CN111100822B - 一株类肺炎克雷伯菌及其在制备微生物燃料电池中的应用 - Google Patents
一株类肺炎克雷伯菌及其在制备微生物燃料电池中的应用 Download PDFInfo
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Abstract
一株类肺炎克雷伯菌及其在制备微生物燃料电池中的应用,属于微生物燃料电池领域。本发明所述类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203菌株保藏于中国普通微生物菌种保藏中心,保藏编号为CGMCC No.19001。类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203具备产电性能,可在微生物燃料电池中应用。本发明拓展了产电微生物的范围,探究了该菌株产生电子中介体和形成生物膜进行胞外电子转移的能力,并对该菌产生的电子中介体进行了进一步的检测。
Description
技术领域
本发明涉及微生物燃料电池领域,具体涉及一株类肺炎克雷伯菌及其在制备微生物燃料电池中的应用。
背景技术
21世纪,能源短缺是世界各国普遍关注的问题,能源问题已经成为人类社会共同面临的巨大挑战,亟需寻找新的能源技术。在此背景下,可再生能源的开发与研究作为解决能源问题的重要途径,日益得到各国的重视。可再生能源主要包括太阳能、风能和生物质能的开发利用。其中,生物质能因为清洁、低碳、原料丰富等特点,被誉为“第四大”能源,近年来得到了快速发展。微生物燃料电池(Microbial fucl cell,MFC)因能以微生物为催化剂,通过降解有机底物,将化学能转化为电能的特性,得到了科研工作者的广泛关注。
微生物燃料电池工作原理主要是电化学活性微生物通过利用底物进行代谢,产生电子、质子及其他代谢产物,电子和质子传递至阴极产生还原反应。微生物燃料电池的电化学性能主要取决于三个因素:电池构建,运行环境和系统的生物组成部分。其中,电化学活性微生物是提高微生物燃料电池产电性能的关键。
电化学活性微生物主要通过生物膜接触、纳米导线和电子中介体进行胞外电子转移(Extracellular Electron Transfer,EET)。类肺炎克雷伯菌作为一种被广泛研究的致病性菌,其电化学活性的研究极少,并且在已有资料中,未见关于类肺炎克雷伯菌电子中介体及其电子中介体代谢途径的研究。
发明内容
解决的技术问题:针对目前对于类肺炎克雷伯菌电化学活性及其电子中介体相关研究较少的现状,本发明提供一株类肺炎克雷伯菌及其在制备微生物燃料电池中的应用,所述类肺炎克雷伯菌具有产生电子中介体进行胞外电池传递的能力,能够应用于微生物燃料电池的制备。
技术方案:一株类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203,其保藏编号为CGMCC No.19001。本发明所述类肺炎克雷伯菌SP.203来自徐州污水厂附近活性污泥。在湿地微生物燃料电池(CW-MFC)运行四周期后,从电池阳极富集电化学活性菌,经分离纯化及厌氧培养筛选后得到。该菌株为兼性厌氧菌、杆状、革兰氏阴性。
上述的类肺炎克雷伯菌SP.203在制备微生物燃料电池及检测该菌电子中介体中的应用。
作为优选,所述微生物燃料电池是质子交换膜间隔的双室微生物燃料电池,阴极与阳极均为载铂碳纸,质子交换膜为N117。
作为优选,具体应用步骤如下:
步骤一.将类肺炎克雷伯菌SP.203接种于柠檬酸铁培养基中,于厌氧培养箱中培养,到对数期中期;
步骤二.将步骤一培养后的菌接入微生物燃料电池的阳极室,微生物燃料电池的阳极室包括阳极、阳极液和类肺炎克雷伯菌SP.203,类肺炎克雷伯菌悬浮生长在阳极液中,或附着在阳极表面;
步骤三.接种后,运行微生物电池一直到输出电压达到最大值,在每周期输出电压降到200 mV以下时进行换液,更新阳极液和阴极液,换液时,取各个周期的阳极液离心后的上清,进行循环伏安法分析;
步骤四. 取产生最大电压并保持稳定时的上清,进行气相色谱-质谱联用技术分析,即可检测类肺炎克雷伯菌SP.203代谢的电子中介体。
