钝齿棒杆菌厌氧发酵产琥珀酸的研究
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摘要
琥珀酸是一种广泛用于食品、医药及化工产品重要的四碳二元羧酸。目前,琥珀酸主要采用化学法合成,容易造成环境污染,且其发展受到石化资源短缺的限制。可再生资源微生物发酵制备琥珀酸是一种最具发展潜力的绿色工艺模式,不仅摆脱了对石化原料的依赖,而且开辟了温室气体CO2利用的新途径。近年来,国内外学者对微生物发酵制备琥珀酸进行了大量的研究,卓有成效。但是,仍然存在菌株代谢网络不完善、无法有效降低代谢副产物、琥珀酸生产强度低等问题。
Corynebacterium crenatum是工业发酵制备谷氨酸的主要菌株,已有的研究发现,改变C.crenatum氨基酸发酵培养基溶氧水平,会对该菌株的代谢流分布产生较大的影响,厌氧发酵条件下,有相当部分碳流向琥珀酸发酵途径。本文基于Corynebacterium crenatum CICC20219厌氧发酵特性研究,以提高琥珀酸产量和生产强度为主要目标,通过对胞内碳代谢关键酶活测定,并结合代谢抑制分析,分析了C. crenatum在厌氧条件下的代谢网络,计算出琥珀酸形成的代谢通量分布；通过基因工程方法对其代谢网络进行针对性改造,有效降低代谢副产物的生成,提高了琥珀酸产量。研究并优化了琥珀酸厌氧发酵pH调控工艺,提高了生产强度。主要结论如下：
(1)通过对比C. crenatum在有氧和厌氧两种条件下的代谢产物,并结合关键酶活测定,研究C. crenatum厌氧发酵产琥珀酸特性,分析其产酸的能力。将C. crenatum在厌氧条件下培养,发现C. crenatum细胞不增殖,但可以吸收利用葡萄糖产乳酸、琥珀酸等有机酸。C crenatum厌氧发酵22h内共产生了19.8g/L琥珀酸和46g/L乳酸,琥珀酸和乳酸的产量分别为0.27g/g和0.62g/g。将C. crenatum进行两阶段培养,发现在有氧阶段时,C.crenatum生物量迅速增加,而琥珀酸和乳酸的积累量很少,在通入二氧化碳转为厌氧发酵后,C.crenatum的生物量不再增加,而琥珀酸和乳酸的浓度开始迅速增加,分别达到了15g/L和51g/L。富集C. crenatum细胞进行高密度厌氧发酵时,乳酸和琥珀酸的生产强度与C. crenatum的生物量呈线性关系,生物量为60g/L时,乳酸和琥珀酸的积累浓度在2h内分别达到了59g/L和23g/L,生产强度分别达到了29.5g/(L·h)和11.5g/(L·h)。将C. crenatum进行循环厌氧发酵,C. crenatum在10批共100h的循环发酵时间内利用780g葡萄糖,生成310g乳酸和423g琥珀酸,糖酸转化率在90%以上,乳酸与琥珀酸生产强度分别为3.1g/(L·h)和4.2g/(L-h)。 C. crenatum厌氧发酵产酸的特性表明C. crenatum具有持续、高强度产琥珀酸的能力,这一结论为进一步利用C.crenatum发酵产琥珀酸奠定了基础。
(2)通过对C. crenatum代谢途径中关键酶活性分析,发现该菌株在厌氧条件下的磷酸烯醇式丙酮酸羧化酶和苹果酸脱氢酶的酶活较高,异柠檬酸裂合酶的酶活较弱,检测不到异柠檬酸脱氢酶的酶活。通过分析抑制剂对C. crenatum厌氧发酵产琥珀酸的影响,发现当培养基中的丙二酸浓度由0g/L增加到2g/L时,琥珀酸的终浓度下降了43%,而氟乙酸对该菌株产琥珀酸的影响很小。通过研究氧化还原电位(ORP)对C. crenatum厌氧发酵产琥珀酸的影响,发现当培养基的ORP由-40mv降低为-300mv时,发酵液中琥珀酸的终浓度由14.4g/L增加到21.5g/L,同比增加了50%,琥珀酸产量也由0.18g/g上升到0.31g/g。结合酶活分析结果及抑制剂、ORP对发酵结果的影响,表明C. crenatum产琥珀酸的代谢途径主要是C4途径和乙醛酸循环。参考Corynebacterium glutamicum基因组信息,得出C. crenatum在厌氧条件下的代谢网络模型,代谢通量计算结果表明C4途径及乙醛酸循环是C. crenatum产琥珀酸的主要代谢途径,经由C4途径生产的琥珀酸占整个琥珀酸产量的93%,为对C. crenatum代谢途径进行针对性改造提供了理论支持。
(3)乳酸是C. crenatum厌氧发酵产琥珀酸的主要副产物。以C. crenatum基因组为模板PCR扩增山ldhA基因,构建了ldhA基因敲除载体pMKA,并电转化C. crenatum,质粒pMKA与C. crenatum基因组进行基因重组,敲除了C. crenatum的IdhA基因,获得突变株C. crenatum AldhA。与原始菌株相比,突变株的葡萄糖消耗速度下降50%,乳酸脱氢酶酶活和乳酸产量均为0,琥珀酸的产量由0.22g/g上升到0.62g/g。通过敲除IdhA基因达到了消除乳酸生成、增加琥珀酸产量的目的。
(4)通过表达ppc和pyc基因,可以强化C. crenatum磷酸烯醇式丙酮酸与丙酮酸羧化途径,将更多的碳引向C4途径。以C. crenatum基因组为模板PCR扩增出磷酸烯醇式丙酮酸羧化酶基因ppc和丙酮酸羧化酶基因pyc,构建了ppc与pyc基因表达质粒pJA、pJC。分别以表达质粒为载体在C. crenatum△AldhA内过表达ppc与pyc基因,获得菌株C. crenatum△ldhA(pJA)与C. crenatum△ldhA(pJC)。与C. crenatum△ldhA相比,C. crenatum△ldhA(pJA)的磷酸烯醇式丙酮酸羧化酶活力比对照株增加1.5倍,葡萄糖的消耗速度和琥珀酸的产量并没有明显的变化,表明磷酸烯醇式丙酮酸羧化酶不是C. crenatum△ldhA产琥珀酸的限速酶。过量表达pyc后,C.crenatum△ldhA(pJC)胞内丙酮酸羧化酶活力比C. crenatum△ldhA增加8倍,葡萄糖消耗速度增加了46%,琥珀酸的产量由0.64g/g增加到0.68g/g,同比增加了7.8%。
