王草三种降解糖化技术的研究
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摘要
王草是C4植物,具有植株高大、生长速度快、高生物质产量、多年生等特点,是我国产量最高的热带牧草,因此以王草为原料制取生物燃料乙醇具有巨大的潜力。在利用王草木质纤维原料生成乙醇的过程中,最重要是将高度结晶度和难溶性木质纤维素降解为糖类,再经微生物的发酵作用制取燃料乙醇。
本论文以热研4号王草(Pennisetum purpureum×P. typhoideum cv. Reyan No.4)为原料,对化学法降解、生物法降解及综合法降解工艺进行了研究,选择适宜的反应条件获得最大还原糖产量,并与麦秆和甘蔗渣进行比较,以期为后续戊糖、己糖发酵制取燃料乙醇提供基础和依据。
试验结果总结如下：
1、以还原糖得率作为指标,研究了酸浓度(v/v)、时间、固液比(g/mL)、温度对还原糖得率的影响。在单因素实验的基础上通过二次通用旋转组合试验设计建立了稀硫酸最佳处理工艺,获得了稀硫酸最佳处理工艺参数为：稀硫酸浓度3%,水解温度117℃,水解时间15 min；最佳工艺条件下的还原糖得率分别为王草33.08%；甘蔗渣31.98%；麦秆28.56%。
2、研究了含不同原料(王草、麦秆、甘蔗渣)的混合发酵培养基中木霉菌(Trichoderma sp.)的诱变菌株H28、赭绿青霉(Penicillium ochro-chloron) SP1、粗毛栓菌(Trametes gallica sp.)的诱变菌株T906分别产纤维素酶、木聚糖酶和漆酶的情况。结果表明,菌株H28和SP1都能较好利用王草产酶,其中H28产羧甲基纤维素酶的最高酶活为80.91U/mL, SP1产木聚糖酶的最高酶活为104.36U/mL。菌株T906能较好利用麦秆产漆酶,其漆酶活力在第1ld达到24118.52 U/L。而三株菌对甘蔗渣的利用效果不好,酶活不高。根据在混合发酵培养基中,酶活力随时间的变化规律,确定三菌混合糖化的最佳时间。结果表明,单独培养8d的T906与单独培养4d的H28、SP1混合,对原料的糖化效率最高,分别为：王草5.85%；麦秆5.44%；甘蔗渣3.60%。
3、利用H28所产的纤维素酶对稀硫酸处理后的三种样品进行水解,测定了水解后的还原糖含量并计算糖化效率。结果表明,对三种底物进行水解的最适条件为：纤维素酶使用量15 U/g底物,于50-C,酶解72 h。在此条件下,底物酶水解得率分别为：麦秆29.47%；王草17.64%；甘蔗渣15.39%。
The king grass, which is C4 plants with tall, fast growth, high biomass material yield, perennial and other features, is the highest production of tropical grasses in our country. The biological fuel ethanol made from king grass has great potential.In the process of ethanol in the use of king grass wooden fiber as raw material, the most important key point is lignocellulose which high crystallinity and hard woodiness degraded to sugars, and microbial fermentation after.
In this research, king grass (Pennisetum purpureum×P. typhoideum cv. Reyan No.4) was used as materials in the degradation techniques of chemical, bioanalysis and comprehensive, choose appropriate reaction conditions for maximum reducing sugar production, and compared with wheat-straw and bagasse, which providing foundation and depending of making fuel ethanol from sugar and subsequent sugar fermentation.
The results were as follows:
1. The influence of acid concentration, treatment time, solid-to-liquid ratio and temperature on reducing sugar were studied. A general rotatory composed test was done to optimize the process of dilute sulfuric acid pretreatment. Based on the single factor, the results showed that the best pretreatment process was acid concentration was 3%, at 117℃for 15 min, reducing sugars of king grass, bagasse, wheat-straw were 33.08%,31.98%,28.56% respectively under this condition.
2. The production of carboxy methyl cellulose(CMCase), xylanase and laccase by Trichoderma inductive-mutant strain H28, Penicillium ochro-chloron, named SP1 and Trametes gallica inductive-mutant strain T906 was studied, respectively, in the mixed fermentation medium contained different materials, such as king grass, wheat-straw and bagasse. Results showed that, H28 and SP1 could use king grass producing enzymes. The highest enzyme activity of carboxy methyl cellulose secreted by H28 was 80.91 U/mL, while the xylanase from SP1 reached 104.36 U/mL. T906 could use wheat-straw producing laccase, and the laccase activity was 24118.52 U/L in the eleventh day. Whereas, the effect of bagasse is not good, and the enzyme activity is not high. According to the variation of enzyme production of H28, SP1 and T906 with time in the mixture, the saccharification efficiency was highest when the culture of T906 for 8 days, the culture of H28 and SP1 which had fermented for 4 days, was mix together. After co-cultured, the saccharification efficiencies of king grass and wheat-straw was 5.85% and 5.44% respectively, while that of bagasse was only 3.6%.
