采用Klebsiella pneumoniae生物合成2，3-丁二醇过程分析
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摘要
2,3-丁二醇作为重要的化工原料具有广泛用途。生物法较化学法具有安全、环保、反应条件温和、原料可再生等优点,已成为当前研究开发的热点。目前许多研究聚焦在采用木质纤维素水解液进行发酵,然而这些研究中2,3-丁二醇浓度、生产强度都无法与葡萄糖发酵结果相比。本文综合考察了菌种、底物消耗及培养条件等因素对克雷伯氏肺炎杆菌(Klebsiella.pneumoniae)采用模拟木质纤维素水解液合成2,3-丁二醇的影响特点及作用规律。
为了提高菌株2,3-丁二醇产量,以紫外诱变为手段,采用产酸量和产物耐受性为目标筛选突变菌株,最终获得一株编号为Klebsiella pneunoniae UV-86(K.pneumoniae UV-86)遗传稳定的突变株。此菌株产物得率0.438g/g,比原菌提高1.22倍,2,3-丁二醇脱氢酶比酶活提高38.6%。表明该筛选方法有效。
对底物类型及影响2,3-丁二醇合成的温度、pH进行研究,结果表明菌株底物代谢顺序为葡萄糖先于木糖。两者代谢的较优温度均为30℃,pH均为6.0。添加乙酸能够促进细胞生长,而乙酸添加量为1g/L时产物量达到最高。
以葡萄糖和木糖混合物为底物,研究不同供氧对发酵过程影响,结果表明生物量随供氧增加而增加。供氧对菌株消耗葡萄糖影响较小。而代谢木糖能力随供氧增加而增强。微氧条件下菌株生产能力最强,2,3-丁二醇产量和产率均在1.5vvm达到最高为30.1g/L和0.485g/(Lh)。木糖代谢阶段乙酸生成量显著增加。并据此提出了可能的供氧调控策略。
2,3-butanediol was an important chemical because it has a large number of industrial applications. Comparing with the chemical process, the biological process was safe, environmental friendly, mild reaction condition and could use renewable resources. So it has been paid more and more attentions. Nowadays, many research efforts have focused on using low price substrates lignocellulosic hydrolysate to produce the 2,3-butanediol. However, both the 2,3-butanediol concentration and the productivity were lower than those of glucose. This paper investigated the screening of producing strains, the sugars fermented and operating conditions applied on 2,3-butanediol fermentation by Klebsiella pneumoniae with synthetic medium of rice straw hydrolysate.
In order to improve strain to produce 2,3-butanediol with high yield, UV mutagenesis with acid-producing and product tolerance capability was established to select positive mutant bacteria. A strain named Klebsiella pneumoniae UV-86 (K.pneumoniae UV-86) with the highest 2,3-butanediol production capability and heritage stability was chosen as the target strain for the following investigations. The yield of 2,3-butanediol by K.pneumoniae UV-86 was 0.438g/g, which was 1.22 times to that of the original strain. The specific activity of 2, 3-butanediol dehydrogenase was increased by 38.6%. The results demonstrated the effective and efficient high throughput screening technique.
The sugars fementated, temperature and pH were investigated for the bio-production of 2, 3-butanediol. It was found that the order of the preferential sugar utilization was glucose>xylose. The optimal temperature and pH for bacterial fermentation with glucose and xylose were 30℃and 6.0, respectively. The cell growth was improved by adding acetate and the maximum 2,3-butanediol concentration was obtained by acetate adding concentration 1g/L.
In order to investigate the influence of dissolved oxygen concentration on 2, 3-butanediol bioproduction process, the cofermentation of glucose and xylose was investigated. It was found that high oxygen supply favoured cell mass formation. The influence of oxygen on glucose consumption was not significant. But it had a significant impact on xylose consumption and the average specific xylose consumption rate was improved with the oxygen supply. The micro-aerobic condition favoured formation of 2, 3-butanediol. The maximum 2,3-butanediol concentration 30.1g/L and productivity 0.485g/ (L h) were both obtained at 1.5vvm. The accumulation of acetate was enhanced during the xylose metabolism process. According to these results, the oxygen supply control strategy was proposed.
为了提高菌株2,3-丁二醇产量,以紫外诱变为手段,采用产酸量和产物耐受性为目标筛选突变菌株,最终获得一株编号为Klebsiella pneunoniae UV-86(K.pneumoniae UV-86)遗传稳定的突变株。此菌株产物得率0.438g/g,比原菌提高1.22倍,2,3-丁二醇脱氢酶比酶活提高38.6%。表明该筛选方法有效。
对底物类型及影响2,3-丁二醇合成的温度、pH进行研究,结果表明菌株底物代谢顺序为葡萄糖先于木糖。两者代谢的较优温度均为30℃,pH均为6.0。添加乙酸能够促进细胞生长,而乙酸添加量为1g/L时产物量达到最高。
以葡萄糖和木糖混合物为底物,研究不同供氧对发酵过程影响,结果表明生物量随供氧增加而增加。供氧对菌株消耗葡萄糖影响较小。而代谢木糖能力随供氧增加而增强。微氧条件下菌株生产能力最强,2,3-丁二醇产量和产率均在1.5vvm达到最高为30.1g/L和0.485g/(Lh)。木糖代谢阶段乙酸生成量显著增加。并据此提出了可能的供氧调控策略。
2,3-butanediol was an important chemical because it has a large number of industrial applications. Comparing with the chemical process, the biological process was safe, environmental friendly, mild reaction condition and could use renewable resources. So it has been paid more and more attentions. Nowadays, many research efforts have focused on using low price substrates lignocellulosic hydrolysate to produce the 2,3-butanediol. However, both the 2,3-butanediol concentration and the productivity were lower than those of glucose. This paper investigated the screening of producing strains, the sugars fermented and operating conditions applied on 2,3-butanediol fermentation by Klebsiella pneumoniae with synthetic medium of rice straw hydrolysate.
In order to improve strain to produce 2,3-butanediol with high yield, UV mutagenesis with acid-producing and product tolerance capability was established to select positive mutant bacteria. A strain named Klebsiella pneumoniae UV-86 (K.pneumoniae UV-86) with the highest 2,3-butanediol production capability and heritage stability was chosen as the target strain for the following investigations. The yield of 2,3-butanediol by K.pneumoniae UV-86 was 0.438g/g, which was 1.22 times to that of the original strain. The specific activity of 2, 3-butanediol dehydrogenase was increased by 38.6%. The results demonstrated the effective and efficient high throughput screening technique.
The sugars fementated, temperature and pH were investigated for the bio-production of 2, 3-butanediol. It was found that the order of the preferential sugar utilization was glucose>xylose. The optimal temperature and pH for bacterial fermentation with glucose and xylose were 30℃and 6.0, respectively. The cell growth was improved by adding acetate and the maximum 2,3-butanediol concentration was obtained by acetate adding concentration 1g/L.
In order to investigate the influence of dissolved oxygen concentration on 2, 3-butanediol bioproduction process, the cofermentation of glucose and xylose was investigated. It was found that high oxygen supply favoured cell mass formation. The influence of oxygen on glucose consumption was not significant. But it had a significant impact on xylose consumption and the average specific xylose consumption rate was improved with the oxygen supply. The micro-aerobic condition favoured formation of 2, 3-butanediol. The maximum 2,3-butanediol concentration 30.1g/L and productivity 0.485g/ (L h) were both obtained at 1.5vvm. The accumulation of acetate was enhanced during the xylose metabolism process. According to these results, the oxygen supply control strategy was proposed.
引文
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