CN113249373A - 一种直流电场刺激重组大肠杆菌提高氢气效率的方法 - Google Patents
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Abstract
本发明公开了一种直流电场刺激重组大肠杆菌提高氢气效率的方法,包括:筛选高效产氢的重组大肠杆菌,构建直流电场刺激重组大肠杆菌产氢的电极;重组大肠杆菌在外加直流电场刺激作用下,24h产生的氢气总量与对照组比较,增加了74.38%。本发明提供的直流电场刺激重组大肠杆菌提高氢气量的方法,对生物制氢的工业化生产和大规模应用具有重大意义。
Description
技术领域
本发明涉及一种直流电场刺激重组大肠杆菌提高氢气效率的方法,属于生物制氢技术领域。
背景技术
随着经济社会的迅猛发展,人类社会对于能源的需求量持续增长,而化石能源开发利用过程中会产生大量的有毒气体。能源短缺、环境污染,包括温室效应引起的全球变暖,臭氧层破坏,酸雨和生物多样性的减少,最终危及人类的生命和健康。由此,对清洁可再生能源的开发利用以缓解能源短缺和环境污染问题已变得迫在眉睫。氢气作为一种高效环保的能源,是世界能源转型的一个重大战略方向,被广泛看做未来能源系统中的重要媒介。生物制氢因其高效、绿色、低成本受到了越来越多的关注和重视,并且极具发展潜力。发展生物制氢技术的会带来显著的经济效益、环境效应和社会效应。制氢原料来源非常广泛,可以利用木材废料、农作物、农业生产上的残留物、生活垃圾、污水污泥和动物粪便等,而生物质资源丰富,是重要的可再生能源,可解决能源危机,同时实现废物利用,改善环境,是制氢工业新的发展方向。
而大肠杆菌具有较强环境适应能力、生长繁殖迅速、底物利用率高、高效且洁净的产氢等特点。产氢大肠杆菌被认为是一种低能耗、无污染、可持续发展的技术,在生物制氢领域具有广泛的应用前景。
发明内容
本发明提供一种直流电场刺激重组大肠杆菌提高氢气效率的方法,促进了重组大肠杆菌对有机物的降解,提高了氢气的产生速率和产量。
本发明提供一种直流电场刺激重组大肠杆菌提高氢气效率的方法,通过外加直流电场刺激重组大肠杆菌,促进细胞增殖,增加细菌细胞内总蛋白含量;增强细菌ATP酶活性,为重组大肠杆菌降解代谢提供电子,一定程度提升了细胞生长能力和代谢水平,促进重组大肠杆菌的厌氧发酵产氢。该方法可以有效提高生物氢的产量,推动生物制氢技术的发展和应用,而生物制氢技术的应用会带来显著的经济效益、环境效应和社会效应。
本发明提供一种直流电场刺激重组大肠杆菌提高氢气效率的方法,包括以下步骤:
(1)通过实验筛选能够高效产氢的重组大肠杆菌,并将其保存在-70℃冰箱中;
(2)将电极材料预处理后,所有材料在121℃高压灭菌20分钟,在超净工作台上将电极装入厌氧瓶,并连接好导线,将灭菌之后的产氢培养基(115 ℃高压灭菌20分钟)倒入,并加入氨苄,最终浓度为0.1mg/mL;
(3)将活化好的重组大肠杆菌以5% (v/v)的比例接种到含氨苄的培养基的厌氧瓶中,将电源正极与厌氧瓶的阳极相连,负极与阴极相连,然后打开电源,通入30mA的直流电流,对照组除了不通入电流,其他条件一致;
(4)在120rpm,37℃的恒温振荡培养箱中,培养至菌体的OD600nm值达到0.7,添加异丙基硫代半乳糖苷(IPTG)至最终浓度1mM,然后在继续培养30分钟后静置培养,观察气泡;
(5)每隔2h对氢气的产量进行检测。
上述方法中,所述外加直流电场使重组大肠杆菌增产氢气的阴阳极均为7cm×2cm×0.1 cm的碳布。
上述方法中,所述外加直流电场使重组大肠杆菌增产氢气电源输出电压范围为0~30 V,电流输出范围为0~5 A。
本发明的优点和有益效果:
本发明增强了重组大肠杆菌对底物的利用率,提高了氢气产量,具有以下实质性特点和显著的效果:
(1)本发明的优点在于操作简单,成本低,在外加直流电场的作用下,重组大肠杆菌的生长明显加快;
(2)外加直流电场,可以不损伤细胞的情况下在一定范围内增强细菌膜的通透性,改变菌体的形态,促进重组大肠杆菌的生长活力和代谢水平而提高氢气的产量;