(5)通过选择合适的pH及中和剂,可以提高琥珀酸的生产效率以降低生物法生产琥珀酸的生产成本。pH优化结果显示,pH为6.8时的发酵效果优于其它pH值。pH为6.8时,消耗的葡萄糖和琥珀酸的生产强度达到最大,分别为43.6g/L和1.88g/(L-h),发酵液中琥珀酸的终浓度也达到最大,为30g/L. Mg(OH)2对C. crenatum AldhA(pJC)发酵产琥珀酸过程中的pH调控效果优于Ca(OH)2. KOH和NaOH。Mg(OH)2作为中和剂时,琥珀酸的产量达到最大,葡萄糖的消耗速度与琥珀酸的生产强度也明显高于Ca(OH)2、KOH和NaOH作为中和剂时的发酵结果。当pH为6.8,以Mg(OH)2作为中和剂,C.crenatum△ldhA(pJC)在22h内共消耗78.5g/L葡萄糖,发酵液中琥珀酸的终浓度达到53.8g/L,琥珀酸的平均生产强度为2.45g/(L-h)。通过优化琥珀酸厌氧发酵pH调控工艺,获得了适于C. crenatum AldhA(pJC)厌氧发酵产琥珀酸的pH和中和剂,为进一步扩大发酵规模提供技术支持。
C. crenatum厌氧发酵产琥珀酸是生物法制备琥珀酸产业中的新工艺。C. crenatum具有高密度、高强度持续发酵产琥珀酸的能力。基于琥珀酸生产强度和产酸培养基中所含的营养元素,C. crenatum厌氧发酵产琥珀酸的工艺比其它琥珀酸发酵工艺更具有优势。通过本研究实施,掌握了C. crenatum厌氧发酵产琥珀酸的特点,可为工业化生产琥珀酸提供理论与实际意义。
As a C4-dicarboxylic acid, succinic acid is valuable due to its widely use in food, chemical and pharmaceutical industries. It is currently produced primarily from fossil fuels by chemical synthetic process. This process causes environmental pollution easily and the development is limited by the shortage of fossil fuels. Therefore, the biological synthetic process for succinic acid is more economical and acceptable. The commercial production of succinic acid by biological process will become one sustainable model of development due to the consumption of greenhouse gas-CO2, meanwhile, the microbial transformation of renewable resources to produce succinic acid can get rid of the dependence on petrochemical raw materials. Scholars have done a lot of effective research on the microbial production of succinic acid, however, there are still some problems to solve, such as the unclear metabolic network and the formation of by-product, particularly the low succinic acid productivity.
Corynebacterium crenatum is one of the main glutamic acid production microorganisms used in the practical production for many years. It is known that the metabolic flux will be changed during the production of glutamic acid when the medium dissolved oxygen is insufficient. When the culture condition is changed to anaerobic, the carbon flow was directed into the succinic acid biosynthesis pathway. In this report, in order to enhance the succinic acid yield and productivity, the features of succinic acid production by Corynebacterium