玉米秸秆为原料燃料乙醇制备的关键问题研究
摘要
木质纤维素是地球上最丰富、最廉价的可再生资源,被认为是最重要的乙醇生产的后续资源物质。但在以木质纤维素为原料进行乙醇制备方面仍存在原料预处理技术难、纤维素酶水解成本高和木糖尚无法被利用产酒精等问题,纤维素乙醇的价格尚无法与粮食乙醇竞争,解决纤维素乙醇制备过程中的关键技术已成为全世界共识。本论文针对目前制约木质纤维素物质生产乙醇的主要技术瓶颈,在原料预处理、纤维素降解菌群在纤维素酒精生产中的应用和木糖利用方面进行以下研究:
根据微波加热的基本原理,结合预处理的目标和特点,将微波引入秸秆的预处理过程,对不同的吸波介质进行了比较和选择,确定水是对纤维素酶毒性最小、预处理效果较好且环保经济的吸波介质。优化出微波预处理玉米秸秆的工艺参数为液固比为58:1,微波功率为630 W,处理时间为5.5分钟,在该条件下,微波预处理玉米秸秆的酶解率可达到49.12%。应用原子力显微镜(AFM)观察微波水预处理前后的秸秆表面形貌,结合本研究中预处理前后秸秆成分的变化结果,发现微波预处理是利用水分子在秸秆表面“钻孔”从而增大秸秆与纤维素酶的接触面积,也使得纤维素酶容易进入秸秆内部进行水解从而达到提高酶解效率的目的。
从自然环境中分离筛选出高效产酶且酶活稳定的纤维素降解菌株FLZ6和FLZ10,采用形态学、生理生化和微生物细胞膜脂肪酸特异性分析,鉴定FLZ6为地衣芽孢杆菌(Bacillus-licheniformis),FLZ10为毛壳菌(Chaetomium spp)。应用FLZ6和FLZ10构建纤维素降解菌群代替纤维素酶使用,进行同时糖化发酵(SSF)生产乙醇的研究,通过测定反应体系中的乙醇含量,对SSF工艺进行了优化,最终确定了SSF工艺的最佳条件为:反应温度37℃,初始pH 6.0,纤维素降解菌群和酵母菌的比例为1:1,反应时间36小时。在该条件下,获得了13.1 g/100 g秸秆的乙醇。
从富含丰富的腐烂纤维素的土壤中分离筛选出一株木糖发酵酵母菌株FL-20-2。采用形态学、生理生化和分子生物学方法将这株菌鉴定为假丝酵母属中的热带假丝酵母(Candida tropicalis)。在确定最优发酵培养基组份(木糖50 g/L,酵母粉0.5 g/L,KH2PO4 2.5 g/L,MgSO4. 7H2O 0.4 g/L)的基础上,进行菌株代谢特性分析。发现FL20-2在以木糖为唯一碳源时,pH 5.5,温度35℃,接种量为10%的厌氧条件下培养96 h,乙醇产量最高,为其利用木糖生成乙醇理论产量的32.4%,此时伴有31.05 g/L的木糖醇生成。在以木糖和葡萄糖的混合糖为碳源时,相同发酵条件下,木糖和葡萄糖转化的乙醇产量分别为其理论产量的37.7%和79.2%,同时伴有30.2 g/L的木糖醇产生。采用纤维素降解菌群和热带假丝酵母混合,对经过微波预处理的玉米秸秆进行乙醇制备研究,在纤维素降解菌群和热带假丝酵母菌按体积比1:1,10%接种量,温度为40℃,时间84 h条件下,乙醇产量为17.9 g/100g玉米秸秆,同时伴有10.05 g/100 g玉米秸秆的木糖醇生成。
通过对假丝酵母菌属(Candida sp.)木糖还原酶基因序列保守区域的分析,设计了一系列引物,以近平滑假丝酵母(Candida parapsilosis)的总RNA为模板,采用RACE(Rapid Amplification of cDNA Ends)的方法成功获取了Candida parapsilosis木糖还原酶基因,其开放阅读框长度为954 bp,使用Blastn程序进行检索后发现与白色假丝酵母(Candida albicans)的醛糖还原酶(aldose reductase ,XM715658)同源性最高,相似性为80%,通过进化关系分析认定本文发现了一种新的木糖还原酶基因,其登录号为:EF033247。选用巴斯德毕赤酵母表达载体pGAPZαA为载体,将获得的木糖还原酶基因开放阅读框克隆该表达载体pGAP启动子下,构建分泌型重组质粒,该重组质粒同源重组到毕赤酵母基因组上进行稳定表达,表达产物采用镍离子亲和层析柱进行纯化,得到约36-kDa蛋白条带。酶学性质的分析表明,重组蛋白具有较高的木糖催化效率(kcat/Km = 61 /mM/min)和不同于其它木糖还原酶的辅酶特性,其依赖于NADH的催化效率(kcat/Km = 41.5 /μM/min)大于依赖于NADPH的催化效率(kcat/Km = 2.2 /μM/ min)。
本论文通过上述研究,基本确定了以玉米秸秆为原料进行乙醇制备的工艺过程,采用本论文的技术,可获得17.9 g乙醇/100g玉米秸秆这一较高的乙醇产量;同时通过对木糖利用关键酶-木糖还原酶的辅酶特性研究,有望解决木糖发酵乙醇途径中的辅酶不平衡问题,为提高木糖发酵生产酒精的产量,进而进一步增加玉米秸秆的乙醇产量奠定基础。
Lignocellulose is one of the most abundant and inexpensive renewable resources on the earth. Thus far, although the mechanisms and technologies that using lignocellulose in ethanol production have been recognized, many aspects including the pretreatment of raw material, decrease of enzyme costs and utilization of xylose still need to be further improved. Due to the high cost, the ethanol produced from Lignocellulose is less competitive with that produced from corn. So, it is widely realized that significant effort should be driven into the development of low-cost cellulosic ethanol.In this study, three factors including pretreatment of ligncellulose, utilization of cellulosic degradation community instead of enzyme and utilization of xylose addressed below were investigated.