作为优选,所述步骤一中柠檬酸铁培养基组成如下:胰蛋白胨、酵母提取物、氯化钠、柠檬酸铁、琼脂和蒸馏水,其中胰蛋白胨、酵母提取物、氯化钠、柠檬酸铁、琼脂的质量比为10:5:10:6.7:10,胰蛋白胨与蒸馏水的比值为10 g:1000 mL。
作为优选,所述步骤二中阳极液组分如下: KCl、NH4Cl、KH2PO4、MgCl2·6H2O、无水CaCl2、微量元素、柠檬酸钠和蒸馏水,其中,KCl、NH4Cl、KH2PO4、MgCl2·6H2O和无水CaCl2的质量比为0.1:0.25:0.05:0.015:0.015,KCl与蒸馏水的比值为0.1 g:1000 mL,微量元素与蒸馏水的体积比为3:1000,柠檬酸钠与蒸馏水的比值为20mmol:1L,微量元素组分如下:(NH4)2SO4、MgSO4·7H2O、MnSO4·2H2O、H3BO2、CaCl2·6H2O、CuCl2·2H2O、NiCl2·6H2O、ZnCl2、FeCl3·6H2O、Na2MoO4·2H2O和蒸馏水,其中(NH4)2SO4、MgSO4·7H2O、MnSO4·2H2O、H3BO2、CaCl2·6H2O、CuCl2·2H2O、NiCl2·6H2O、ZnCl2、FeCl3·6H2O和Na2MoO4·2H2O的质量比为5600:2000:200:3:2.4:1:2:5:10:0.4,(NH4)2SO4和蒸馏水的比值为5.6 g:1000 mL。
作为优选,所述步骤三中循环伏安法测定时的参数为:扫描电压-1~1V,扫描速率为0.05V/s。
有益效果:本发明提供了一种具有高电化学活性的类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203,将其应用于微生物燃料电池,拓展了电化学活性微生物的范围。所述类肺炎克雷伯菌SP.203是一种革兰氏阴性、兼性厌氧的细菌,具备较高的电化学活性,具备产生丰富的电子中介体以及形成生物膜的能力。该菌能够通过生物膜以及电子中介体途径,将代谢有机底物产生的电子传递给微生物燃料电池的阳极,实现有机底物的降解并产生电能。
目前关于类肺炎克雷伯菌的研究大多集中在其致病性上,本发明从类肺炎克雷伯菌的电化学活性为切入点,深入研究了其相关代谢与产电机制。类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203与其他电化学活性菌相比,具备产生丰富的电子中介体及形成生物膜的能力,拥有高效的电子传递能力。例如,希瓦氏菌S12以乳酸钠为底物,在工作体积为100 mL的微生物燃料电池中,产生的最大功率密度为55.72 mW/m2。而在同等工作体积中的微生物燃料电池中,类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203以柠檬酸钠为底物,产生的最大功率密度为90.69 mW/m2,约为希瓦氏菌S12的1.6倍。
保藏信息:本发明的菌种保藏日期为:2019年11月25日,保藏编号为:CGMCCNo.19001。分类命名为:类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203,保藏单位名称为中国普通微生物菌种保藏中心,保藏单位地址:北京市朝阳区北辰西路,中国科学院微生物研究所,邮编:100101。
附图说明
图1为实施例2中类肺炎克雷伯菌SP.203的产电能力验证结果图。
图2为实施例3中微生物燃料电池运行中产生的氧化还原物质检测结果图(循环伏安分析,CV)。
图3为实施例3中微生物燃料电池阳极液上清的循环伏安法分析曲线图。
图4为实施例3中运行四周期后阳极生物膜电镜图,图中a为第一周期末生物膜电镜图,e为第三周期末生物膜电镜图。
图5为实施例4中微生物燃料电池产生物质检测(气相色谱-质谱联用,GC-MS)图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明。应当理解,此处所描述的具体实施例仅用于解释本发明,而非限定本发明。
实施例1:类肺炎克雷伯菌SP.203的筛选和鉴定