crenatum CICC202019under anaerobic conditions were investigated, and the intracellular key enzyme activity was analyzed. On the basis of above results and combined with metabolic inhibition analysis, the metabolic net of C. crenatum under anaerobic condition was constructed. Meanwhile, the metabolic net was modified by genetically engineering for decreasing the production of by-product and enhancing succinic acid productivity. In order to improve succinic acid productivity, the pH control process was also optimized. The main conclusions and results are as follows.
(1) The characters of lactic acid and succinic acid production by C. crenatum were studied by comparing the products and enzyme activities under different aerobic conditions. When C. crenatum cells were cultured under anaerobic conditions, the cells could produce succinic acid and lactic acid from glucose even through their proliferation was arrested. About19.8g/L succinic acid and46g/L lactic acid were produced in22h, with the yield of succinic acid and lactic acid as0.27g/g and0.62g/g, respectively. In the dual-phase fermentation of C. crenatum, the cells proliferated quickly in the aerobic phase without the production of succinic acid and lactic acid. When aerobic conditions were changed to anaerobic phase by aerating with CO2gas, the concentration of succinic acid and lactic acid increased significantly, which were15g/L and51g/L, respectively. The results of high-cell-density fermentation indicated that a proximate linear relationship existed between cell concentration and the production rates of lactic acid and succinic acid. When dry cell concentration was60g/L,59g/L of lactic acid and23g/L of succinic acid were produced within2h. The productivity of lactic acid and succinic acid was29.5g/(L-h) and11.5g/(L-h), respectively. During cell-recycling repeated fermentation, about310g lactic acid and423g succinic acid were produced from780g glucose in100h. The volumetric productivities of succinic acid and lactic acid could maintain above4.2g/(L-h) and3.1g/(L-h) for at least100h. The characters of acid production by C. crenatum demonstrated that C. crenatum cells had sustained succinic acid production ability and high acid volumetric productivity under anaerobic conditions, those conclusions in this part are beneficial to produce succinic acid subsequently with C. crenatum.
