CN108103136A - 一种通过电化学系统强化微生物菌体生产丁二酸的方法 - Google Patents
一种通过电化学系统强化微生物菌体生产丁二酸的方法 Download PDFInfo
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
本发明公开了一种通过电化学系统强化微生物菌体生产丁二酸的方法及其应用,包括菌种活化、种子培养和厌氧发酵步骤,所述厌氧发酵采用电化学发酵,在发酵培养基中添加电子载体,所述电子载体的浓度为0.1‑1.0 mmol/L。所述电化学发酵中,阳极微生物菌体为产琥珀酸放线杆菌(Actinobacillus succinogenes),阴极微生物菌体为大肠埃希氏菌(Escherichia coli)。本发明将微生物燃料电池与微生物电解池偶联引入微生物发酵体系中,用于平衡及调控胞内的还原力水平,包括降低阳极微生物产琥珀酸放线杆菌(Actinobacillus succinogenes)还原力水平,增强还原型底物甘油的消耗与微生物生长,增加丁二酸的合成,在此基础上产生电能以提高阴极微生物大肠埃希氏菌(Escherichia coli)的还原力水平,增加丁二酸的合成。
Description
技术领域
本发明涉及一种通过电化学系统强化微生物菌体生产丁二酸的方法及其应用,属于生物化工技术领域。
背景技术
在厌氧条件下发酵产丁二酸的过程中,还原力水平对丁二酸的产量有着至关重要的影响。在以葡萄糖为唯一碳源时,NADH被大量的消耗用于合成还原性产物丁二酸,造成了还原力的缺失;而在以甘油为唯一碳源时,微生物的生长过程会产生额外的NADH,造成NADH的大量积累,使胞内还原力过剩。两种情况都会抑制微生物菌体的生长,减少目的代谢产物丁二酸积累量。
为了平衡胞内还原力水平,恢复菌体生长的同时增加还原型产物的收率。 ASingh等(Metabolic Engineering,2011,13(1):76)通过在大肠杆菌内过量阻断积累副产物的代谢途径及激活乙醛酸途径,减少了胞内NADH的消耗,增强了胞内NADH再生,使得丁二酸的产量提高了60%。Claire Vieille等通过引入异源还原性途径增加NADH的消耗,同时一些研究过程中采用微厌氧条件发酵,恢复了产琥珀酸放线杆菌利用甘油代谢生长的能力。但是这些代谢工程的改造,均会加重菌株的代谢负担,造成目的产物的收率不高。
在电化学系统中,微生物燃料电池与微生物电解池均可以达成电子在微生物菌体和电极之间的传递。如电化学活性菌株可通过合成导电鞭毛或纳米导线或者向胞外分泌电子载体,非电化学活性菌株可外源添加电子载体(中性红、核黄素等)。微生物菌体通过在电极上获得或失去电子,以达到细胞代谢和还原力水平的平衡。虽然电子载体与微生物菌体间的电子传递机理尚不明确,Kracke F等 (Frontiers in Microbiology,2015,6:575)研究了多种微生物及电子载体在电化学系统中的应用,为此提供了实验依据。
发明内容
本发明的目的是提供一种通过电化学系统强化微生物菌体生产丁二酸的方法及其应用,将微生物燃料电池与微生物电解池偶联引入微生物发酵体系中,用于平衡胞内的还原力水平,包括利用还原性底物甘油而造成的高还原力的释放,及以葡萄糖为底物时的还原力缺失的补充,消除细胞生长的抑制,提高丁二酸的产量。
为了实现本发明的技术目的,本发明采用的技术方案如下:
一种通过电化学系统强化微生物菌体生产丁二酸的方法,包括菌种活化、种子培养、厌氧发酵,所述厌氧发酵采用电化学发酵,在发酵培养基中添加电子载体。所述电子载体的浓度为0.01-1.0mmol/L。
所述电子载体为具有氧化还原对特性的化合物,可以为化学型电子载体,也可以选择生物型电子载体,包括但不限于中性红和核黄素。
更优选地,电化学系统中阳极发酵培养基中性红1mM或者核黄素1mM;阴极发酵培养基添加中性红0.05mM,或者核黄素0.1mM。
所述阳极菌株为任意可在厌氧条件下生长并可发酵积累丁二酸产物的菌株,包括但不限于产琥珀酸放线杆菌(Actinobacillus succinogenes)。
所述阴极菌株为任意可在厌氧条件下生长并可发酵积累丁二酸产物的菌株,包括但不限于大肠埃希氏菌(Escherichia coli)。
所述电化学发酵中,选用石墨碳毡作为阴阳两极电极,Ag/AgCl(饱和KCl) 作为参比电极,通过电子载体介导电子在微生物胞内和电极之间的传递。
电化学发酵时,阴、阳两极室内填充的发酵培养基中包含不同浓度的电子载体,并通过外电阻将阴阳两极室联通。
所述电化学系统为采用气密性良好的H-cell型电化学装置,通过外加电子载体协助电子在细胞与电极之间的传递。