本发明所述类肺炎克雷伯菌SP.203的筛选过程如下:在徐州污水处理厂附近取厌氧活性污泥于灭菌后的PE管中,存放在4℃冰箱中。将取来的活性污泥与污水混匀,加入构建好的CW-MFC中。CW-MFC由一个圆柱形聚丙烯酸塑料室(内径:28 cm;高度:50 cm;体积:30L左右,从下到上分为四层(底层、阳极层、中间层、阴极层)。底层(5 cm高)和中层(20 cm高)铺满砾石,砾石的平均粒径为0.51 mm,平均孔隙率约为0.2。阳极层(10 cm高)和阴极层(10cm高)由活性炭颗粒和不锈钢网组成。经4周期运行后,富集阳极上的微生物。将富集的微生物接种于配置好的柠檬酸铁培养基,在厌氧培养箱进行培养,柠檬酸铁培养基成分(溶于1000 mL蒸馏水):胰蛋白胨(10 g);酵母提取物(5 g);氯化钠(10 g);柠檬酸铁(6.7 g);琼脂(10 g)。挑取黑色单菌落进行多次划线纯化,得到纯化菌株。
对上述获得的菌株进行形态鉴定及分子鉴定,结果如下:
形态鉴定结果:该菌为短杆状,革兰氏染色为阴性,有荚膜。
分子鉴定结果:该菌16S rRNA测序信息提交GenBank,登录号为MN900629。通过16SrRNA的同源性分析表明,该菌与Klebsiella quasipneumoniae subsp. quasipneumoniae(01A030 T)相似性达到99.71%。
根据以上的鉴定结果,该菌属于类肺炎克雷伯菌。
实施例2:类肺炎克雷伯菌SP.203的电化学活性检测
构建双室微生物燃料电池,阴极和阳极均为载铂碳纸,阴极液和阳极液均为50mL。阴极液成分为:0.1M Na2HPO4(9.5 mL),0.1M Na2HPO4(40.5 mL),K3Fe(CN)6(0.823g);阳极液成分为(溶于1000 mL蒸馏水):KCl(0.1 g),NH4Cl(0.25 g),KH2PO4(0.05 g), MgCl2·6H2O(0.015 g),无水CaCl2(0.015 g),微量元素(每50 mL阳极液加入3 mL),20 mM柠檬酸钠;微量元素成分为(溶于1000 mL蒸馏水):(NH4)2SO4(5.6 g),MgSO4·7H2O(2 g),MnSO4·2H2O(200 mg),H3BO2(3 mg),CaCl2·6H2O(2.4 mg),CuCl2·2H2O(1 mg),NiCl2·6H2O(2mg),ZnCl2(5 mg),FeCl3·6H2O(10 mg),Na2MoO4·2H2O(0.4 mg)。将实施例1中制备得到的类肺炎克雷伯菌SP.203菌株接入阳极室,类肺炎克雷伯菌悬浮生长在阳极液中,或附着在阳极表面,用高速信号采集器进行连续电压测定。
微生物燃料电池启动过程中,外电路的电阻保持2000 Ω;运行微生物电池一直到电压达到最大值并保持稳定,在每周期输出电压低于200 mV时,进行换液,更新阳极液和阴极液。在实验前,实验装置及所用溶液都进行高压蒸汽灭菌,且加溶液和接种过程均在超净台完成。
电压输出如图1所示,图中MFC-1曲线为对照组(不接入菌株做空白),MFC-2为实验组,微生物燃料电池于31℃培养箱中运行700小时,最大输出电压为606 mV。
实施例3:类肺炎克雷伯菌SP.203的胞外电子转移特性测定
同实施例2,将电化学活性菌类肺炎克雷伯菌SP.203接入阳极室,运行微生物燃料电池一直到电压达到最大值(一般为第三周期)并保持稳定。进行循环伏安法分析,循环伏安法为三电极循环伏安法,玻碳电极作为工作电极,钛电极为对电极,Ag/AgCl电极作为参比电极。扫描电压为-1V~1V,扫描速率为0.05 V/s梯度扫描,结果如图2所示。图中MFC-1曲线为对照组(不接入菌株做空白),MFC-2为实验组,从图中可看出MFC-2具有氧化还原峰,说明可通过电子中介体进行胞外电子转移。
同实施例2,将电化学活性菌类肺炎克雷伯菌SP.203接入阳极室,运行微生物燃料电池一直到电压达到最大值,在每周期电压降到200 mV以下时进行换液。换液时,取阳极液进行离心,离心后将类肺炎克雷伯菌SP.203接入电池阳极,并留存阳极液上清,冰箱4℃保存。对上清液进行循环伏安法分析,结果如图3所示。从图中可以看出在第三周期(3rd),出现明显的氧化还原峰,与图1第三周期电压最高结果一致,说明类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203依赖于电子中介体进行胞外电子转移。