(2) Through analyzing the key enzyme activity in metabolic pathway, it was found that the phosphoenolpyruvate carboxylase and malate dehydrogenase enzyme activity in anaerobic conditions were higher than the value detected in aerobic conditions, meantime, isocitrate lyase enzyme activity was low. However, the isocitric dehydrogenase enzyme activity could not be detected. The effect of inhibitor on succinic acid production indicated that succinic acid concentration decreased43%with propane diacid concentration increasing from0to2g/L, while fluoroacetic acid had no obvious effect on succinic acid production. From the effect of ORP on succinic acid production, it was found that when reducing the ORP level from-40mv to-300mv, succinic acid concentration increased from14.4g/L to21.5g/L, meanwhile, the yield of succinic acid increased from0.18g/g to0.31g/g. The conclusions stated above in this part indicated that succinic acid was produced through C4pathway and glyoxylate cycle. Combined with Corynebacterium glutamicum genome information, the metabolic network of C. crenatum under anaerobic conditions was constructed. Metabolic flux calculation results indicated that the C4pathway was the main metabolic pathway for succinic acid production, the succinic acid produced by C4pathway accounted for93%of the whole succinic acid produced.
(3) Lactic acid is the primary by-product when C. crenatum is used to produce succinic acid. The effect of lactate dehydrogenase(1dhA) knockout on the succinic acid production was studied. The IdhA gene was amplified and the knockout plasmid pMKA was constructed and electrotransformed. Compared with the wild strain, the lactate dehydrogenase (LDH) enzyme activity in mutant could not be detected, and glucose consumed by mutant decreased50%. The yield of succinic acid produced by mutant is0.62g/g, which is higher than the value (0.22g/g) obtained from the wild strain. The biosynthetic pathway was removed to enhance the yield of succinic acid by knocking out the1dhA gene.
(4) The effect of ppc and pyc overexpression on the succinic acid production in1dhA mutant was investigated. Although the phosphoenolpyruvate carboxylase activity increased by1.5fold when ppc gene was overexpressed through pJA expression vector, but.the glucose consumption rate and the yield of succinic acid had no obvious change, implying that the phosphoenolpyruvate carboxylase was not the rate-limiting enzymes in the production of succinic acid by C. crenatum AldhA. The pyruvate carboxylase activity increased by8fold when pyc gene was overexpressed through pJC expression vector and the glucose consumption rate increased46%compared with C. crenatum AldhA, meanwhile, the yield of succinic acid increased from0.64g/g to0.68g/g.
(5) The succinic acid biological production cost can be reduced by improving succinic acid production efficiency through optimizing appropriate pH and neutralizing agent. The results indicated, when the pH was6.8, the glucose consumed and succinic acid productivity was the maximum compared with the other pH, which were43.6g/L and1.88g/(L-h), respectively. The succinic acid concentration in broth was also the maximum, which achieved to30g/L. The fermentation efficiency obtained at pH6.8was better than that of the other pH. For controlling the pH during C. crenatum AldhA(pJC) fermentation, Mg(OH)2was superior to Ca (OH)2、KOH and NaOH. When Mg(OH)2as neutralizing agent, the yield of succinic acid reached the maximum, the glucose consumption rate and succinic acid productivity were obviously higher than that of Ca (OH)2, KOH and NaOH. When pH was6.8and with Mg (OH)2as neutralizing agent, there was78.5g/L glucose was consumed within22h, and the final succinic acid concentration reached53.8g/L, with the average succinic acid productivity as of2.45g/(L-h). Through optimizing the pH control process during succinic acid anaerobic fermentation, the suitable pH and neutralizing agent for C. crenatum△IdhA (pJC) anaerobic fermentation were obtained, which can provide technical support to large scale fermentation.