所述阳极发酵培养基为玉米浆干粉5-10g/L,酵母粉5-15g/L,乙酸钠 1.0-2.0g/L,NaCl 0.5-2.0g/L,CaCl20.1-0.5g/L,MgCl20.1-0.5g/L,NaH2PO41.0-2.0 g/L,Na2HPO40.1-0.5g/L,K2HPO41.0-5.0g/L,碳源甘油,甘油浓度为10~40g/L;优选为甘油10g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L, CaCl20.2g/L,MgCl20.2g/L,NaH2PO41.6g/L,Na2HPO40.3g/L,K2HPO43g/L。
所述阴极发酵培养基为一水合柠檬酸3g/L,Na2HPO4·12H2O 4g/L,KH2PO4 8g/L,(NH4)2HPO48g/L,NH4Cl 0.2g/L,(NH4)2SO40.75g/L,MgSO4·7H2O 1g/L, CaCl2·2H2O10.0mg/L,ZnSO4·7H2O 0.5mg/L,CuCl2·2H2O 0.25mg/L, MnSO4·H2O 2.5mg/L,CoCl2·6H2O 1.75mg/L,H3BO30.12mg/L,Al2(SO4)31.77 mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1mg/L,生物素2mg/L,维生素 B120mg/L,葡萄糖30g/L。
本发明所述的菌种活化、种子培养步骤是常规的放线杆菌菌种活化方法及种子培养方法,阳极以产琥珀酸放线杆菌NJ113(Actinobacillus succinogenes NJ113) 为例说明菌种活化及种子培养步骤:产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株经固体平板培养基活化后,37℃条件下,在厌氧血清瓶中培养12-14小时后转接于种子培养基,在37℃,200转/分钟的条件下培养6-8 小时得到种子液;
优选地,所述固体平板培养基和种子培养基的配方为:葡萄糖20g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl20.2g/L,MgCl2 0.2g/L,NaH2PO41.6g/L,Na2HPO40.3g/L,K2HPO43g/L,琼脂粉15-20g/L。
优选地,所述发酵培养基中添加单一电子载体。
本发明所述的菌种活化、种子培养步骤是常规的菌种活化方法及种子培养方法,阴极以大肠埃希氏菌AFP111(Escherichia coliAFP111)为例说明菌种活化及种子培养步骤:本发明中将大肠埃希氏菌AFP111(Escherichia coliAFP111)菌株经固体平板培养基活化后,37℃,有氧条件下培养12-14小时后转接于种子培养基,在37℃,200转/分钟的条件下培养6-8小时得到种子液。
优选地,所述固体平板培养基和种子培养基的配方为:蛋白胨10g/L,酵母粉5g/L,NaCl 5g/L,琼脂粉15-20g/L。
本发明的另一目的是提供上述通过电化学系统强化微生物菌体生产丁二酸方法的应用。
本发明通过采用电化学调控手段,动态调控了胞内还原力水平,与单纯厌氧条件下产丁二酸实验相比,其有益效果在于:
本发明所用大肠埃希氏菌AFP111(Escherichia coliAFP111)可以在发酵培养基中,以葡萄糖为唯一碳源在纯厌氧条件下生长、合成并积累丁二酸:在厌氧条件下发酵48小时后丁二酸积累量可达10.11g/L;产琥珀酸放线杆菌NJ113 (Actinobacillussuccinogenes NJ113)可以在发酵培养基中,以甘油为唯一碳源在纯厌氧条件下生长、合成并积累丁二酸:在厌氧条件下发酵48小时后,甘油消耗达0.3g/L,丁二酸积累量达1.07g/L。将大肠埃希氏菌AFP111(Escherichia coliAFP111)和产琥珀酸放线杆菌NJ113(Actinobacillus succinogenes NJ113)进行偶联电化学发酵时,大肠埃希氏菌AFP111(Escherichia coliAFP111)的丁二酸积累量增加到了14.92g/L,相比于对照组丁二酸的产量提高了47.6%。而产琥珀酸放线杆菌NJ113(Actinobacillus succinogenes NJ113)恢复了厌氧条件下利用甘油的能力,丁二酸积累量达到了9.71g/L。
附图说明
图1.H-cell电化学装置示意图。
