CN111048816B - 一种驱动非产电微生物产电方法 - Google Patents
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Abstract
本发明提供了一种驱动非产电微生物产电方法,将非产电微生物和辅助产电微生物共培养,然后接种于微生物燃料电池的阳极,加入电子供体,可以实现非产电微生物产电。
Description
技术领域
本发明涉及一种驱动非产电微生物产电方法,属于生物电化学技术领域。
背景技术
生物电化学系统(Bioelectrochemical systems,BESs)是一种交叉学科的前沿技术,随着全球淡水资源和可利用能源日益剧减,BESs的运用受到了广泛的关注。它是微生物燃料电池(MFCs)、微生物电解电池(MECs)和微生物电合成电池(MESs)等技术的总称。微生物燃料电池利用微生物的催化作用,将燃料中的化学能转化为电能,同时又可以处理废水的新型技术,具有显著的环境效益和经济效益。近年来,相继有多种产电微生物菌株被发现,大大拓展了微生物燃料电池的应用领域,如直接制造生物传感器、处理废水产电等。微生物燃料电池的研究与应用,对缓解当前的能源危机有重大意义,其发展潜力很大。
目前关于MFC的研究多集中在提高产电微生物的产电能力和去污效能方面,研究的绝大多数微生物为地杆菌属(Geobacter)和希瓦氏菌属(Shewanella)等。产电微生物与MFC阳极电极间存在呼吸作用,即:微生物在产能代谢中底物降解释放出的电子,通过呼吸链即电子传递链最终传递给外源电子受体(如阳极电极)。
然而,并未有研究报道非产电微生物也能实现产电。Clostridium intestinale是梭菌属中一种非产电的微生物,通过发酵(不完全氧化)果糖、蔗糖等底物进行生长繁殖,释放的电子不经过电子传递链而直接传递给内源性中间代谢产物,利用底物水平磷酸化(Substrate level phosphorylation)产能。由于发酵过程只是部分氧化底物,因而产能效率相对较低。若能实现微生物的代谢从发酵向呼吸转化,将有望将非产电微生物引入生物电化学系统中,实现对阳极催化的作用,从而推进对生物电化学系统(Bioelectrochemicalsystems,BESs)的研究。但是,现有技术中还没有实现非产电微生物产电的方法。
发明内容
本发明提供了一种驱动非产电微生物产电方法,可以有效解决上述问题。
本发明是这样实现的:
一种驱动非产电微生物产电方法,将非产电微生物和辅助产电微生物共培养,然后接种于微生物燃料电池的阳极,加入电子供体。
作为进一步改进的,所述非产电微生物为Clostridium intestinale,所述辅助产电微生物为Geobacter sulfurreducens。
作为进一步改进的,所述非产电微生物和辅助产电微生物在共培养前还需分别进行厌氧培养。
作为进一步改进的,所述共培养的接种量按照体积分数为5~10%;接种前,所述非产电微生物和辅助产电微生物的OD600为0.4~0.6。
作为进一步改进的,所述共培养还需每3~5天接种传代,传到第5~7代。
作为进一步改进的,所述共培养的培养温度为28~32℃。
作为进一步改进的,所述电子供体为乙醇。
作为进一步改进的,所述电子供体的终浓度为10~20mM。
作为进一步改进的,所述微生物燃料电池为ITO三电极体系微生物燃料电池。
作为进一步改进的,所述共培养的培养基的配方为MgCl2·7H2O0.05~0.15g/L,CaCl2·2H2O 0.02~0.06g/L,NaCO3·H2O 0.4~0.6g/L,NaHCO31.6~2.0g/L,Fumaric Acid4.50~4.84g/L,100X NB Salts Mix 8~12mL/L,1Mm Na2SeO 40.0~42.0mL,NB MineralElixir 8~12mL/L,DL维生素溶液10~20mL/L,其余为水。
本发明的有益效果是:
本发明公开的一种驱动非产电微生物实现产电的方法,以辅助产电微生物Geobacter sulfurreducens作为驱动力,驱动非产电微生物Clostridium intestinale的代谢途径从发酵转向呼吸,从而改善了自身的代谢通路,并使不能代谢乙醇的Clostridiumintestinale与Geobacter sulfurreducens都能够在以乙醇作为电子供体的条件下产电,促进了彼此的生长代谢,电流最大达0.4mA。
本发明突破了对非产电微生物的传统认识,同时扩大了Geobactersulfurreducens与Clostridium intestinale的底物范围与生态位,很大程度上提升了对生物电化学系统的研究,因此可作为一种参考方法进行大规模的推广应用。
附图说明
为了更清楚地说明本发明实施方式的技术方案,下面将对实施方式中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1是本发明实施例2中的Clostridium intestinale与Geobactersulfurreducens及共培养的产电图。