Based on mechanism of microwave, microwave is introduced into corn stover pretreatment. Several microwave absorbers were used and compared. Water is determined to be the best microwave absorber in pretreatment. By Design Expert software showed that the optimal operational parameters were: liquid to solid ratio 58:1, power of microwave 630 W, pretreatment time 5.5 min. Under this condition, the enzyme degradation percentage can be reached to 49.12%. Images obtained by AFM showed that micropores were formed on the surface of corn stover after pretreatment. Considered with results obtained above, Microwave pretreatment enlarged the interface between solids and enzyme due to the micro pores water molecule drilled, which also facilitates enzyme entrance into interiors of solids.
Cellulosic degradation strains FLZ6 and FLZ10 were isolated from natural environment. Morphologic analysis, physiological-biochemical characterization and cell membrane fatty acid analysis revealed that the FLZ6 belongs to Bacillus-licheniformis, and the FLZ10 belongs to Chaetomium spp. FLZ6 and FLZ10 were chosed in Simultaneous sacchariferous fermentation (SSF) tests. By testing the ethanol production, the optimal parameters of SSF process were obtained as: temperature 37°C, original pH 6.0, the ratio of cellulose decomposing strains and yeast 1:1, retention time 36 h. Under this condition, the production rate was 13.1 g ethanol per 100 g corn stover.
A xylose fermentive yeast FL-20-2 were isolated from humus soil.Using morphologic analysis, physiological-biochemical characterization and molecular biological methods, the yeast was identified as Candida tropicalis. The ethanol production rate was 32.4% of the theoretical percentage, accompanied with 31.05 g/L xylitol production with pH 5.5, temperature 35°C, inoculation percentage 10% and fermentation time 96 h. When xylose and glucose were mixed as carbon sources, the ethanol production rates were 37.7% and 79.2% of their theoretical rates respectively, with 30.2 g/L xylitol produced.
A degenerate primers and other primers ware designed according to the result of homologous analysis of nucleotide sequences encoding XR of Candida sp. Gene of xylose reductase (XR) from Candida parapsilosis was amplified by RACE technology from the total RNA of Candida parapsilosis. The open reading frame of Candida parapsilosis xyl1 gene is 954bp. A BLAST search showed that this sequence contained homology with Candida albicans aldose reductase XM_715658 and the homologous rate was 80%. This gene should be a new XR gene(Genebank accession:EF033247). The open reading frame of xyl1 from Candida parapsilosis was inserted into the downstream of the alpha-mating factor signal of the P.pastoris expression vector pGAPZαA .Under the control of promoter pGAP (glyceraldehydes 3-phosphate dehydrogenase)the recombinant protein can secreted express into culture medium. The products were purified using nickel affinity chromatography and an approximate 36-kDa protein band was obtained. Enzyme character analysis indicated that the recombinant XR showed high catalytic efficiency (kcat/Km =61 mM-1 min-1) for D-xylose and showed unusual coenzyme specificity with greater catalytic efficiency with NADH-dependent (kcat/Km=41.5μM-1 min-1 ) than with NADPH-dependent (kcat/Km=2.2μM-1 min-1).