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenes NJ113)胞内的NADH 将电子传递给通过某种方式进如胞内周至空间的氧化型电子载体,还原型载体在电极阳极表面被氧化,电子通过外电路从阳极传递到阴极,在阴极表面还原氧化型的电子载体,氧化型电子载体通过某种方式进入周至空间,将电子传递给NAD+,形成NADH。
具体实施方式
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenes NJ113)为本实验室所有,在中国专利局或国际专利组织承认的中国微生物菌种保藏管理委员会普通微生物中心进行了专利程序保藏,保藏编号:CGMCC No.1716。
大肠埃希氏菌AFP111(Escherichia coliAFP111)菌种由D.P.Clark教授惠赠,保存于本实验室,申请人保证从本申请日起二十年内向公众发放该生物材料。
本发明中将产琥珀酸放线杆菌NJ113菌株,大肠埃希氏菌AFP111菌株通过固体平板培养基培养后接种至种子培养基中培养得到种子液;然后将种子液接种到发酵培养基中,并通过电化学调控强化菌株的代谢性能。
产琥珀酸放线杆菌NJ113菌株固体平板培养基和种子培养基的配方为:葡萄糖20g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl2 0.2g/L,MgCl20.2g/L,NaH2PO41.6g/L,Na2HPO40.3g/L,K2HPO43g/L,琼脂粉 15-20g/L。
大肠埃希氏菌AFP111菌株固体平板培养基和种子培养基的配方为:蛋白胨 10g/L,酵母粉5g/L,NaCl 5g/L,琼脂粉15-20g/L。
产琥珀酸放线杆菌NJ113菌株经固体平板培养基活化后转接至厌氧血清瓶, 37℃,厌氧条件下培养12-14小时后转接于种子培养基,在37℃,200转/分钟的条件下培养6-8小时得到种子液;大肠埃希氏菌AFP111菌株经固体平板培养基活化后转接至试管,37℃,有氧条件下培养12-14小时后转接于种子培养基,在37℃,200转/分钟的条件下培养6-8小时得到种子液
将产琥珀酸放线杆菌NJ113菌株种子液和大肠埃希氏菌AFP111菌株种子液按照6-10%(v/v)的接种量分别接种于含有发酵培养基的H-cell电化学装置的阳极室和阴极室中,于37℃进行厌氧发酵。在发酵过程中每隔一段时间进行无菌取样,对样品离心处理后测定甘油、葡萄糖及有机酸浓度。
根据以下实施例,可以更好的理解本发明。实施案例中所描述的具体的物料配比,工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
本实施例说明将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株,大肠埃希氏菌AFP111(Escherichia coliAFP111)进行厌氧发酵的方法及其应用。
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)厌氧发酵方法如下:
将冻存管中的产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)作为出发菌株,按上述所述方法获得种子液,以6%(v/v)的接种量接种于含有 450mL发酵培养基的700mL恒化器装置中。向反应器内持续通入无菌二氧化碳以维持厌氧环境。发酵过程中定时无菌取样,检测培养装置中菌体的密度;将样品离心后保留上清,通过高效液相色谱检测有机酸含量。
所述发酵培养基组成为:甘油10g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl20.2g/L,MgCl20.2g/L,NaH2PO41.6g/L, Na2HPO40.3g/L,K2HPO43g/L。
大肠埃希氏菌AFP111(Escherichia coliAFP111)厌氧发酵方法如下:
将冻存管中的大肠埃希氏菌AFP111(Escherichia coliAFP111)作为出发菌株,按上述所述方法获得种子液,将二级种子液以10%(v/v)的接种量接种于含有 450mL发酵培养基的700mL恒化器装置中,向恒化器内持续通入无菌二氧化碳以维持厌氧环境。发酵过程中定时无菌取样,检测培养装置中菌体的密度;将样品离心后保留上清,通过高效液相色谱检测有机酸含量。