A new succinic acid production bioprocess by C. crenatum was investigated in mineral medium under anaerobic conditions. The results indicated that C. crenatum cells can produce succinic acid with sustained acid production ability and high acid volumetric productivity. The biological process was more efficient than some of the conventional fermentations in terms of volumetric productivity and dispensability of complex nutrients. Through this project, the characteristics of succinic acid production by C. crenatum under anaerobic conditions were known, the obtained results in this paper have theoretical and practical significance for succinic acid industrial production.
Corynebacterium crenatum是工业发酵制备谷氨酸的主要菌株,已有的研究发现,改变C.crenatum氨基酸发酵培养基溶氧水平,会对该菌株的代谢流分布产生较大的影响,厌氧发酵条件下,有相当部分碳流向琥珀酸发酵途径。本文基于Corynebacterium crenatum CICC20219厌氧发酵特性研究,以提高琥珀酸产量和生产强度为主要目标,通过对胞内碳代谢关键酶活测定,并结合代谢抑制分析,分析了C. crenatum在厌氧条件下的代谢网络,计算出琥珀酸形成的代谢通量分布；通过基因工程方法对其代谢网络进行针对性改造,有效降低代谢副产物的生成,提高了琥珀酸产量。研究并优化了琥珀酸厌氧发酵pH调控工艺,提高了生产强度。主要结论如下：
(1)通过对比C. crenatum在有氧和厌氧两种条件下的代谢产物,并结合关键酶活测定,研究C. crenatum厌氧发酵产琥珀酸特性,分析其产酸的能力。将C. crenatum在厌氧条件下培养,发现C. crenatum细胞不增殖,但可以吸收利用葡萄糖产乳酸、琥珀酸等有机酸。C crenatum厌氧发酵22h内共产生了19.8g/L琥珀酸和46g/L乳酸,琥珀酸和乳酸的产量分别为0.27g/g和0.62g/g。将C. crenatum进行两阶段培养,发现在有氧阶段时,C.crenatum生物量迅速增加,而琥珀酸和乳酸的积累量很少,在通入二氧化碳转为厌氧发酵后,C.crenatum的生物量不再增加,而琥珀酸和乳酸的浓度开始迅速增加,分别达到了15g/L和51g/L。富集C. crenatum细胞进行高密度厌氧发酵时,乳酸和琥珀酸的生产强度与C. crenatum的生物量呈线性关系,生物量为60g/L时,乳酸和琥珀酸的积累浓度在2h内分别达到了59g/L和23g/L,生产强度分别达到了29.5g/(L·h)和11.5g/(L·h)。将C. crenatum进行循环厌氧发酵,C. crenatum在10批共100h的循环发酵时间内利用780g葡萄糖,生成310g乳酸和423g琥珀酸,糖酸转化率在90%以上,乳酸与琥珀酸生产强度分别为3.1g/(L·h)和4.2g/(L-h)。 C. crenatum厌氧发酵产酸的特性表明C. crenatum具有持续、高强度产琥珀酸的能力,这一结论为进一步利用C.crenatum发酵产琥珀酸奠定了基础。
(2)通过对C. crenatum代谢途径中关键酶活性分析,发现该菌株在厌氧条件下的磷酸烯醇式丙酮酸羧化酶和苹果酸脱氢酶的酶活较高,异柠檬酸裂合酶的酶活较弱,检测不到异柠檬酸脱氢酶的酶活。通过分析抑制剂对C. crenatum厌氧发酵产琥珀酸的影响,发现当培养基中的丙二酸浓度由0g/L增加到2g/L时,琥珀酸的终浓度下降了43%,而氟乙酸对该菌株产琥珀酸的影响很小。通过研究氧化还原电位(ORP)对C. crenatum厌氧发酵产琥珀酸的影响,发现当培养基的ORP由-40mv降低为-300mv时,发酵液中琥珀酸的终浓度由14.4g/L增加到21.5g/L,同比增加了50%,琥珀酸产量也由0.18g/g上升到0.31g/g。结合酶活分析结果及抑制剂、ORP对发酵结果的影响,表明C. crenatum产琥珀酸的代谢途径主要是C4途径和乙醛酸循环。参考Corynebacterium glutamicum基因组信息,得出C. crenatum在厌氧条件下的代谢网络模型,代谢通量计算结果表明C4途径及乙醛酸循环是C. crenatum产琥珀酸的主要代谢途径,经由C4途径生产的琥珀酸占整个琥珀酸产量的93%,为对C. crenatum代谢途径进行针对性改造提供了理论支持。