Based on the research above, ethanol production process using corn stover was determined. Using this technology, a ethanol yield of 17.9 g ethanol/100 g corn stover had been achieved. Simultaneously, based on the research on the coenzyme characteristics of xylose reducase (key enzyme), it is promising that the problem of coenzyme imbalance during xylose fermentation can be solved, which could further increase the ethanol output from corn stover.
根据微波加热的基本原理,结合预处理的目标和特点,将微波引入秸秆的预处理过程,对不同的吸波介质进行了比较和选择,确定水是对纤维素酶毒性最小、预处理效果较好且环保经济的吸波介质。优化出微波预处理玉米秸秆的工艺参数为液固比为58:1,微波功率为630 W,处理时间为5.5分钟,在该条件下,微波预处理玉米秸秆的酶解率可达到49.12%。应用原子力显微镜(AFM)观察微波水预处理前后的秸秆表面形貌,结合本研究中预处理前后秸秆成分的变化结果,发现微波预处理是利用水分子在秸秆表面“钻孔”从而增大秸秆与纤维素酶的接触面积,也使得纤维素酶容易进入秸秆内部进行水解从而达到提高酶解效率的目的。
从自然环境中分离筛选出高效产酶且酶活稳定的纤维素降解菌株FLZ6和FLZ10,采用形态学、生理生化和微生物细胞膜脂肪酸特异性分析,鉴定FLZ6为地衣芽孢杆菌(Bacillus-licheniformis),FLZ10为毛壳菌(Chaetomium spp)。应用FLZ6和FLZ10构建纤维素降解菌群代替纤维素酶使用,进行同时糖化发酵(SSF)生产乙醇的研究,通过测定反应体系中的乙醇含量,对SSF工艺进行了优化,最终确定了SSF工艺的最佳条件为:反应温度37℃,初始pH 6.0,纤维素降解菌群和酵母菌的比例为1:1,反应时间36小时。在该条件下,获得了13.1 g/100 g秸秆的乙醇。
从富含丰富的腐烂纤维素的土壤中分离筛选出一株木糖发酵酵母菌株FL-20-2。采用形态学、生理生化和分子生物学方法将这株菌鉴定为假丝酵母属中的热带假丝酵母(Candida tropicalis)。在确定最优发酵培养基组份(木糖50 g/L,酵母粉0.5 g/L,KH2PO4 2.5 g/L,MgSO4. 7H2O 0.4 g/L)的基础上,进行菌株代谢特性分析。发现FL20-2在以木糖为唯一碳源时,pH 5.5,温度35℃,接种量为10%的厌氧条件下培养96 h,乙醇产量最高,为其利用木糖生成乙醇理论产量的32.4%,此时伴有31.05 g/L的木糖醇生成。在以木糖和葡萄糖的混合糖为碳源时,相同发酵条件下,木糖和葡萄糖转化的乙醇产量分别为其理论产量的37.7%和79.2%,同时伴有30.2 g/L的木糖醇产生。采用纤维素降解菌群和热带假丝酵母混合,对经过微波预处理的玉米秸秆进行乙醇制备研究,在纤维素降解菌群和热带假丝酵母菌按体积比1:1,10%接种量,温度为40℃,时间84 h条件下,乙醇产量为17.9 g/100g玉米秸秆,同时伴有10.05 g/100 g玉米秸秆的木糖醇生成。
通过对假丝酵母菌属(Candida sp.)木糖还原酶基因序列保守区域的分析,设计了一系列引物,以近平滑假丝酵母(Candida parapsilosis)的总RNA为模板,采用RACE(Rapid Amplification of cDNA Ends)的方法成功获取了Candida parapsilosis木糖还原酶基因,其开放阅读框长度为954 bp,使用Blastn程序进行检索后发现与白色假丝酵母(Candida albicans)的醛糖还原酶(aldose reductase ,XM715658)同源性最高,相似性为80%,通过进化关系分析认定本文发现了一种新的木糖还原酶基因,其登录号为:EF033247。选用巴斯德毕赤酵母表达载体pGAPZαA为载体,将获得的木糖还原酶基因开放阅读框克隆该表达载体pGAP启动子下,构建分泌型重组质粒,该重组质粒同源重组到毕赤酵母基因组上进行稳定表达,表达产物采用镍离子亲和层析柱进行纯化,得到约36-kDa蛋白条带。酶学性质的分析表明,重组蛋白具有较高的木糖催化效率(kcat/Km = 61 /mM/min)和不同于其它木糖还原酶的辅酶特性,其依赖于NADH的催化效率(kcat/Km = 41.5 /μM/min)大于依赖于NADPH的催化效率(kcat/Km = 2.2 /μM/ min)。
本论文通过上述研究,基本确定了以玉米秸秆为原料进行乙醇制备的工艺过程,采用本论文的技术,可获得17.9 g乙醇/100g玉米秸秆这一较高的乙醇产量;同时通过对木糖利用关键酶-木糖还原酶的辅酶特性研究,有望解决木糖发酵乙醇途径中的辅酶不平衡问题,为提高木糖发酵生产酒精的产量,进而进一步增加玉米秸秆的乙醇产量奠定基础。
Lignocellulose is one of the most abundant and inexpensive renewable resources on the earth. Thus far, although the mechanisms and technologies that using lignocellulose in ethanol production have been recognized, many aspects including the pretreatment of raw material, decrease of enzyme costs and utilization of xylose still need to be further improved. Due to the high cost, the ethanol produced from Lignocellulose is less competitive with that produced from corn. So, it is widely realized that significant effort should be driven into the development of low-cost cellulosic ethanol.In this study, three factors including pretreatment of ligncellulose, utilization of cellulosic degradation community instead of enzyme and utilization of xylose addressed below were investigated.
