木糖产L-乳酸耐热菌种的选育及利用玉米芯水解液发酵的研究
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摘要
木糖广泛存在于林业及农业废弃物中,利用木糖发酵生产L-乳酸可有效利用再生资源。本研究通过富集、平板筛选、液体发酵筛选、高浓度木糖驯化、复筛、遗传稳定性试验从自然界中得到两株耐高温、高效利用木糖发酵L-乳酸的优良菌株L-8、L-20。
在摇瓶发酵的基础上对L-8、L-20的发酵条件进行了优化,以木糖为唯一碳源发酵L-乳酸,对不同的木糖浓度、发酵温度、接种量、种龄、发酵培养基装载量和发酵时间进行了单因素发酵试验,确定了适宜的发酵培养基木糖浓度为8%,最适发酵温度是52℃,适宜的接种量为10%,适宜的种龄为14h,250ml的摇瓶适宜的装液量为150ml(160rpm),碳酸钙的适宜添加量为50g/L,最佳发酵时间为72h。
本研究还对玉米芯半纤维素的稀硫酸水解进行研究,以期获得稀硫酸水解玉米芯的最佳水解条件。通过对硫酸浓度、水解时间、水解温度和料液比对玉米芯水解单因素试验。得到了各单因素较佳的水解条件:硫酸浓度2%、水解时间30 min、水解温度120℃和料液比1:10。在这样的条件下水解玉米芯,测定其中还原糖的含量最高达到49.35g/L。通过薄层层析法分析水解液中糖的组成,其中主要的糖类为木糖,还含有少量的葡萄糖。
利用最佳水解条件对玉米芯进行水解,水解液经过处理后用筛选所得2株菌株L-8和L-20进行玉米芯水解液L-乳酸发酵实验,在水解液浓缩倍数为2倍时,菌株L-8能够充分利用玉米芯水解液中的葡萄糖和木糖来发酵生产L-乳酸,其L-乳酸产量达到67.06g/L,糖酸转化率为91.03%。
Xylose is widespread in forest and agricultural residues.Xylose can be fermented to L-lactic acid which is an effective exploitation of renewable materials.In this study,through strain screening method,two Thermophilus Bacteria strain L-8 and L-20 that can high-efficient convert xylose to L-lactic acid were obtained.
Through single factor experiment on shaking flask fermentation,the conditions of fermentation were experimented.The Optimized fermentation condition determined that:the best xylose concentration is 8%,the suitable fermentation temperature is 52℃,the inoculum size is 10%,the inoculum age is 14h, the media value is 150ml/250ml,the CaCO_3 suitable quantity is 50g/L,fermentation time is 72 hours.
In this study,the conditions of corn cob hydrolysis by dilute sulfuric acid were experimented too.The Optimized hydrolysis condition determined that:the concentration of dilute sulfuric acid of 2%,the hydrolysis reaction time of 30 min and and hydrolysis reaction temperature of 120℃and the ratio of solid/liquid of 1/10.Under the Optimized hydrolysis condition,the concentration of reducing sugar was 49.35g/L.Through thin-layer chromatography of corn cob hydrolysis,the main monosaccharide was xylose.
After fermentation for 72 hours using corn cob hydrolysate,L-8 produced 67.06g of L-lactic acid/L, and the conversion rate respectively were 91.03%.
在摇瓶发酵的基础上对L-8、L-20的发酵条件进行了优化,以木糖为唯一碳源发酵L-乳酸,对不同的木糖浓度、发酵温度、接种量、种龄、发酵培养基装载量和发酵时间进行了单因素发酵试验,确定了适宜的发酵培养基木糖浓度为8%,最适发酵温度是52℃,适宜的接种量为10%,适宜的种龄为14h,250ml的摇瓶适宜的装液量为150ml(160rpm),碳酸钙的适宜添加量为50g/L,最佳发酵时间为72h。
本研究还对玉米芯半纤维素的稀硫酸水解进行研究,以期获得稀硫酸水解玉米芯的最佳水解条件。通过对硫酸浓度、水解时间、水解温度和料液比对玉米芯水解单因素试验。得到了各单因素较佳的水解条件:硫酸浓度2%、水解时间30 min、水解温度120℃和料液比1:10。在这样的条件下水解玉米芯,测定其中还原糖的含量最高达到49.35g/L。通过薄层层析法分析水解液中糖的组成,其中主要的糖类为木糖,还含有少量的葡萄糖。
利用最佳水解条件对玉米芯进行水解,水解液经过处理后用筛选所得2株菌株L-8和L-20进行玉米芯水解液L-乳酸发酵实验,在水解液浓缩倍数为2倍时,菌株L-8能够充分利用玉米芯水解液中的葡萄糖和木糖来发酵生产L-乳酸,其L-乳酸产量达到67.06g/L,糖酸转化率为91.03%。
Xylose is widespread in forest and agricultural residues.Xylose can be fermented to L-lactic acid which is an effective exploitation of renewable materials.In this study,through strain screening method,two Thermophilus Bacteria strain L-8 and L-20 that can high-efficient convert xylose to L-lactic acid were obtained.
Through single factor experiment on shaking flask fermentation,the conditions of fermentation were experimented.The Optimized fermentation condition determined that:the best xylose concentration is 8%,the suitable fermentation temperature is 52℃,the inoculum size is 10%,the inoculum age is 14h, the media value is 150ml/250ml,the CaCO_3 suitable quantity is 50g/L,fermentation time is 72 hours.
In this study,the conditions of corn cob hydrolysis by dilute sulfuric acid were experimented too.The Optimized hydrolysis condition determined that:the concentration of dilute sulfuric acid of 2%,the hydrolysis reaction time of 30 min and and hydrolysis reaction temperature of 120℃and the ratio of solid/liquid of 1/10.Under the Optimized hydrolysis condition,the concentration of reducing sugar was 49.35g/L.Through thin-layer chromatography of corn cob hydrolysis,the main monosaccharide was xylose.
After fermentation for 72 hours using corn cob hydrolysate,L-8 produced 67.06g of L-lactic acid/L, and the conversion rate respectively were 91.03%.
引文
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