所述发酵培养基的配方为:一水合柠檬酸3g/L,Na2HPO4·12H2O 4g/L, KH2PO48g/L,(NH4)2HPO48g/L,NH4Cl 0.2g/L,(NH4)2SO40.75g/L,MgSO4·7H2O 1g/L,CaCl2·2H2O10.0mg/L,ZnSO4·7H2O 0.5mg/L,CuCl2·2H2O 0.25mg/L,MnSO4·H2O 2.5mg/L,CoCl2·6H2O1.75mg/L,H3BO30.12mg/L, Al2(SO4)31.77mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1mg/L,生物素2 mg/L,维生素B120mg/L,葡萄糖30-40g/L。
检测的有机酸浓度和电量如表1所示:
表1:对照组厌氧发酵48h后底物消耗及有机酸产量
实施例2
本实施例说明将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法及其应用。
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法如下:
将冻存管中的产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)和大肠埃希氏菌AFP111(Escherichia coliAFP111)作为出发菌株,按上述所述方法获得种子液后,将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113) 种子液以6%(v/v)接种量接种于H-cell电化学装置的阳极室中,并添加1mM 中性红,向阳极室内持续通入无菌二氧化碳以维持厌氧环境。将大肠埃希氏菌 AFP111(Escherichia coliAFP111)种子液按10%(v/v)接种量接种于H-cell电化学装置的阴极室中,并添加0.05mM中性红,向阴极室内持续通入无菌二氧化碳以维持厌氧环境。发酵过程中定时无菌取样,检测培养装置中菌体的密度;将样品离心后保留上清,通过高效液相色谱检测有机酸含量。
所述阳极发酵培养基组成为:甘油10g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl20.2g/L,MgCl20.2g/L,NaH2PO41.6g/L, Na2HPO40.3g/L,K2HPO43g/L。所述阴极发酵培养基的配方为:一水合柠檬酸3 g/L,Na2HPO4·12H2O 4g/L,KH2PO48g/L,(NH4)2HPO48g/L,NH4Cl 0.2g/L, (NH4)2SO40.75g/L,MgSO4·7H2O 1g/L,CaCl2·2H2O 10.0mg/L,ZnSO4·7H2O 0.5 mg/L,CuCl2·2H2O 0.25mg/L,MnSO4·H2O 2.5mg/L,CCl2·6H2O 1.75mg/L, H3BO30.12mg/L,Al2(SO4)31.77mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1 mg/L,生物素2mg/L,维生素B120mg/L,葡萄糖30g/L。
检测的有机酸浓度和电量如表2所示:
表2:厌氧发酵48h后底物的消耗量及有机酸的产量
实施例3
本实施例说明将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法及其应用。
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法如下:
将冻存管中的产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)和大肠埃希氏菌AFP111(Escherichia coliAFP111)作为出发菌株,按上述所述方法获得种子液后,将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113) 种子液以6%(v/v)接种量接种于H-cell电化学装置的阳极室中,并添加1mM 中性红,向阳极室内持续通入无菌二氧化碳以维持厌氧环境。将大肠埃希氏菌 AFP111(Escherichia coliAFP111)种子液按10%(v/v)接种量接种于H-cell电化学装置的阴极室中,并添加0.1mM核黄素,向阴极室内持续通入无菌二氧化碳以维持厌氧环境。发酵过程中定时无菌取样,检测培养装置中菌体的密度;将样品离心后保留上清,通过高效液相色谱检测有机酸含量。
所述阳极发酵培养基组成为:甘油10g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl20.2g/L,MgCl20.2g/L,NaH2PO41.6g/L, Na2HPO40.3g/L,K2HPO43g/L。