(3)乳酸是C. crenatum厌氧发酵产琥珀酸的主要副产物。以C. crenatum基因组为模板PCR扩增山ldhA基因,构建了ldhA基因敲除载体pMKA,并电转化C. crenatum,质粒pMKA与C. crenatum基因组进行基因重组,敲除了C. crenatum的IdhA基因,获得突变株C. crenatum AldhA。与原始菌株相比,突变株的葡萄糖消耗速度下降50%,乳酸脱氢酶酶活和乳酸产量均为0,琥珀酸的产量由0.22g/g上升到0.62g/g。通过敲除IdhA基因达到了消除乳酸生成、增加琥珀酸产量的目的。
(4)通过表达ppc和pyc基因,可以强化C. crenatum磷酸烯醇式丙酮酸与丙酮酸羧化途径,将更多的碳引向C4途径。以C. crenatum基因组为模板PCR扩增出磷酸烯醇式丙酮酸羧化酶基因ppc和丙酮酸羧化酶基因pyc,构建了ppc与pyc基因表达质粒pJA、pJC。分别以表达质粒为载体在C. crenatum△AldhA内过表达ppc与pyc基因,获得菌株C. crenatum△ldhA(pJA)与C. crenatum△ldhA(pJC)。与C. crenatum△ldhA相比,C. crenatum△ldhA(pJA)的磷酸烯醇式丙酮酸羧化酶活力比对照株增加1.5倍,葡萄糖的消耗速度和琥珀酸的产量并没有明显的变化,表明磷酸烯醇式丙酮酸羧化酶不是C. crenatum△ldhA产琥珀酸的限速酶。过量表达pyc后,C.crenatum△ldhA(pJC)胞内丙酮酸羧化酶活力比C. crenatum△ldhA增加8倍,葡萄糖消耗速度增加了46%,琥珀酸的产量由0.64g/g增加到0.68g/g,同比增加了7.8%。
(5)通过选择合适的pH及中和剂,可以提高琥珀酸的生产效率以降低生物法生产琥珀酸的生产成本。pH优化结果显示,pH为6.8时的发酵效果优于其它pH值。pH为6.8时,消耗的葡萄糖和琥珀酸的生产强度达到最大,分别为43.6g/L和1.88g/(L-h),发酵液中琥珀酸的终浓度也达到最大,为30g/L. Mg(OH)2对C. crenatum AldhA(pJC)发酵产琥珀酸过程中的pH调控效果优于Ca(OH)2. KOH和NaOH。Mg(OH)2作为中和剂时,琥珀酸的产量达到最大,葡萄糖的消耗速度与琥珀酸的生产强度也明显高于Ca(OH)2、KOH和NaOH作为中和剂时的发酵结果。当pH为6.8,以Mg(OH)2作为中和剂,C.crenatum△ldhA(pJC)在22h内共消耗78.5g/L葡萄糖,发酵液中琥珀酸的终浓度达到53.8g/L,琥珀酸的平均生产强度为2.45g/(L-h)。通过优化琥珀酸厌氧发酵pH调控工艺,获得了适于C. crenatum AldhA(pJC)厌氧发酵产琥珀酸的pH和中和剂,为进一步扩大发酵规模提供技术支持。
C. crenatum厌氧发酵产琥珀酸是生物法制备琥珀酸产业中的新工艺。C. crenatum具有高密度、高强度持续发酵产琥珀酸的能力。基于琥珀酸生产强度和产酸培养基中所含的营养元素,C. crenatum厌氧发酵产琥珀酸的工艺比其它琥珀酸发酵工艺更具有优势。通过本研究实施,掌握了C. crenatum厌氧发酵产琥珀酸的特点,可为工业化生产琥珀酸提供理论与实际意义。
As a C4-dicarboxylic acid, succinic acid is valuable due to its widely use in food, chemical and pharmaceutical industries. It is currently produced primarily from fossil fuels by chemical synthetic process. This process causes environmental pollution easily and the development is limited by the shortage of fossil fuels. Therefore, the biological synthetic process for succinic acid is more economical and acceptable. The commercial production of succinic acid by biological process will become one sustainable model of development due to the consumption of greenhouse gas-CO2, meanwhile, the microbial transformation of renewable resources to produce succinic acid can get rid of the dependence on petrochemical raw materials. Scholars have done a lot of effective research on the microbial production of succinic acid, however, there are still some problems to solve, such as the unclear metabolic network and the formation of by-product, particularly the low succinic acid productivity.