图2为本发明的实施例3的Clostridium intestinale与Geobactersulfurreducens共培养三电极体系中,对附着在ITO电极上的电活性生物膜进行活死染色后激光共聚焦显微镜下的图。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚,下面将结合本发明实施方式中的附图,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式是本发明一部分实施方式,而不是全部的实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。因此,以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施方式。基于本发明中的实施方式,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式,都属于本发明保护的范围。
本发明实施例中所述NBAF培养基、NBEF共培养培养基与CT培养基的配方如表1所示。
表1培养基的组成
*每升100X NB Salts Mix溶液中含有4g KH2PO4,22g K2HPO4,20g NH4Cl,38g KCl,36g NaCl,余量为水。
**每升NB Mineral Elixir溶液中含有2.14g NTA,0.1g MnCl2*4H2O,0.3g FeSO4*7H2O,0.17g CoCl2*6H2O,0.1g ZnSO4*7H2O,0.03g CuCl2*2H2O,0.005g AlK(SO4)2*12H2O,0.005g H3BO3,0.09g Na2MnO4*2H2O,0.11g NiSO4*6H2O,0.02g Na2WO4*2H2O,余量为水。
***每升DL维生素溶液中含有0.002g维生素H,0.005g维生素B5,0.0001g维生素B12,0.005g对氨基苯甲酸,0.005g a-硫辛酸,0.005g烟酸,0.005g维生素B1,0.005g核黄素,0.01g Pyridoxine HCl,0.002g叶酸,余量为水。
****每升DL微量元素溶液中含有1.5g NTA Trisodium Salt,3g MgSO4,0.5gMnSO4*H2O,1g NaCl,0.1g FeSO4*7H2O,0.1g CaCl2*2H2O,0.1g CoCl2*6H2O,0.13g ZnCl2,0.01g CuSO4*5H2O,0.01g AlK(SO4)2*12H2O,0.01g H3BO3,0.025g Na2MoO4*2H2O,0.024gNiCl2*6H2O,0.025g Na2WO4*2H2O,余量为水。
本发明实施例中所述FWNN电解液的配方如表2所示。
表2电解液的组成
*每升DL维生素溶液中含有0.002g维生素H,0.005g维生素B5,0.0001g维生素B12,0.005g对氨基苯甲酸,0.005g a-硫辛酸,0.005g烟酸,0.005g维生素B1,0.005g核黄素,0.01g Pyridoxine HCl,0.002g叶酸,余量为水。
**每升DL微量元素溶液中含有1.5g NTA Trisodium Salt,3g MgSO4,0.5g MnSO4*H2O,1g NaCl,0.1g FeSO4*7H2O,0.1g CaCl2*2H2O,0.1g CoCl2*6H2O,0.13g ZnCl2,0.01gCuSO4*5H2O,0.01g AlK(SO4)2*12H2O,0.01g H3BO3,0.025g Na2MoO4*2H2O,0.024g NiCl2*6H2O,0.025g Na2WO4*2H2O,余量为水。
Clostridium intestinale与Geobacter sulfurreducens均购买于美国典型培养物保藏中心,保藏号分别为ATCC BAA-1027和ATCC 51573。
实施例1:本发明Clostridium intestinale与Geobacter sulfurreducens的共培养
(1)将Clostridium intestinale培养于pH为6.5的10mL CT培养基中,30℃恒温培养;
(2)将Geobacter sulfurreducens培养于pH为7.0的10mL NBAF培养基中,30℃恒温培养;
(3)当(1)与(2)中Clostridium intestinale与Geobacter sulfurreducens的OD600值为0.5时,分别取0.5mL菌液接种于10mL的NBF培养基中,并加入乙醇,乙醇终浓度为20mM,30℃恒温培养;
(4)以5天为一个传代单位,将(3)中的共培养往下传5代,到第六代(T6)。
实施例2:本发明ITO三电极体系微生物燃料电池的构建与产电考察
(1)选取30mL的双室三电极微生物燃料电池,切取50mm*50mm的ITO作为阳极电极,用硅橡胶粘ITO,每组实验做3个重复;
