纤维素乙醇及乳酸耦合炼制过程与机理研究
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摘要
木质纤维原料是规模生物炼制的潜在原料,目前以纤维生物炼制过程成本较高。本论文以糠醛渣(FR)、木薯渣(CR)两种工业废弃物为主要原料,以玉米作为辅助原料,通过生物方法转化为乙醇和乳酸(本文中均为L(+)-乳酸),开发混合原料和产品联产两种策略来提高过程效率和过程经济,从木质素、纤维素酶和发酵微生物相互作用出发探讨相应的促进机理。主要结论如下：
FR为原料采用混菌发酵方式联产乙醇和乳酸,乙醇在54h前生成。产品组成受温度和底物浓度影响,温度、底物浓度和接种质量比都会影响纤维素转化率。混菌发酵能取得较高纤维利用率,9%FR经过混菌发酵120h,得到乙醇9.02g/L和乳酸18.9g/L,酸醇摩尔比1.07,纤维素利用率86.8%。混菌发酵需要添加额外营养,序列发酵混合原料,在乙醇发酵和乳酸发酵之间加入一定处理能实现零化学品消耗联产。序列发酵120h,得到乙醇22.4g/L和47.6g/L乳酸,比混菌发酵提高148%和152%,酸醇摩尔比1.08,多糖利用率86.9%。
脱除木质素和添加表面活性剂都能降低木质素的负面影响。碱性过氧化氢脱除FR木质素,脱除度80.2%,乙醇转化率提高最高(72.1%),脱除度37.3%,乳酸浓度提高最高(48.5%)。皂荚皂素具有比合成表面活性剂更高价效,利用酵母制得的蛋白类表面活性剂具有生物兼容性。酵母在121℃下处理30min,酵母64.4%氮、83%碳水化合物保存在固相中(YHS),仅有2.3%氮溶于液相(YHL)。YHL的溶质主要是具有和纤维素酶分子量相近的非催化蛋白质,这些蛋白易于被木质素吸附,其和纤维素酶形成的竞争性吸附是YHL酶促机理。糠醛渣水解体系中,最佳蛋白用量是11.8-23.5mg/g糠醛渣,该用量下每克木质素至少吸附0.63mgYHL氮,提高酶水解率14.4%。
木质素、纤维素酶和发酵微生物存在以下相互作用：酵母对纤维素酶影响不大,乳酸菌会消耗纤维素酶,木质素和营养物质会缓解乳酸菌对纤维素酶的同化。苯环类抑制物对乳酸菌的抑制作用大于酵母。活酵母和活乳酸菌对彼此都会产生竞争,死酵母是乳酸菌的营养来源,二者虽然不代谢对方的发酵产品,但是各自代谢产品会抑制对方发酵活力。
混合原料能极大提高转化率和产品浓度,过程主要影响因素取决于产品种类。混合原料乙醇过程底物比是过程主要影响因素。优化条件下,混合原料乙醇发酵乙醇浓度66.0g/L～73.1g/L,多糖利用率73.3%～90.3%。混合原料乳酸发酵,营养添加、底物比和碳氮比都是过程主要影响因素,可行的营养来源是水热处理酵母细胞。当碳氮比介于20-63时,乳酸浓度40.7g/L～79.5g/L,多糖利用率81.5%～87.1%。
Lignocelluloses are the most promising feedstocks for biorefinery, but the requirement for depolymerisation of cellulose makes cellulosic bioconversion cost-inefficient. In this study, industrial wastes, including cassava residues (CR) and furfural residues (FR), with corn were bio-converted into ethanol and L(+)-lactic acid. Integration process using multiple materials and producing multiple products were developed to cost-efficiently use these wastes. The main conclusions were as follows:
A fermentation system using the mixed cultures of yeast and lactic acid bacteria (MSSF) was designed to mainly produce ethanol before54h and lactic acid after54h from FR. The substrate loading and temperature influence the acid/alcohol molar ratio. However, substrate loading, temperature and the cell mass ration have effect on the cellulose conversion ratio of MSSF, which is always higher than those of sole SSF. MSSF obtained9.02g/L of ethanol and18.9g/L of lactic acid at120h using substrate loading of9%, corresponded to1.07of acid/alcohol molar ratio and86.8%of cellulose conversion ratio. One bottleneck of MSSF is the need of nutrient addition. The sequential production of ethanol and lactic acid can be improved by using starchy materials and cellulosic materials. Some treatment added between ethanol fermentation and lactic acid fermentation makes nutrient addition unnecessary. Under the selected conditions, the integrated process using mixed materials coproduce22.4g/L ethanol and47.6g/L lactic acid at120h, corresponded to acid/alcohol ratio of1.08and a polysaccharide utilization ratio of86.9%.
