基于玉米秸秆乙醇化的氨化预处理与酶解工艺研究
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摘要
我国是一个能源消耗大国,能源的短缺将会严重危害我国的经济发展和国家安全。因此开发新型能源以代替逐渐枯竭的传统能源将是我国今后能源产业的发展方向。生物燃料具有可再生,无污染的特点,因此备受各国的重视。木质纤维素是地球上最丰富、最廉价的可再生资源,被认为是最重要的生物燃料酒精生产的后续资源物质。相对于以玉米和大豆等粮食为原料的第一代生物燃料来说,以秸秆等植物纤维为原料的纤维素乙醇被称为第二代生物燃料,其最大优势在于避免了“与人争粮、与粮争地”的风险。因此,利用生物技术将木质纤维素转化成乙醇,具有重要的社会及经济意义。其中主要技术途径之一是利用生物技术将植物纤维原料转化成单糖,主要分为二步：1、原料预处理；2、纤维素的酶水解。目前预处理技术不尽人意,存在糖得率低、技术工艺复杂等问题,而纤维素的酶水解又存在水解速度慢、酶用量大等缺点。这两方面同时制约了纤维原料生物转化的发展。本论文以农业废弃物玉米秸秆为原料,对纤维原料的预处理方式及最优处理条件的筛选、酶解糖化条件的优化、超声波和金属离子对酶解的影响进行了研究,主要结果如下
采用氨水浸泡预处理、加压氨解预处理、超声波辅助氨水预处理三种方式处理玉米秸秆。氨水浸泡预处理的最优方案为：氨水浓度15%,时间39h,温度40℃,基质浓度162.5g／L,粒径0.5mm。在此条件下的预处理还原糖得率为3.23%,木质素去除率为61.20%。对应的酶解还原糖得率为45.28%,比未处理的12.51%,提高了32.77%。加压氨解预处理的最优方案为：氨水浓度15%,时间6min,压力0.075MPa,基质浓度150g/L,粒径1mm。在此条件下的预处理还原糖得率为1.74%,木质素去除率为43.30%。对应的酶解还原糖得率为21.36%,提高了8.85%。超声波辅助氨水预处理的最优方案为：氨水浓度12.5%,时间24min,功率240W,基质浓度187.5g／L,温度70℃,粒径0.5mm。在此条件下的预处理还原糖得率为1.51%,木质素去除率为32.20%。对应的酶解还原糖得率为40.33%,提高了27.82%。将最优处理结果进行比较并综合经济效益因素后认为,超声波辅助氨水预处理所需时间短、能耗低、效果较好,是比较理想的预处理方式。
采用经超声波辅助氨水预处理后的玉米秸秆为原料,在环境温度为50℃条件下进行酶解。基质浓度因素对还原糖得率影响极显著(p<0.01),酶解时间对还原糖得率影响显著(p<0.05)。酶解最优方案为：pH值5,基质浓度60g／L,酶用量47mg／g(干秸秆),酶解时间30h。在此条件下的酶解还原糖得率为49.00%,比未预处理玉米秸秆的酶解还原糖得率提高了36.49%。
一定程度的超声波辐射对酶解有促进作用。当酶解基质浓度为60g／L、酶用量为47mg／g(干秸秆)时,最适超声波功率为39W,超声波时间为9min。此时酶解还原糖得率为56.55%,比没有超声波参与酶解时提高了7.55%。
K+、Na+,Ca2+、Mg2+、Zn2+对纤维素酶起激活作用,有助于预处理产物的酶解,提高酶解还原糖得率。其中Zn2+对纤维素酶的激活作用最明显,其浓度为1.5mg／ml(酶液)时激活作用最大。Fe3+、Al3+则对纤维素酶起抑制作用。
Energy shortage would be harm Chinese Economy and national security seriously.Therefore, replace traditional energy sources by new energy sources will be the development direction of the energy industry in the future. The biofuel receive national attention lately, for it has characteristics of renewable and pollution-free.The lignocellulose is the most abundant and cheapest renewable resources on the earth, and supposed to be the most important resources for fuel alcohol production. Relative to the first generation material of biofuel such as corn and bean, the straw fiber were identified as the second generation material of biofuel, which the most advantage is avoided the risk of be hard up of foodstuff. Therefore, the social and economic significance is very important to transform cellulose into ethanol by biological technology.There are two steps to convert cellulosic substrate to monosaccharide, namely,pretreatment of materials and enzymatic hydrolysis of cellulose.The technology of pretreatment is unsatisfactory at present. The development of cellulose bioconversion is limited by many problems in technology of stalks ethanol such as low rate of sugar conversion, process complication, low hydrolysis rate and large enzyme dosages. In this thesis, optimization conditions and handling methods of pretreatment of corn stalk as the fiber material, optimization conditions of enzymatic saccharification, the impact of ultrasound and metal ions on the enzyme has been studied.The results are shown as follows:
Study is carried out on pretreatments of ammonia immersion, pammonolysis under pressure and ultrasound-assisted ammonia immersion to deal with corn stalks. The optimal scheme of pretreatment of ammonia immersion is shown as follows:Under the conditions of ammonia concentration is 15%,time is 39h, temperature is 40℃,substrate concentration is 162.5g/L, and stalk diameter is 0.5mm, the saccharifying efficiency is 3.23%,and lignin removal rate is 61.20%. Corresponding hydrolytic enzyme of reducing sugar is 45.28%, increases 32.77% than the unprocessed(12.51%).The optimal scheme of pretreatment of pammonolysis under pressure is shown as follows:Under the conditions of ammonia concentration is 15%.time is 6min. the pressure is 0.075MPa. substrate concentration is 150g/L, and stalk diameter is 1mm, the saccharifying efficiency is 1.74%. and lignin removal rate