Corynebacterium crenatum is one of the main glutamic acid production microorganisms used in the practical production for many years. It is known that the metabolic flux will be changed during the production of glutamic acid when the medium dissolved oxygen is insufficient. When the culture condition is changed to anaerobic, the carbon flow was directed into the succinic acid biosynthesis pathway. In this report, in order to enhance the succinic acid yield and productivity, the features of succinic acid production by Corynebacterium crenatum CICC202019under anaerobic conditions were investigated, and the intracellular key enzyme activity was analyzed. On the basis of above results and combined with metabolic inhibition analysis, the metabolic net of C. crenatum under anaerobic condition was constructed. Meanwhile, the metabolic net was modified by genetically engineering for decreasing the production of by-product and enhancing succinic acid productivity. In order to improve succinic acid productivity, the pH control process was also optimized. The main conclusions and results are as follows.
(1) The characters of lactic acid and succinic acid production by C. crenatum were studied by comparing the products and enzyme activities under different aerobic conditions. When C. crenatum cells were cultured under anaerobic conditions, the cells could produce succinic acid and lactic acid from glucose even through their proliferation was arrested. About19.8g/L succinic acid and46g/L lactic acid were produced in22h, with the yield of succinic acid and lactic acid as0.27g/g and0.62g/g, respectively. In the dual-phase fermentation of C. crenatum, the cells proliferated quickly in the aerobic phase without the production of succinic acid and lactic acid. When aerobic conditions were changed to anaerobic phase by aerating with CO2gas, the concentration of succinic acid and lactic acid increased significantly, which were15g/L and51g/L, respectively. The results of high-cell-density fermentation indicated that a proximate linear relationship existed between cell concentration and the production rates of lactic acid and succinic acid. When dry cell concentration was60g/L,59g/L of lactic acid and23g/L of succinic acid were produced within2h. The productivity of lactic acid and succinic acid was29.5g/(L-h) and11.5g/(L-h), respectively. During cell-recycling repeated fermentation, about310g lactic acid and423g succinic acid were produced from780g glucose in100h. The volumetric productivities of succinic acid and lactic acid could maintain above4.2g/(L-h) and3.1g/(L-h) for at least100h. The characters of acid production by C. crenatum demonstrated that C. crenatum cells had sustained succinic acid production ability and high acid volumetric productivity under anaerobic conditions, those conclusions in this part are beneficial to produce succinic acid subsequently with C. crenatum.
