【作者】 孙敏；

【导师】 俞汉青； 盛国平；

【作者基本信息】 中国科学技术大学 ， 应用化学， 2009， 博士

【摘要】 微生物燃料电池(MFC)利用微生物作为反应主体,将燃料的化学能直接转化为电能。MFC在处理废水的同时能够利用微生物进行发电,是一种具有良好前景的环境污染控制与清洁能源生产的新技术。本课题以MFC为研究对象,集成应用电化学、分析化学以及微生物学的多种方法和手段,深入探索了电活性微生物的产电机制及在电化学诱导下的催化行为,系统研究了MFC在硫化物去除以及强化废水生物制氢方面的应用。主要研究内容和研究结果如下:1、研究了短暂电场刺激对于启动阶段的混合菌群MFC性能的影响。结果表明,短暂电场刺激可以通过作用于阳极电活性微生物而影响MFC的电池性能。+1 V的正电场可以加速MFC的启动,石英晶体微天平实验证实该电场下微生物通过电泳机制加速在电极上的吸附,且正电场下微生物可以得到较高的代谢能量:-1 V和-5 V的负电场可以使MFC具有较好的产电性能,这是由于负电场可以增强电活性微生物膜的氧化还原能力,同时负电势也有利于厌氧细菌的生长。较高的电场刺激延缓甚至破坏MFC的启动,这是因为高电场强度下由于电空穴效应使细菌死亡所致。短暂的电场刺激可以作为电活性微生物富集驯化的辅助强化手段。2、构建以硫化物为基质的MFC,通过间歇实验研究了硫化物在MFC中的氧化过程。通过对硫氧化产物的分析以及功能微生物菌群的鉴定,阐明了硫化物在MFC中的氧化途径以及微生物的催化作用。结果表明,在微生物催化下MFC获得较高的持续电流;MFC中由S(-Ⅱ)经过S(0)向S(+Ⅵ)的转化可以通过电化学反应完成,而微生物的参与可以加速该过程;依靠单一的电化学机制,硫首先氧化成为单质硫和多硫化物,接着进一步氧化成为硫代硫酸盐和连四硫酸盐;在反应的初始阶段,微生物可以催化硫化物向单质硫的氧化,并使MFC具有较高的电流;MFC中微生物的催化作用主要表现在催化单质硫向硫代硫酸盐和硫酸盐的转化,而这也是MFC具有较长持续电流的主要原因:硫酸盐的生成是微生物催化作用的特殊标志;在MFC的阳极检测到硫氧化细菌和硫酸盐还原细菌,这两类细菌在还原硫的生物催化氧化方面扮演着重要角色。对于MFC阳极电极上和沉积物中的微生物菌群构建了16S rRNA基因克挛目狻Ｍü治龊捅冉掀湎妇郝浣峁沟淖槌汕榭?进一步揭示了MFC阳极细菌的多样性以及阳极反应的复杂性。研究结果表明,这两个位置的微生物具有相似性,但也有很大的不同;硫氧化细菌主要存在于电极上,而大部分硫酸盐还原细菌则在沉积物中;沉积物中的微生物多样性高于电极,表明沉积物中发生的反应比电极上发生的反应复杂:除了硫氧化细菌和硫酸盐还原细菌这两类硫功能微生物,MFC阳极室也存在很多其它种类的微生物,这些微生物与硫功能微生物相互协作,共同完成MFC中硫的氧化以及相关的产电过程。3、构建了微生物电解池-微生物燃料电池(MEC-MFC)产氢耦合系统,并实现了以乙酸和丙酸为基质的生物产氢。与传统的MEC相比,MEC-MFC耦合系统具有以下优势:1)由于MEC的辅助能量来源由MFC提供,产氢所需要的能量完全由MEC和MFC中的基质提供,降低了辅助能源的成本;2)对MFC而言,其电能输出后被直接利用辅助产氢,因而节省了电能的储存装置,并减少了相应的能量损失。对MEC和MFC之间的相互关系进行了研究,证实耦合系统中MEC和MFC两个电池的4个电极反应相互影响,系统的产氢效率受控于效率最低的电极反应。而高效的产氢系统要求4个电极反应均以较高的效率进行。通过改变负载电阻以及电助MFC的数目实现了耦合系统产氢效率的调控。结果表明,不同的负载电阻可以改变MEC的输入功率从而影响其产氢效率;系统的氢气产生速率和氢气转化率均随着负载电阻的升高而下降,这是由于高的负载电阻分担较多的MFC的功率输出;通过采用多个串联MFC作为MEC的辅助电源可以提高氢气产生速率及MEC中底物转化为氢气的转化率,但是系统总体氢气转化率则随着MFC数目的增多而下降;由于MFC内阻的差异,并联MFC电池组无法提高系统的产氢效率;采用串联MFC电池组作为MEC辅助能源,可以有效地强化耦合体系的产氢。4、研究了MFC中S.oneidensis MR-1以葡萄糖为基质的电子传递以及能量代谢途径。首先采用高效液相色谱法(HPLC)和三维荧光光谱法(EEM)的测定,证实MR-1在生长过程中可以分泌核黄素类物质至细胞外;通过对野生型及细胞色素c类基因缺失型菌株产电能力以及分泌核黄素能力的比较,确定核黄素无法直接进入细胞内得到电子并将其携带至胞外,而只是在附着态微生物和电极之间起到辅助电子传递的作用;细胞膜上由cymA、mtrA和mtrB调控的酶系是电子由细胞内向胞外传递所必须的。通过对MFC中葡萄糖代谢产物及变化趋势的分析,并结合S.oneidensisMR-1相关基因组学的研究结果,解析了葡萄糖在MFC中的代谢途径。S.oneidensis MR-1在MFC中通过ED厌氧途径代谢成为丙酮酸,并进一步厌氧代谢生成甲酸、乙醇、乙酸、丙酸和丁酸,其中丙酮酸、甲酸和丁酸可以被进一步用于产电;S.oneidensis MR-1的产电过程是非生长相关型的,在前期葡萄糖快速被消耗其能量主要用于菌体生长,而电流则是细菌在进一步利用葡萄糖的代谢产物甲酸和丁酸过程中产生的。更多还原

【Abstract】 Microbial fuel cell（MFC） is an electrochemical cell that directly captures the energy contained in bio-convertible substrates in the form of electricity.It has attracted increasing interests to employ MFCs to produce "green" energy from wastes. In an MFC,the microorganisms that completely oxidize organic compounds with an electrode as the sole electron acceptor are so-called exoelectrogens.The exoelectrogens play a key role in the anode oxidation by facilitating electron transfer between the electron donor and the electrode.In this thesis,the electron transfer mechanisms,substrate metabolism and exoelectrogens behavior in MFCs were investigated.Also,the functions of MFCs were further extended for their utilization in sulfide removal and biohydrogen production.In chapter 2,the influence of a transient external electric