根据文献,电化学活性菌的胞外电子转移主要有三种方式:生物膜接触传递,纳米导线辅助传递,和依赖电子中介体传递。图4公开了类肺炎克雷伯菌SP.203胞外电子传递的另外一种测试结果。按实施例2接种类肺炎克雷伯菌SP.203,微生物燃料电池设置对照组(不贴膜)和实验组(贴膜,0.22 μm微滤膜)。微生物可以附着在阳极表面形成生物膜,在电池阳极表面贴光滑的微滤膜可以避免微生物在阳极表面附着。从图4可以看出不贴膜的微生物燃料电池阳极生物膜生长良好,且经测定,不贴膜的微生物燃料电池产电性能更好。结果表明,类肺炎克雷伯菌SP.203可以通过生物膜接触进行胞外电子转移。类肺炎克雷伯菌SP.203具有产生丰富电子中介体和形成生物膜的能力,与电化学活性菌中的模式菌希瓦氏菌MR-1相比,该菌的产电能力与其相似,但胞外电子转移机制不同,通过相关基因改造、接入混菌,如将该菌分泌电子中介体的编码基因过表达等方法,提高微生物燃料电池的产电能力。
实施例4:类肺炎克雷伯菌SP.203代谢产物测定
本实施例公开类肺炎克雷伯菌SP.203产电过程中的代谢产物,揭示该菌产生的电子中介体。如实施例2所示将类肺炎克雷伯菌SP.203菌株接入微生物燃料电池,运行至产生最大电压并保持稳定时,取阳极液进行气相色谱-质谱联用技术分析。送样分析前进行预处理,处理方式如下:先使用旋转蒸发仪浓缩阳极液,浓缩到原体积的1/5。浓缩完毕后,向每份样品中加入95 vt.%乙醇,摇晃,出现白色沉淀,静置过夜。过夜后离心去除沉淀,留上清液。将处理好的样品继续浓缩为原体积的1/5,回收乙醇。浓缩完成后,进行萃取实验,萃取剂为乙酸乙酯。使用分液漏斗进行分离,萃取3~4遍即可。最后将收集的乙酸乙酯层使用旋转蒸发仪浓缩为1 mL,送样检测。结果如图5所示。结果表明,类肺炎克雷伯菌SP.203主要通过2,6-二叔丁基对苯二酚(2,6-DTBHQ)和2,6-二叔丁基苯醌(2,6-DTBBQ)这一对氧化还原对的氧化还原反应进行电子转移。类肺炎克雷伯菌SP.203具有产生大量苯酚苯醌的能力,可通过基因组学、蛋白质组学和代谢组学联合分析,研究该菌产生电子中介体的完整代谢途径及相关编码基因簇,进一步提高电化学活性菌的产电能力。
Claims (4)
1.一株类肺炎克雷伯菌(Klebsiella quasipneumoniae)SP.203,其保藏编号为CGMCCNo.19001。
2.根据权利要求1所述的类肺炎克雷伯菌SP.203在制备微生物燃料电池及检测该菌电子中介体中的应用。
3.根据权利要求2所述的应用,其特征在于,所述微生物燃料电池是质子交换膜间隔的双室微生物燃料电池,双室微生物燃料电池中阳极室包括阳极、阳极液和类肺炎克雷伯菌SP.203,双室微生物燃料电池的阴极与阳极均为载铂碳纸,质子交换膜为N117。
4.根据权利要求3所述的应用,其特征在于,所述阳极液组分如下: KCl、NH4Cl、KH2PO4、MgCl2·6H2O、无水CaCl2、微量元素、柠檬酸钠和蒸馏水,其中,KCl、NH4Cl、KH2PO4、MgCl2·6H2O和无水CaCl2的质量比为0.1:0.25:0.05:0.015:0.015,KCl与蒸馏水的比值为0.1 g:1000 mL,微量元素与蒸馏水的体积比为3:1000,柠檬酸钠与蒸馏水的比值为20mmol:1L,微量元素组分如下:(NH4)2SO4、MgSO4·7H2O、MnSO4·2H2O、H3BO2、CaCl2·6H2O、CuCl2·2H2O、NiCl2·6H2O、ZnCl2、FeCl3·6H2O、Na2MoO4·2H2O和蒸馏水,其中(NH4)2SO4、MgSO4·7H2O、MnSO4·2H2O、H3BO2、CaCl2·6H2O、CuCl2·2H2O、NiCl2·6H2O、ZnCl2、FeCl3·6H2O和Na2MoO4·2H2O的质量比为5600:2000:200:3:2.4:1:2:5:10:0.4,(NH4)2SO4和蒸馏水的比值为5.6 g:1000 mL。
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