(2) Through analyzing the key enzyme activity in metabolic pathway, it was found that the phosphoenolpyruvate carboxylase and malate dehydrogenase enzyme activity in anaerobic conditions were higher than the value detected in aerobic conditions, meantime, isocitrate lyase enzyme activity was low. However, the isocitric dehydrogenase enzyme activity could not be detected. The effect of inhibitor on succinic acid production indicated that succinic acid concentration decreased43%with propane diacid concentration increasing from0to2g/L, while fluoroacetic acid had no obvious effect on succinic acid production. From the effect of ORP on succinic acid production, it was found that when reducing the ORP level from-40mv to-300mv, succinic acid concentration increased from14.4g/L to21.5g/L, meanwhile, the yield of succinic acid increased from0.18g/g to0.31g/g. The conclusions stated above in this part indicated that succinic acid was produced through C4pathway and glyoxylate cycle. Combined with Corynebacterium glutamicum genome information, the metabolic network of C. crenatum under anaerobic conditions was constructed. Metabolic flux calculation results indicated that the C4pathway was the main metabolic pathway for succinic acid production, the succinic acid produced by C4pathway accounted for93%of the whole succinic acid produced.
(3) Lactic acid is the primary by-product when C. crenatum is used to produce succinic acid. The effect of lactate dehydrogenase(1dhA) knockout on the succinic acid production was studied. The IdhA gene was amplified and the knockout plasmid pMKA was constructed and electrotransformed. Compared with the wild strain, the lactate dehydrogenase (LDH) enzyme activity in mutant could not be detected, and glucose consumed by mutant decreased50%. The yield of succinic acid produced by mutant is0.62g/g, which is higher than the value (0.22g/g) obtained from the wild strain. The biosynthetic pathway was removed to enhance the yield of succinic acid by knocking out the1dhA gene.
(4) The effect of ppc and pyc overexpression on the succinic acid production in1dhA mutant was investigated. Although the phosphoenolpyruvate carboxylase activity increased by1.5fold when ppc gene was overexpressed through pJA expression vector, but.the glucose consumption rate and the yield of succinic acid had no obvious change, implying that the phosphoenolpyruvate carboxylase was not the rate-limiting enzymes in the production of succinic acid by C. crenatum AldhA. The pyruvate carboxylase activity increased by8fold when pyc gene was overexpressed through pJC expression vector and the glucose consumption rate increased46%compared with C. crenatum AldhA, meanwhile, the yield of succinic acid increased from0.64g/g to0.68g/g.
(5) The succinic acid biological production cost can be reduced by improving succinic acid production efficiency through optimizing appropriate pH and neutralizing agent. The results indicated, when the pH was6.8, the glucose consumed and succinic acid productivity was the maximum compared with the other pH, which were43.6g/L and1.88g/(L-h), respectively. The succinic acid concentration in broth was also the maximum, which achieved to30g/L. The fermentation efficiency obtained at pH6.8was better than that of the other pH. For controlling the pH during C. crenatum AldhA(pJC) fermentation, Mg(OH)2was superior to Ca (OH)2、KOH and NaOH. When Mg(OH)2as neutralizing agent, the yield of succinic acid reached the maximum, the glucose consumption rate and succinic acid productivity were obviously higher than that of Ca (OH)2, KOH and NaOH. When pH was6.8and with Mg (OH)2as neutralizing agent, there was78.5g/L glucose was consumed within22h, and the final succinic acid concentration reached53.8g/L, with the average succinic acid productivity as of2.45g/(L-h). Through optimizing the pH control process during succinic acid anaerobic fermentation, the suitable pH and neutralizing agent for C. crenatum△IdhA (pJC) anaerobic fermentation were obtained, which can provide technical support to large scale fermentation.
A new succinic acid production bioprocess by C. crenatum was investigated in mineral medium under anaerobic conditions. The results indicated that C. crenatum cells can produce succinic acid with sustained acid production ability and high acid volumetric productivity. The biological process was more efficient than some of the conventional fermentations in terms of volumetric productivity and dispensability of complex nutrients. Through this project, the characteristics of succinic acid production by C. crenatum under anaerobic conditions were known, the obtained results in this paper have theoretical and practical significance for succinic acid industrial production.
引文
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