is 43.30%.Corresponding hydrolytic enzyme of reducing sugar is 21.36%, increases 8.85% than the unprocessed.The optimal scheme of pretreatment of ultrasound-assisted ammonia immersion is shown as follows:Under the conditions of ammonia concentration is 12.5%,ultrasonic time is 24min.ultrasonic power is 240W,substrate concentration is 187.5g/L,temperature is 70℃. and stalk diameter is 0.5mm, the saccharifying efficiency is 1.51%,and lignin removal rate is 32.20%. Corresponding hydrolytic enzyme of reducing sugar is 40.33%,increases 27.82% than the unprocessed.The results of optimal schemes and the factor of economic are considered that the pretreatment of ultrasound-assisted ammonia immersion is an ideal pretreatment method required for a short time, low energy consumption and the effect is better.
Corn straw as material that deal with pretreatment of ultrasound-assisted ammonia immersion to carry out enzymatic hydrolysis under the conditions of temperature is 50℃.The factor of substrate concentration affected the reducing sugar yield significantly (p<0.01),hydrolysis time affected that as follow (p<0.05).Optimal scheme of enzymatic hydrolysis is shown as follows: Under the condition of pH value is 5,substrate concentration is 60g/L,enzyme dosage is 47mg/g (dry straw), and enzymatic hydrolysis time is 30h, the saccharifying efficiency of enzymatic hydrolysis is 49.00%, which increase 36.49% more than enzymatic hydrolysis without pretreatment.
An appropriate degree of ultrasonic radiation plays an activation role in enzyme.Under condition of the substrate concentration is 60g/L, enzyme dosage is 47mg/g (dry straw),the optimal ultrasonic power is 39W. and ultrasonic time is 9min. Under the condition of these, reducing sugar yield of enzymatic is 56.55%.which increase 7.55% more than enzymatic hydrolysis without ultrasound.
K+. Na+,Ca2+,Mg2+, and Zn2+ play an activation role in enzyme,improve the enzymatic hydrolysis yield of reducing sugar, of which Zn2+ is the most significant one. and the saccharifying efficiency has maximum activation when the Zn2+ concentration is 1.5mg/ml(liquid),Fe3-,Al3- play an restrain role in enzyme.
采用氨水浸泡预处理、加压氨解预处理、超声波辅助氨水预处理三种方式处理玉米秸秆。氨水浸泡预处理的最优方案为：氨水浓度15%,时间39h,温度40℃,基质浓度162.5g／L,粒径0.5mm。在此条件下的预处理还原糖得率为3.23%,木质素去除率为61.20%。对应的酶解还原糖得率为45.28%,比未处理的12.51%,提高了32.77%。加压氨解预处理的最优方案为：氨水浓度15%,时间6min,压力0.075MPa,基质浓度150g/L,粒径1mm。在此条件下的预处理还原糖得率为1.74%,木质素去除率为43.30%。对应的酶解还原糖得率为21.36%,提高了8.85%。超声波辅助氨水预处理的最优方案为：氨水浓度12.5%,时间24min,功率240W,基质浓度187.5g／L,温度70℃,粒径0.5mm。在此条件下的预处理还原糖得率为1.51%,木质素去除率为32.20%。对应的酶解还原糖得率为40.33%,提高了27.82%。将最优处理结果进行比较并综合经济效益因素后认为,超声波辅助氨水预处理所需时间短、能耗低、效果较好,是比较理想的预处理方式。
采用经超声波辅助氨水预处理后的玉米秸秆为原料,在环境温度为50℃条件下进行酶解。基质浓度因素对还原糖得率影响极显著(p<0.01),酶解时间对还原糖得率影响显著(p<0.05)。酶解最优方案为：pH值5,基质浓度60g／L,酶用量47mg／g(干秸秆),酶解时间30h。在此条件下的酶解还原糖得率为49.00%,比未预处理玉米秸秆的酶解还原糖得率提高了36.49%。
一定程度的超声波辐射对酶解有促进作用。当酶解基质浓度为60g／L、酶用量为47mg／g(干秸秆)时,最适超声波功率为39W,超声波时间为9min。此时酶解还原糖得率为56.55%,比没有超声波参与酶解时提高了7.55%。
K+、Na+,Ca2+、Mg2+、Zn2+对纤维素酶起激活作用,有助于预处理产物的酶解,提高酶解还原糖得率。其中Zn2+对纤维素酶的激活作用最明显,其浓度为1.5mg／ml(酶液)时激活作用最大。Fe3+、Al3+则对纤维素酶起抑制作用。
Energy shortage would be harm Chinese Economy and national security seriously.Therefore, replace traditional energy sources by new energy sources will be the development direction of the energy industry in the future. The biofuel receive national attention lately, for it has characteristics of renewable and pollution-free.The lignocellulose is the most abundant and cheapest renewable resources on the earth, and supposed to be the most important resources for fuel alcohol production. Relative to the first generation material of biofuel such as corn and bean, the straw fiber were identified as the second generation material of biofuel, which the most advantage is avoided the risk of be hard up of foodstuff. Therefore, the social and economic significance is very important