field on the performance of an MFC inoculated with mixed cultures was investigated.Different positive and negative electric fields were applied to a set of anodes.During the start-up period,the MFCs imposed by the electric fields of +1 V,-1 V,and -5 V obtained higher current densities than the control.The MFC exposed to the electric field of +1 V had the highest maximum power density of 73.5 mW m^-2 after 96-hr operation.On the other hand,the electric fields of +5 and +10 V delayed and even destroyed the MFC start-up.The electric field of-10 V initially induced a higher power output,but later had a detrimental effect on the MFC performance.An electrochemical quartz crystal microbalance test demonstrates that the electrophoresis was involved in the attachment/detachment of exoelectrogens on the electrode during the electric field application period.Furthermore,the application of negative electric field was proven to enhance the catalytic activity of the exoelectrogens,which was partially responsible for its influence on the MFC performance.In chapter 3,the sulfide oxidation process in MFCs was explored through identifying the sulfur species evolution and microbial communities.The results demonstrate that both electrochemical reactions and microbial catalysis were involved in such a complex sulfide oxidation process in the MFC anode.The microbe-assisted sulfide oxidation generated a higher persistent current density than the sulfide oxidation via single electrochemical reactions only.Three valence states of S（-Ⅱ）,S （0） and S（+Ⅵ） were discovered from the sulfide oxidation,and S₀,S_x^2-,S₄O₆^2-, S₂O₃^2-,and SO₄^2- were detected as the intermediates.The sulfur-oxidizing bacteria and sulfate-reducing bacteria were found in the MFC anode.Based on the sulfur speciation and microbial community analysis,the sulfide oxidation pathways in the MFC were proposed.The oxidation of sulfide to S₀/S_x^2- and further to S₄O₆^2-/S₂O₃^2- occurred spontaneously as electrochemical reactions,and electricity was generated. The formation of S₀/S_x^2- and S₂O₃^2- was accelerated by the bacteria in the MFC anode, and SO₄^2- was generated because of a microbial catalysis.The microbe-assisted production of S₂O₃^2- and SO₄^2- resulted in a persistent current from the MFC.Molecular biological techniques were applied to analyze and compare the microbial diversity on the electrode and in the sediment of the anode of the sulfide-fed MFC.The microbial community in the sediment exhibited higher diversity than that on the electrode.The population on the electrode consisted mainly ofα-Proteobacteria,β-Proteobacteria,γ-Proteobacteria and Firmicutes.In addition to four phyla above,δ-Proteobacteria,Firmicutes and Gemmatimonadetes were also found in the sediment.The sulfur-oxidizing bacteria were in much more abundance on the electrode than in the sediment.On the contrary,the sulfate-reducing bacteria preferably grew in the sediments.Besides the sulfur-related bacteria,Acinebobacter sp.was found to be rich in the MFC anode.The exoelectrogens-containing species Pseudomonas