3. The king grass, wheat-straw and bagasse, which was after pretreated with dilute sulfuric acid were hydrolyzed by the cellulose secreted by H28. Resules showed that, it was saitable to hydrolyze the three mterials with 15U/g per gram material, at 50℃for 72h. In this way, the saccharfication efficiency of wheat-straw, king grass and bagasse was about 29.47%,17.64% and 15.39% respectively.
本论文以热研4号王草(Pennisetum purpureum×P. typhoideum cv. Reyan No.4)为原料,对化学法降解、生物法降解及综合法降解工艺进行了研究,选择适宜的反应条件获得最大还原糖产量,并与麦秆和甘蔗渣进行比较,以期为后续戊糖、己糖发酵制取燃料乙醇提供基础和依据。
试验结果总结如下：
1、以还原糖得率作为指标,研究了酸浓度(v/v)、时间、固液比(g/mL)、温度对还原糖得率的影响。在单因素实验的基础上通过二次通用旋转组合试验设计建立了稀硫酸最佳处理工艺,获得了稀硫酸最佳处理工艺参数为：稀硫酸浓度3%,水解温度117℃,水解时间15 min；最佳工艺条件下的还原糖得率分别为王草33.08%；甘蔗渣31.98%；麦秆28.56%。
2、研究了含不同原料(王草、麦秆、甘蔗渣)的混合发酵培养基中木霉菌(Trichoderma sp.)的诱变菌株H28、赭绿青霉(Penicillium ochro-chloron) SP1、粗毛栓菌(Trametes gallica sp.)的诱变菌株T906分别产纤维素酶、木聚糖酶和漆酶的情况。结果表明,菌株H28和SP1都能较好利用王草产酶,其中H28产羧甲基纤维素酶的最高酶活为80.91U/mL, SP1产木聚糖酶的最高酶活为104.36U/mL。菌株T906能较好利用麦秆产漆酶,其漆酶活力在第1ld达到24118.52 U/L。而三株菌对甘蔗渣的利用效果不好,酶活不高。根据在混合发酵培养基中,酶活力随时间的变化规律,确定三菌混合糖化的最佳时间。结果表明,单独培养8d的T906与单独培养4d的H28、SP1混合,对原料的糖化效率最高,分别为：王草5.85%；麦秆5.44%；甘蔗渣3.60%。
3、利用H28所产的纤维素酶对稀硫酸处理后的三种样品进行水解,测定了水解后的还原糖含量并计算糖化效率。结果表明,对三种底物进行水解的最适条件为：纤维素酶使用量15 U/g底物,于50-C,酶解72 h。在此条件下,底物酶水解得率分别为：麦秆29.47%；王草17.64%；甘蔗渣15.39%。
The king grass, which is C4 plants with tall, fast growth, high biomass material yield, perennial and other features, is the highest production of tropical grasses in our country. The biological fuel ethanol made from king grass has great potential.In the process of ethanol in the use of king grass wooden fiber as raw material, the most important key point is lignocellulose which high crystallinity and hard woodiness degraded to sugars, and microbial fermentation after.
In this research, king grass (Pennisetum purpureum×P. typhoideum cv. Reyan No.4) was used as materials in the degradation techniques of chemical, bioanalysis and comprehensive, choose appropriate reaction conditions for maximum reducing sugar production, and compared with wheat-straw and bagasse, which providing foundation and depending of making fuel ethanol from sugar and subsequent sugar fermentation.
The results were as follows:
1. The influence of acid concentration, treatment time, solid-to-liquid ratio and temperature on reducing sugar were studied. A general rotatory composed test was done to optimize the process of dilute sulfuric acid pretreatment. Based on the single factor, the results showed that the best pretreatment process was acid concentration was 3%, at 117℃for 15 min, reducing sugars of king grass, bagasse, wheat-straw were 33.08%,31.98%,28.56% respectively under this condition.
2. The production of carboxy methyl cellulose(CMCase), xylanase and laccase by Trichoderma inductive-mutant strain H28, Penicillium ochro-chloron, named SP1 and Trametes gallica inductive-mutant strain T906 was studied, respectively, in the mixed fermentation medium contained different materials, such as king grass, wheat-straw and bagasse. Results showed that, H28 and SP1 could use king grass producing enzymes. The highest enzyme activity of carboxy methyl cellulose secreted by H28 was 80.91 U/mL, while the xylanase from SP1 reached 104.36 U/mL. T906 could use wheat-straw producing laccase, and the laccase activity was 24118.52 U/L in the eleventh day. Whereas, the effect of bagasse is not good, and the enzyme activity is not high. According to the variation of enzyme production of H28, SP1 and T906 with time in the mixture, the saccharification efficiency was highest when the culture of T906 for 8 days, the culture of H28 and SP1 which had fermented for 4 days, was mix together. After co-cultured, the saccharification efficiencies of king grass and wheat-straw was 5.85% and 5.44% respectively, while that of bagasse was only 3.6%.
3. The king grass, wheat-straw and bagasse, which was after pretreated with dilute sulfuric acid were hydrolyzed by the cellulose secreted by H28. Resules showed that, it was saitable to hydrolyze the three mterials with 15U/g per gram material, at 50℃for 72h. In this way, the saccharfication efficiency of wheat-straw, king grass and bagasse was about 29.47%,17.64% and 15.39% respectively.
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