所述阴极发酵培养基的配方为:一水合柠檬酸3g/L,Na2HPO4·12H2O 4g/L,KH2PO48g/L,(NH4)2HPO48g/L,NH4Cl 0.2g/L,(NH4)2SO40.75g/L, MgSO4·7H2O 1g/L,CaCl2·2H2O 10.0mg/L,ZnSO4·7H2O 0.5mg/L,CuCl2·2H2O 0.25mg/L,MnSO4·H2O 2.5mg/L,CoCl2·6H2O 1.75mg/L,H3BO30.12mg/L, Al2(SO4)31.77mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1mg/L,生物素2 mg/L,维生素B120mg/L,葡萄糖30g/L。
检测的有机酸浓度和电量如表3所示:
表3:厌氧发酵48h后底物的消耗量及有机酸的产量
实施例4
本实施例说明将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法及其应用。
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法如下:
将冻存管中的产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)和大肠埃希氏菌AFP111(Escherichia coliAFP111)作为出发菌株,按上述所述方法获得种子液后,将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113) 种子液以6%(v/v)接种量接种于H-cell电化学装置的阳极室中,并添加1mM 核黄素,向阳极室内持续通入无菌二氧化碳以维持厌氧环境。将大肠埃希氏菌 AFP111(Escherichia coliAFP111)种子液按10%(v/v)接种量接种于H-cell电化学装置的阴极室中,并添加0.05mM中性红,向阴极室内持续通入无菌二氧化碳以维持厌氧环境。发酵过程中定时无菌取样,检测培养装置中菌体的密度;将样品离心后保留上清,通过高效液相色谱检测有机酸含量。
所述阳极发酵培养基组成为:甘油10g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl20.2g/L,MgCl20.2g/L,NaH2PO41.6g/L, Na2HPO40.3g/L,K2HPO43g/L。
所述阴极发酵培养基的配方为:一水合柠檬酸3g/L,Na2HPO4·12H2O 4g/L,KH2PO48g/L,(NH4)2HPO48g/L,NH4Cl 0.2g/L,(NH4)2SO40.75g/L, MgSO4·7H2O 1g/L,CaCl2·2H2O 10.0mg/L,ZnSO4·7H2O 0.5mg/L,CuCl2·2H2O 0.25mg/L,MnSO4·H2O 2.5mg/L,CoCl2·6H2O 1.75mg/L,H3BO30.12mg/L, Al2(SO4)31.77mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1mg/L,生物素2 mg/L,维生素B120mg/L,葡萄糖30g/L。
检测的有机酸浓度和电量如表4所示:
表4:厌氧发酵48h后底物的消耗量及有机酸的产量
实施例5
本实施例说明将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法及其应用。
产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)菌株和大肠埃希氏菌AFP111(Escherichia coliAFP111)进行偶联电化学厌氧发酵的方法如下:
将冻存管中的产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113)和大肠埃希氏菌AFP111(Escherichia coliAFP111)作为出发菌株,按上述所述方法获得种子液后,将产琥珀酸放线杆菌NJ113(Actinobacillus succinogenesNJ113) 种子液以6%(v/v)接种量接种于H-cell电化学装置的阳极室中,并添加1mM 核黄素,向阳极室内持续通入无菌二氧化碳以维持厌氧环境。将大肠埃希氏菌 AFP111(Escherichia coliAFP111)种子液按10%(v/v)接种量接种于H-cell电化学装置的阴极室中,并添加0.1mM核黄素,向阴极室内持续通入无菌二氧化碳以维持厌氧环境。发酵过程中定时无菌取样,检测培养装置中菌体的密度;将样品离心后保留上清,通过高效液相色谱检测有机酸含量。
