碳源和氮源，大幅度降低了生产成本，并在30 m~3发酵罐中成功地完成了放大试验，纤维素酶活力高达8.16 FPIU／mL(331.7 FPIU／g纤维素)，为纤维素酶的工业化应用打下了良好的基础。
筛选到一个纤维二糖酶高产菌株(黑曲霉Asperillus niger LR-12)，对其固态发酵条件进行了优化，纤维二糖酶活力达到了国际先进水平(438.3 CBIU／g干曲)。研究发现，该菌的孢子中富含纤维二糖酶，采用海藻酸钙凝胶包埋孢子可将纤维二糖酶有效固定。包埋后的黑曲霉孢子在酶解温度(50℃)下不会萌发，但可以在凝胶珠内缓慢地释放纤维二糖酶，与直接固定酶蛋白的传统方法相比，这种新型的固定化技术具有操作经济简便、对酶活力无破坏、固定化酶的半衰期长等优点。该固定化酶在纤维素酶的协同降解过程中具有明显的促进作用。
对纤维素酶解过程中底物性质、底物浓度、纤维素酶用量及酶系组成等关键因子进行了研究。由于里氏木霉纤维素酶系中纤维二糖酶活力很低(CBA／FPA为0.03)，对经稀酸预处理后的玉米芯纤维底物的酶解得率仅为67.4％。通过添加纤维二糖酶，将CBA／FPA增加到0.42，酶解得率可提高至83.6％。进一步利用里氏木霉纤维素酶和固定化纤维二糖酶的协同作用，纤维原料的酶解得率可高达88.2％。这方面的研究结果对于深入了解纤维素酶的协同降解机制具有重要意义。
采用海藻酸钙凝胶包埋固定德氏乳酸杆菌，固定化细胞与游离细胞相比，发酵时间缩短，乳酸得率提高，并能有效地利用纤维原料水解液进行乳酸发酵，乳酸得率可达90.7％。
将纤维原料的酶解、固定化纤维二糖酶和固定化乳酸杆菌的作用有机耦联，构建成新型的三级串联式生物反应器，该反应器可有效解除纤维二糖和葡萄糖对纤维素酶的反馈抑制作用，促进纤维原料的酶水解，乳酸浓度达55.2g/L，纤维素对乳酸的转化率高达90.6％。该反应器性能稳定，反应效率高，固定化酶和固定化细胞可以重复使用，便于自动化控制，对于优化反应工艺，降低操作成本等具有重要意义。采用分批添料式协同酶解发酵工艺，可提高纤维底物的终浓度和产物乳酸的终浓度(达105.2g／L)，有效提高了酶的利用率和乳酸生产效率。
浙江大学博士学位论文
摘要
定在海藻酸钙凝胶珠中，并将这种共固定化体系与纤维原料的酶解过程相祸合，
构建成新型的串联式生物反应器。在共固定化体系的协同作用下，纤维原料对乳
酸的转化率可达91.5%。本研究工作有利于简化工艺流程，减少设备投资，提高
反应效率，国内外至今尚未见到相同的研究报道。
在纤维素酶基因的克隆与表达方面做了一些探索性的研究工作:将纤维素酶
ES基因在克隆质粒pDS引上酶切分离后，连接到表达质粒pSE420上，进一步将
含有目的基因的表达质粒转入宿主细胞E.coll’JM109中，发酵液中的CMC酶活
力达到了31.6U/mL，成功地实现了基因的表达和胞外分泌。这一工作为进一步
构建高效产酶菌株打下了坚实的基础。
本文在固定化纤维二糖酶的制备、利用固定化纤维二糖酶协同降解纤维原
料、利用三级串联式生物反应器优化纤维原料协同糖化发酵过程、共固定化反应
体系的构建，以及利用共固定化体系组建串联式生物反应器协同降解纤维原料发
酵生产乳酸等方面的研究工作具有明显的特色与创新，有关研究结果不仅有重要
的学术价值，而且在促进可再生纤维素资源的转化利用、推动国民经济的可持续
发展等方面具有深远意义。
Cellulosic material is the most abundant renewable carbon source in the world. Cellulose may be hydrolyzed using cellulase to produce glucose, and glucose can be used for lactic acid production. The utilization of renewable biomass can not only save the foodstuff but also reduce the environmental pollution.
Since the production of cellulase was the major contribution to the bioconversion process, the submerged fermentation by Trichoderma reesei ZU~04 was studied. Using corn cob residue from xylose factory as the carbon source, the obtained cellulase activity was comparable with that using purified cellulose on the same cellulose basis. The bean cake was used as nitrogen source instead of peptone or yeast extract with the C/N ratio 10.0. The fermentation was scaled up in a 30 m3 stirred fermenter, and the activity of cellulase reached 8.16 FPIU/ml (331.7 FPIU/g cellulose) after 4 days. This research work greatly reduced the production cost, and laid a good foundation for industrial application.
A high yield cellobiase producing strain (Aspergillus niger LR-12) was screened. Using solid-state fermentation, the activity of cellobiase could reach 438.3 CBIU/g dry koji under optimized conditions, and this result was very attractive compared with those reported in recent literature. It was found that the spores of this strain were rich in cellobiase. By entrapping the spores into calcium alginate gels, the cellobiase was immobilized efficiently. The entrapped spores would not germinate at hydrolysis temperature (50 C), but could release cellobiase slowly into the gel beads. Comparing with the traditional immobilization of pure enzyme protein, this new method was more convenient and economical. The activity of enzyme was not destroyed, and the immobilized cellobiase were quite stable with a long half-life. The immobilized cellobiase could accelerate the synergetic hydrolysis process of cellulosic biamass.
During the saccharification of cellulosic material, the main influence factors including character and concentration of substrate, enzyme dosage, and composition of cellulase system were studied. Since the cellulase system from T. reesei was poor in cellobiase (CBA / FPA was 0.03), the hydrolysis yield of corn cob residue was only 67.4%. When cellobiase was added into the system to increase CBA/FPA to 0.42, the hydrolysis yield could be improved to 83.6%. Under the synergetic reaction of T. reesei cellulase and immobilized cellobiase, the yield of hydrolysis was raised to 88.2%.
Lactic acid is an important organic acid and applied in broad fields. Using cellulose instead of starch for lactic acid production was significant for development of national economy. In this work, Lactobacillus delbrueckii was immobilized by
entrapping the cells into calcium alginate gels. Comparing with free cells, the fermentation term of immobilized cells was shortened, and the yield of lactic acid was improved. The immobilized cells could utilize cellulosic hydrolysate to produce lactic acid efficiently, the yield of lactic acid reached 90.7%.
A three-step coupling bioreactor was set up by coupling the cellulose hydrolysis, the immobilized cellobiase and the immobilized L. delbrueckii cells together. In this coupling bioreactor, the feedback inhibition to cellulase reaction caused by the accumulation of cellobiose and glucose was eliminated, the hydrolysis of cellulosic material was promoted, and the yield of lactic acid from cellulose reached 90.6%. The new reactor was stable and efficient, and the immobilized cellobiase and cells could be repeatedly used for a long time. Under fed-batch process, the final concentration of cellulosic substrate and lactic acid were increased to 200 g/L and 105.2 g/1, respectively. The utilization of cellulase and the productive efficiency of lactic acid were both unproved.
Further, the spores of A. niger containing cellobiase and the cells of L. delbrueckii were entrapped together into calcium alginate gels to form a coimmobilized system. The coupling bioreactor with coimmobilized system showed a good p
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