(3)通过外加直流电场辅助手段,在阳极,重组大肠杆菌在降解有机物的同时释放质子并将电子转移到阳极表面,电子通过外电路到达阴极表面,该过程改善了重组大肠杆菌的厌氧发酵环境,有利于重组大肠杆菌对甲酸的分解,促进重组大肠杆菌产氢。
具体实施方式
下面通过实施例来进一步说明本发明,但不局限于以下实施例。
实施例1:
一种直流电场刺激重组大肠杆菌提高氢气效率的方法,包括以下步骤:
(1)通过实验筛选能够高效产氢的重组大肠杆菌,并将其保存在-70℃冰箱中;
(2)将冻存的重组大肠杆菌接种到LB培养基(10g蛋白胨,5g酵母浸出粉,10g氯化钠,1L蒸馏水)中进行过夜培养活化;
(3)电极预处理后,所有材料在121℃高压灭菌20分钟,在超净工作台上将电极放入厌氧瓶,并连接导线。将灭菌之后的培养基(115 ℃高压灭菌20分钟)(葡萄糖20 g,酵母浸出粉5 g,蛋白胨5 g,磷酸氢二钾7 g,L-半胱氨酸盐酸盐0.5 g,磷酸二氢钾5.5 g,硫酸铵1 g,七水硫酸镁0.25 g,二水氯化钙0.021 g,六水硝酸钴0.029 g,六水硫酸亚铁氨0.039 g,烟酸2 mg,亚硒酸钠0.172 mg,氯化镍0.02 mg,微量元素溶液10 mL,蒸馏水1 L)倒入250 mL厌氧瓶4中,其中产氢微量元素溶液配方如下:四水二氯化锰0.5 g,硼酸0.1 g,二水氯化铜1 mg,十二水硫酸铝钾0.01 g,乙二胺四乙酸二钠0.5 g,蒸馏水1 L。并加入氨苄,最终浓度为0.1mg/mL;
(4)将过夜培养活化的重组大肠杆菌用1000~5000 μL的移液枪以5% (v/v)的比例接种到含氨苄的250 mL培养基的厌氧瓶中,将电源正极与厌氧瓶的阳极相连,负极与阴极相连,然后打开电源,通入30mA的直流电流,对照组除了不通入电流,其他条件一致;
(5)在120rpm,37℃的恒温振荡培养箱中培养,每隔两小时取样后稀释适当倍数后测其OD600nm值,当菌体的OD600nm值达到0.7时,用5 mL的一次性无菌注射器添加异丙基硫代半乳糖苷(IPTG)至最终浓度1mM,然后再继续培养30分钟后静置培养,观察气泡;
(6)每隔2h对氢气的产量进行检测,通过检测发现,24h后在外加直流电场的作用下氢气总量较未加电场情况下增加了74.38%。
Claims (4)
1.一种直流电场刺激重组大肠杆菌提高氢气效率的方法,其特征在于:通过外加直流电场刺激重组大肠杆菌,促进细胞增殖,增加细菌细胞内总蛋白含量;增强细菌ATP酶活性,为重组大肠杆菌降解代谢提供电子,提升了细胞生长能力和代谢水平,促进重组大肠杆菌的厌氧发酵产氢。
2.根据权利要求1所述的直流电场刺激重组大肠杆菌提高氢气效率的方法,其特征在于:包括以下步骤:
(1)通过实验筛选高效产氢的重组大肠杆菌,并将其保存在-70℃冰箱中;
(2)将电极材料预处理后,所有材料在121℃高压灭菌20分钟,在超净工作台上将电极放入厌氧瓶中,连接好导线,将灭菌之后的产氢培养基(115 ℃高压灭菌20分钟)倒入,并加入氨苄,最终浓度为0.1mg/mL;
(3)将活化好的重组大肠杆菌以5% (v/v)的比例接种到含氨苄的产氢培养基的厌氧瓶中,将电源正极与厌氧瓶的阳极相连,负极与阴极相连,然后打开电源,通入30mA的直流电流,对照组除了不通入电流,其他条件一致;
(4)在120rpm,37℃的恒温振荡培养箱中,培养至菌体的OD600nm值达到0.7,添加异丙基硫代半乳糖苷(IPTG)至最终浓度1mM,然后在继续培养30分钟后静置培养,观察气泡;
(5)每隔2h对氢气的产量进行检测。
3.根据权利要求1或2所述的直流电场刺激重组大肠杆菌提高氢气效率的方法,其特征在于:所述直流电场刺激重组大肠杆菌产氢气的阴阳极均为7cm×2 cm×0.1 cm的碳布。
4.根据权利要求1或2所述的重组大肠杆菌提高氢气效率的方法,其特征在于:所述直流电场刺激重组大肠杆菌产氢气的电源输出电压范围为0~30 V,电流范围为0~5 A。
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