sp.,Clostridium sp and Ochrobactrum sp.were found both on the electrode and in the sediments.The microbial diversity in the MFC anode further reveals the complexity of the anodic reactions in the sulfide-fed MFC.There might be a syntrophic association among the various bacteria in the MFC anode.In Chapter 4,an MEC-MFC-coupled system was constructed for biohydrogen production,in which hydrogen was produced in an MEC and the extra power was supplied by an MFC.This MEC-MFC-coupled system has a potential for biohydrogen production from wastes,and provides an effective way for in situ utilization of the power generated from MFCs.In this coupled system,hydrogen was successfully produced from acetate and propionate without external electric power supply.The performance of the MEC and the MFC was influenced by each other.A stable system requires the high efficiencies of four half-reactions in both MEC and MFC,and that any reaction at a low efficiency will have a negative effect on the overall system performance.It was demonstrated that the hydrogen production in such an MEC-MFC-coupled system could be manipulated through adjusting the power input on the MEC.The power input of the MEC was regulated by applying different loading resistors connected into the circuit in series.All circuit current,volumetric hydrogen production rate,hydrogen recovery,Coulombic efficiency,and hydrogen yield decreased with the increase in loading resistance.Thereafter,in order to add power supply for hydrogen production in the MEC,additional one or two MFCs were introduced into this coupled system.When the MFCs were connected in series,the hydrogen production was significantly enhanced.In comparison,the parallel connection slightly reduced the hydrogen production.Connecting several MFCs in series was able to effectively increase power supply for hydrogen production,and had a potential to be used as a strategy to enhance hydrogen production in the MEC-MFC-coupled system from wastes.In chapter 5,experiments were conducted to elucidate the electron transfer mechanisms and glucose metabolism of Shewanella oneidensis MR-1 in an MFC.The results show that the S.oneidensis MR-1 secreted flavins into the culture as electron shuttles.The flavins were also secreted in the absence of the anode electrode, implying that they were not deliberately produced for electron transfer.A omcA/mtrC-deficient mutant,which has a poor electricity-producing ability,was observed to secrete a similar level of flavins as the wide type.This suggests that the electron transfer by flavins was not the key mechanisms in the electricity production of MR-1.During the glucose metabolism of MR-1 in an MFC,formate,acetate, propionate,butyrate,pyruvate,and ethanol were identified as the main metabolic products.In this MFC,the glucose was initially metabolized to pyruvate via ED pathway,and then to formate,acetate,propionate,butyrate,and ethanol.The electricity was not produced directly from the glucose decomposition,but was generated from the metabolism of the intermediate products such as formate and butyrate.更多还原