to transform cellulose into ethanol by biological technology.There are two steps to convert cellulosic substrate to monosaccharide, namely,pretreatment of materials and enzymatic hydrolysis of cellulose.The technology of pretreatment is unsatisfactory at present. The development of cellulose bioconversion is limited by many problems in technology of stalks ethanol such as low rate of sugar conversion, process complication, low hydrolysis rate and large enzyme dosages. In this thesis, optimization conditions and handling methods of pretreatment of corn stalk as the fiber material, optimization conditions of enzymatic saccharification, the impact of ultrasound and metal ions on the enzyme has been studied.The results are shown as follows:
Study is carried out on pretreatments of ammonia immersion, pammonolysis under pressure and ultrasound-assisted ammonia immersion to deal with corn stalks. The optimal scheme of pretreatment of ammonia immersion is shown as follows:Under the conditions of ammonia concentration is 15%,time is 39h, temperature is 40℃,substrate concentration is 162.5g/L, and stalk diameter is 0.5mm, the saccharifying efficiency is 3.23%,and lignin removal rate is 61.20%. Corresponding hydrolytic enzyme of reducing sugar is 45.28%, increases 32.77% than the unprocessed(12.51%).The optimal scheme of pretreatment of pammonolysis under pressure is shown as follows:Under the conditions of ammonia concentration is 15%.time is 6min. the pressure is 0.075MPa. substrate concentration is 150g/L, and stalk diameter is 1mm, the saccharifying efficiency is 1.74%. and lignin removal rate is 43.30%.Corresponding hydrolytic enzyme of reducing sugar is 21.36%, increases 8.85% than the unprocessed.The optimal scheme of pretreatment of ultrasound-assisted ammonia immersion is shown as follows:Under the conditions of ammonia concentration is 12.5%,ultrasonic time is 24min.ultrasonic power is 240W,substrate concentration is 187.5g/L,temperature is 70℃. and stalk diameter is 0.5mm, the saccharifying efficiency is 1.51%,and lignin removal rate is 32.20%. Corresponding hydrolytic enzyme of reducing sugar is 40.33%,increases 27.82% than the unprocessed.The results of optimal schemes and the factor of economic are considered that the pretreatment of ultrasound-assisted ammonia immersion is an ideal pretreatment method required for a short time, low energy consumption and the effect is better.
Corn straw as material that deal with pretreatment of ultrasound-assisted ammonia immersion to carry out enzymatic hydrolysis under the conditions of temperature is 50℃.The factor of substrate concentration affected the reducing sugar yield significantly (p<0.01),hydrolysis time affected that as follow (p<0.05).Optimal scheme of enzymatic hydrolysis is shown as follows: Under the condition of pH value is 5,substrate concentration is 60g/L,enzyme dosage is 47mg/g (dry straw), and enzymatic hydrolysis time is 30h, the saccharifying efficiency of enzymatic hydrolysis is 49.00%, which increase 36.49% more than enzymatic hydrolysis without pretreatment.
An appropriate degree of ultrasonic radiation plays an activation role in enzyme.Under condition of the substrate concentration is 60g/L, enzyme dosage is 47mg/g (dry straw),the optimal ultrasonic power is 39W. and ultrasonic time is 9min. Under the condition of these, reducing sugar yield of enzymatic is 56.55%.which increase 7.55% more than enzymatic hydrolysis without ultrasound.
K+. Na+,Ca2+,Mg2+, and Zn2+ play an activation role in enzyme,improve the enzymatic hydrolysis yield of reducing sugar, of which Zn2+ is the most significant one. and the saccharifying efficiency has maximum activation when the Zn2+ concentration is 1.5mg/ml(liquid),Fe3-,Al3- play an restrain role in enzyme.
引文
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