(2)向组装好并灭过菌的ITO三电极微生物燃料电池通入20%:80%的CO2与N2的混合气,然后分别给阳极与阴极换上20mL FWEN与23mL FWNN的电解液,FWEN是通过向FWNN中加入终浓度为20mM的乙醇获得;
(3)分别取3mL实施例1中培养好的Clostridium intestinale、Geobactersulfurreducens以及Clostridium intestinale与Geobacter sulfurreducens共培养菌液,将其加入到(2)的阳极室中,每种菌液做3个重复;
(4)将(3)中的ITO三电极微生物燃料电池接入电化学工作站,并设置阳极电压为0.3V(相对饱和甘汞电极),连续采集电流数据(间隔5s);
结果显示,Clostridium intestinale无法发酵乙醇进行生长,更无法进行呼吸,不产电;Geobacter sulfurreducens无法代谢乙醇进行呼吸,不产电;然而,共培养好的Clostridium intestinale与Geobacter sulfurreducens在该体系中均能很好地存活,并且该生物电化学系统产生约0.4mA的电流(如图1所示)。
实施例3:本发明Clostridium intestinale与Geobacter sulfurreducens共培养ITO三电极体系微生物燃料电池中生物膜的活性考察
(1)当电流达到最大值时,将ITO电极用美工刀切出,并用生理盐水轻轻冲洗两遍;取0.6mL生理盐水,并加各1μL的活死染料,混匀后滴加到ITO电极上,避光染色15分钟,然后用于激光共聚焦显微镜观察;
结果显示,ITO电极上生物膜活性非常好,呈现绿色,并且生物膜厚度较厚(如图2所示)。
以上所述仅为本发明的优选实施方式而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种驱动非产电微生物产电方法,其特征在于:将非产电微生物和辅助产电微生物共培养,然后接种于微生物燃料电池的阳极,加入电子供体;所述非产电微生物为肠梭菌Clostridium intestinale,所述辅助产电微生物为硫还原地杆菌Geobacter sulfurreducens;所述肠梭菌Clostridium intestinale与硫还原地杆菌Geobacter sulfurreducens的保藏号分别为ATCC BAA-1027和ATCC 51573;所述共培养还需每3~5天接种传代,传到第5~7代;所述电子供体为乙醇。
2.根据权利要求1所述的驱动非产电微生物产电方法,其特征在于:所述非产电微生物和辅助产电微生物在共培养前还需分别进行厌氧培养。
3.根据权利要求1所述的驱动非产电微生物产电方法,其特征在于:所述共培养的接种量按照体积分数为5~10%;接种前,所述非产电微生物和辅助产电微生物的OD600为0.4~0.6。
4.根据权利要求1所述的驱动非产电微生物产电方法,其特征在于:所述共培养的培养温度为28~32℃。
5.根据权利要求1所述的驱动非产电微生物产电方法,其特征在于:所述电子供体的终浓度为10~20mM。
6.根据权利要求1所述的驱动非产电微生物产电方法,其特征在于:所述微生物燃料电池为ITO三电极体系微生物燃料电池。
7.根据权利要求1所述的驱动非产电微生物产电方法,其特征在于:所述共培养的培养基的配方为MgCl2·7H2O 0.05~0.15g/L,CaCl2·2H2O 0.02~0.06g/L,NaCO3·H2O 0.4~0.6g/L,NaHCO3 1.6~2.0g/L,富马酸Fumaric Acid 4.50~4.84 g/L,100X NB Salts Mix 8~12mL/L,1mM Na2SeO 40.0~42.0mL,NB Mineral Elixir 8~12mL/L,DL维生素溶液10~20mL/L,其余为水;每升100X NB Salts Mix溶液中含有4 g KH2PO4,22 g K2HPO4,20 g NH4Cl,38 g KCl,36 g NaCl ,余量为水;每升NB Mineral Elixir溶液中含有2.14 g NTA,0.1 gMnCl2·4 H2O,0.3 g FeSO4·7 H2O,0.17 g CoCl2·6 H2O,0.1 g ZnSO4·7 H2O,0.03 gCuCl2·2 H2O,0.005 g AlK(SO4)2·12 H2O,0.005 g H3BO3,0.09 g Na2MnO4·2 H2O,0.11g NiSO4·6 H2O,0.02 g Na2WO4·2 H2O,余量为水;每升DL维生素溶液中含有0.002 g 维生素H,0.005 g维生素B5,0.0001 g维生素B12, 0.005 g 对氨基苯甲酸, 0.005 g a-硫辛酸,0.005 g 烟酸,0.005 g维生素B1,0.005 g 核黄素,0.01 gPyridoxine HCl,0.002 g 叶酸,余量为水。
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