Based on mechanism of microwave, microwave is introduced into corn stover pretreatment. Several microwave absorbers were used and compared. Water is determined to be the best microwave absorber in pretreatment. By Design Expert software showed that the optimal operational parameters were: liquid to solid ratio 58:1, power of microwave 630 W, pretreatment time 5.5 min. Under this condition, the enzyme degradation percentage can be reached to 49.12%. Images obtained by AFM showed that micropores were formed on the surface of corn stover after pretreatment. Considered with results obtained above, Microwave pretreatment enlarged the interface between solids and enzyme due to the micro pores water molecule drilled, which also facilitates enzyme entrance into interiors of solids.
Cellulosic degradation strains FLZ6 and FLZ10 were isolated from natural environment. Morphologic analysis, physiological-biochemical characterization and cell membrane fatty acid analysis revealed that the FLZ6 belongs to Bacillus-licheniformis, and the FLZ10 belongs to Chaetomium spp. FLZ6 and FLZ10 were chosed in Simultaneous sacchariferous fermentation (SSF) tests. By testing the ethanol production, the optimal parameters of SSF process were obtained as: temperature 37°C, original pH 6.0, the ratio of cellulose decomposing strains and yeast 1:1, retention time 36 h. Under this condition, the production rate was 13.1 g ethanol per 100 g corn stover.
A xylose fermentive yeast FL-20-2 were isolated from humus soil.Using morphologic analysis, physiological-biochemical characterization and molecular biological methods, the yeast was identified as Candida tropicalis. The ethanol production rate was 32.4% of the theoretical percentage, accompanied with 31.05 g/L xylitol production with pH 5.5, temperature 35°C, inoculation percentage 10% and fermentation time 96 h. When xylose and glucose were mixed as carbon sources, the ethanol production rates were 37.7% and 79.2% of their theoretical rates respectively, with 30.2 g/L xylitol produced.
A degenerate primers and other primers ware designed according to the result of homologous analysis of nucleotide sequences encoding XR of Candida sp. Gene of xylose reductase (XR) from Candida parapsilosis was amplified by RACE technology from the total RNA of Candida parapsilosis. The open reading frame of Candida parapsilosis xyl1 gene is 954bp. A BLAST search showed that this sequence contained homology with Candida albicans aldose reductase XM_715658 and the homologous rate was 80%. This gene should be a new XR gene(Genebank accession:EF033247). The open reading frame of xyl1 from Candida parapsilosis was inserted into the downstream of the alpha-mating factor signal of the P.pastoris expression vector pGAPZαA .Under the control of promoter pGAP (glyceraldehydes 3-phosphate dehydrogenase)the recombinant protein can secreted express into culture medium. The products were purified using nickel affinity chromatography and an approximate 36-kDa protein band was obtained. Enzyme character analysis indicated that the recombinant XR showed high catalytic efficiency (kcat/Km =61 mM-1 min-1) for D-xylose and showed unusual coenzyme specificity with greater catalytic efficiency with NADH-dependent (kcat/Km=41.5μM-1 min-1 ) than with NADPH-dependent (kcat/Km=2.2μM-1 min-1).
Based on the research above, ethanol production process using corn stover was determined. Using this technology, a ethanol yield of 17.9 g ethanol/100 g corn stover had been achieved. Simultaneously, based on the research on the coenzyme characteristics of xylose reducase (key enzyme), it is promising that the problem of coenzyme imbalance during xylose fermentation can be solved, which could further increase the ethanol output from corn stover.
引文
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