所述阳极发酵培养基组成为:甘油10g/L,玉米浆干粉7.5g/L,酵母粉10g/L,乙酸钠1.36g/L,NaCl 1g/L,CaCl20.2g/L,MgCl20.2g/L,NaH2PO41.6g/L, Na2HPO40.3g/L,K2HPO43g/L。
所述阴极发酵培养基的配方为:一水合柠檬酸3g/L,Na2HPO4·12H2O 4g/L,KH2PO48g/L,(NH4)2HPO48g/L,NH4Cl 0.2g/L,(NH4)2SO40.75g/L, MgSO4·7H2O 1g/L,CaCl2·2H2O 10.0mg/L,ZnSO4·7H2O 0.5mg/L,CuCl2·2H2O 0.25mg/L,MnSO4·H2O 2.5mg/L,CoCl2·6H2O 1.75mg/L,H3BO30.12mg/L, Al2(SO4)31.77mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1mg/L,生物素2 mg/L,维生素B120mg/L,葡萄糖30g/L。
检测的有机酸浓度和电量如表5所示:
表5:厌氧发酵48h后底物的消耗量及有机酸的产量
Claims (10)
1.一种通过电化学系统强化微生物菌体生产丁二酸的方法,包括菌种活化、种子培养、厌氧发酵,其特征在于,所述厌氧发酵采用电化学发酵,在发酵培养基中添加电子载体。
2.根据权利要求1所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电子载体浓度为0.01-1.0mmol/L。
3.根据权利要求1所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,电子载体为具有氧化还原对特性的化合物。
4.根据权利要求3所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电子载体为中性红或者核黄素;电化学系统中阳极发酵培养基添加中性红1mM或者核黄素1mM;阴极发酵培养基添加中性红0.05mM或者核黄素0.1mM。
5.根据权利要求1所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电化学发酵中,阳极微生物菌体为产琥珀酸放线杆菌(Actinobacillussuccinogenes)。
6.根据权利要求1所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电化学发酵中,阴极微生物菌体为大肠埃希氏菌(Escherichia coli)。
7.根据权利要求5所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电化学发酵中,阳极发酵培养基为:玉米浆干粉5-10g/L,酵母粉5-15g/L,乙酸钠1.0-2.0g/L,NaCl 0.5-2.0g/L,CaCl2 0.1-0.5g/L,MgCl2 0.1-0.5g/L,NaH2PO4 1.0-2.0g/L,Na2HPO4 0.1-0.5g/L,K2HPO4 1.0-5.0g/L,碳源甘油,甘油浓度为10g/L。
8.根据权利要求6所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电化学发酵中,阴极发酵培养基为:一水合柠檬酸3g/L,Na2HPO4·12H2O 4g/L,KH2PO4 8g/L,(NH4)2HPO4 8g/L,NH4Cl 0.2g/L,(NH4)2SO4 0.75g/L,MgSO4·7H2O 1g/L,CaCl2·2H2O 10.0mg/L,ZnSO4·7H2O 0.5mg/L,CuCl2·2H2O 0.25mg/L,MnSO4·H2O 2.5mg/L,CoCl2·6H2O 1.75mg/L,H3BO3 0.12mg/L,Al2(SO4)3 1.77mg/L,Na2MoO4·2H2O 0.5mg/L,柠檬酸铁16.1mg/L,生物素2mg/L,维生素B1 20mg/L,葡萄糖30g/L。
9.根据权利要求1所述的通过电化学系统强化微生物菌体生产丁二酸的方法,其特征在于,所述电化学系统采用H-cell型电化学装置,选用石墨碳毡作为阴阳两极电极,Ag/AgCl作为参比电极。
10.权利要求1所述通过电化学系统强化微生物菌体生产丁二酸方法的应用。
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