Delignification and surfactant additions are two ways of reducing the negative effect of lignin on bioconversion. For Alkaline peroxide, a delignification degree of80.2%and37.3%were the optimum delignification degree for ethanol SSF and lactic acid SSF, respectively. Gleditsia Saponin had a better performance in improving ethanol SSF than Tween-20. Protein surfactants can be produced from yeast by hydrothermal treatment. After hydrothermal treatment at121℃for30min,64.4%of yeast nitrogen and83%of yeast carbonhydrate remained in YHS while only2.28%of yeast nitrogen was non-enzymatic proteins dissolved in YHL, which have close molecular weight with cellulases. Competitive adsorption of non-enzymatic proteins and cellulases on lignin is the reason for the enhancement of glucose yield by YHL. The optimum protein loading of FRs was ranged from11.8mg/g FRs to23.5mg/g FRs, resulting in that0.63mg of nitrogen in YHL was absorbed on1gram of lignin and the enzymatic hydrolysis of FRs was increased by14.4%.
Different interactions exist between lignin, cellulases and microorganisms. The effect of yeast on cellulases is not significance while lactic acid bacteria(LAB) consumed cellulases as nutrients. Extra nutrients and lignin reduce the negative effect of LAB on cellulases. Yeast are more tolerated to phenolic components than LAB. LAB and yeast compete with each other in the fermentation by mixed culture, but dead yeast cells provide nutrients for LAB. Though LAB and yeast did not consume ethanol and lactic acid, the two products inhibit lactic acid fermentation and ethanol fermentation.
Using multiple materials obviously increased the yield and the final product concentration of bioconversion process. Whatever the product is ethanol or lactic acid, substrate composition is one of main factors influencing processes using multiple materials. Under optimized conditions of ethanol fermentation using multiple materials, final ethanol concentration and polysaccharide utilization were ranged from66.0g/L to73.1g/L and from73.3%to90.3%, respectively. Nutrients, substrate composition and carbon/nitrogen ratio were the main factors influencing lactic acid fermentation using multiple materials. Yeast treated by hydrothermal treatment was proven to be an effective nitrogen sources. When carbon/nitrogen ratio was between20～63, final lactic acid concentration was ranged from40.7g/L to79.5g/L and polysaccharide utilization was ranged from81.5%to87.1%.
FR为原料采用混菌发酵方式联产乙醇和乳酸,乙醇在54h前生成。产品组成受温度和底物浓度影响,温度、底物浓度和接种质量比都会影响纤维素转化率。混菌发酵能取得较高纤维利用率,9%FR经过混菌发酵120h,得到乙醇9.02g/L和乳酸18.9g/L,酸醇摩尔比1.07,纤维素利用率86.8%。混菌发酵需要添加额外营养,序列发酵混合原料,在乙醇发酵和乳酸发酵之间加入一定处理能实现零化学品消耗联产。序列发酵120h,得到乙醇22.4g/L和47.6g/L乳酸,比混菌发酵提高148%和152%,酸醇摩尔比1.08,多糖利用率86.9%。
脱除木质素和添加表面活性剂都能降低木质素的负面影响。碱性过氧化氢脱除FR木质素,脱除度80.2%,乙醇转化率提高最高(72.1%),脱除度37.3%,乳酸浓度提高最高(48.5%)。皂荚皂素具有比合成表面活性剂更高价效,利用酵母制得的蛋白类表面活性剂具有生物兼容性。酵母在121℃下处理30min,酵母64.4%氮、83%碳水化合物保存在固相中(YHS),仅有2.3%氮溶于液相(YHL)。YHL的溶质主要是具有和纤维素酶分子量相近的非催化蛋白质,这些蛋白易于被木质素吸附,其和纤维素酶形成的竞争性吸附是YHL酶促机理。糠醛渣水解体系中,最佳蛋白用量是11.8-23.5mg/g糠醛渣,该用量下每克木质素至少吸附0.63mgYHL氮,提高酶水解率14.4%。
木质素、纤维素酶和发酵微生物存在以下相互作用：酵母对纤维素酶影响不大,乳酸菌会消耗纤维素酶,木质素和营养物质会缓解乳酸菌对纤维素酶的同化。苯环类抑制物对乳酸菌的抑制作用大于酵母。活酵母和活乳酸菌对彼此都会产生竞争,死酵母是乳酸菌的营养来源,二者虽然不代谢对方的发酵产品,但是各自代谢产品会抑制对方发酵活力。
混合原料能极大提高转化率和产品浓度,过程主要影响因素取决于产品种类。混合原料乙醇过程底物比是过程主要影响因素。优化条件下,混合原料乙醇发酵乙醇浓度66.0g/L～73.1g/L,多糖利用率73.3%～90.3%。混合原料乳酸发酵,营养添加、底物比和碳氮比都是过程主要影响因素,可行的营养来源是水热处理酵母细胞。当碳氮比介于20-63时,乳酸浓度40.7g/L～79.5g/L,多糖利用率81.5%～87.1%。
Lignocelluloses are the most promising feedstocks for biorefinery, but the requirement for depolymerisation of cellulose makes cellulosic bioconversion cost-inefficient. In this study, industrial wastes, including cassava residues (CR) and furfural residues (FR), with corn were bio-converted into ethanol and L(+)-lactic acid. Integration process using multiple materials and producing multiple products were developed to cost-efficiently use these wastes. The main conclusions were as follows:
A fermentation system using the mixed cultures of yeast and lactic acid bacteria (MSSF) was designed to mainly produce ethanol before54h and lactic acid after54h from FR. The substrate loading and temperature influence the acid/alcohol molar ratio. However, substrate loading, temperature and the cell mass ration have effect on the cellulose conversion ratio of MSSF, which is always higher than those of sole SSF. MSSF obtained9.02g/L of ethanol and18.9g/L of lactic acid at120h using substrate loading of9%, corresponded to1.07of acid/alcohol molar ratio and86.8%of cellulose conversion ratio. One bottleneck of MSSF is the need of nutrient addition. The sequential production of ethanol and lactic acid can be improved by using starchy materials and cellulosic materials. Some treatment added between ethanol fermentation and lactic acid fermentation makes nutrient addition unnecessary. Under the selected conditions, the integrated process using mixed materials coproduce22.4g/L ethanol and47.6g/L lactic acid at120h, corresponded to acid/alcohol ratio of1.08and a polysaccharide utilization ratio of86.9%.
Delignification and surfactant additions are two ways of reducing the negative effect of lignin on bioconversion. For Alkaline peroxide, a delignification degree of80.2%and37.3%were the optimum delignification degree for ethanol SSF and lactic acid SSF, respectively. Gleditsia Saponin had a better performance in improving ethanol SSF than Tween-20. Protein surfactants can be produced from yeast by hydrothermal treatment. After hydrothermal treatment at121℃for30min,64.4%of yeast nitrogen and83%of yeast carbonhydrate remained in YHS while only2.28%of yeast nitrogen was non-enzymatic proteins dissolved in YHL, which have close molecular weight with cellulases. Competitive adsorption of non-enzymatic proteins and cellulases on lignin is the reason for the enhancement of glucose yield by YHL. The optimum protein loading of FRs was ranged from11.8mg/g FRs to23.5mg/g FRs, resulting in that0.63mg of nitrogen in YHL was absorbed on1gram of lignin and the enzymatic hydrolysis of FRs was increased by14.4%.
Different interactions exist between lignin, cellulases and microorganisms. The effect of yeast on cellulases is not significance while lactic acid bacteria(LAB) consumed cellulases as nutrients. Extra nutrients and lignin reduce the negative effect of LAB on cellulases. Yeast are more tolerated to phenolic components than LAB. LAB and yeast compete with each other in the fermentation by mixed culture, but dead yeast cells provide nutrients for LAB. Though LAB and yeast did not consume ethanol and lactic acid, the two products inhibit lactic acid fermentation and ethanol fermentation.
Using multiple materials obviously increased the yield and the final product concentration of bioconversion process. Whatever the product is ethanol or lactic acid, substrate composition is one of main factors influencing processes using multiple materials. Under optimized conditions of ethanol fermentation using multiple materials, final ethanol concentration and polysaccharide utilization were ranged from66.0g/L to73.1g/L and from73.3%to90.3%, respectively. Nutrients, substrate composition and carbon/nitrogen ratio were the main factors influencing lactic acid fermentation using multiple materials. Yeast treated by hydrothermal treatment was proven to be an effective nitrogen sources. When carbon/nitrogen ratio was between20～63, final lactic acid concentration was ranged from40.7g/L to79.5g/L and polysaccharide utilization was ranged from81.5%to87.1%.
引文
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