【作者】 张洪；

【导师】 刘颖；

【作者基本信息】 西北农林科技大学 ， 工程硕士（专业学位）， 2022， 硕士

【摘要】 在化石燃料储量短缺、环境污染和全球变暖的大背景下,新能源的开发和应用是目前全世界都在积极投入和研究的领域。微生物将胞内电子传递到胞外的功能启发了人们利用微生物产生能源的想法,即微生物燃料电池（Microbial fuel cells,MFCs）。目前,尽管MFCs还不能实现大规模实际应用,但基于其是一种绿色和环保的新型能源,它仍具有较大的发展潜力。另一方面,纤维素是世界上储量最丰富的可再生多糖,也是资源利用的热点研究对象之一。本研究发现MFCs可以利用纤维素为底物产生电能,pH值的变化对所富集混合菌利用纤维素的产电性能有很大影响,同时所富集混合菌利用纤维素与其它底物的产电性能对比更能揭示出所富集产电混合菌利用纤维素的特点。本论文以实验室自富集的利用可溶性淀粉生长的混合菌为接种源,以羧甲基纤维素钠（CMC）代替淀粉为唯一碳源进行再次驯化,并研究其在不同pH值时的产电性能。同时,对所富集产电混合分别以葡萄糖和乙酸钠为底物时进行可生长pH范围、产电性能、生物膜形貌和微生物群落结构等方面进行了对比研究,主要结论如下:（1）在以CMC为底物时,在生长培养基中以反丁烯二酸为电子受体预富集的混合产电菌在三电极体系中形成的微生物膜的最大输出电流密度为596.3μA/cm²,而经工作电极再富集后其延滞期由约30小时缩短至5小时左右,最大输出电流密度提升至774.2μA/cm²,并且相较于接种源以可溶性淀粉为底物时的最大输出电流密度（528.9μA/cm²）提高到1.46倍。进一步研究所富集混合产电菌利用CMC为底物在不同pH时的生长及产电性能,研究发现以CMC为碳源富集的混合菌在以反丁烯二酸为电子受体时能在pH 4.8-9.0范围内生长,其最大OD_600nm值为0.225-0.527,其中在pH 6.0时达到最大值0.527,且在pH 9.0时为0.225,在pH 4.8时为0.356;在三电极体系中,工作电极在+0.3 V下,pH值从4.8升高到8.0时其最大电流密度从504.25±20.93μA/cm²逐渐增大到850.03±17.22μA/cm²,随后当pH值升高到9.0时最大电流密度降为291.59±19.70μA/cm²,可见所富集混合产电菌利用CMC为底物时在pH为8.0时产电能力最高,同时在pH为4.8和9.0时也具有较好的产电能力,该性能鲜见报道。另外,所富集产电混合菌的产电性能使用双室微生物燃料电池体系进一步做了研究。当阳极室溶液的pH值从4.8增加到5.5、6.0、7.2直到8.0时,该MFCs的最大输出功率密度在随之增大,分别为0.215±0.003 W/m²、0.467±0.038 W/m²、0.494±0.016 W/m²、0.849±0.038 W/m²和1.17±0.016 W/m²,而当pH升高到9.0时电池的最大输出的最大功率密度下降为0.209±0.005 W/m²,这与三电极体系中所富集混合产电菌在pH 8.0时具有最高的产电能力是一致的。这些结果表明,所富集混合产电菌产电性能最优的pH值为8.0。此外,该混合产电菌在pH 4.8和pH 9.0时也具有一定的产电能力,分别为0.215±0.003 W/m²和0.209±0.005 W/m²,尤其是能在pH 4.8时产电,已突破了文献报道的在中性环境中降解纤维素产电的条件限制,这为进一步在弱酸性条件下利用MFCs降解纤维素和产电方面的应用打下了基础。（2）当将CMC换为葡萄糖为底物时,所富集混合产电菌在生长培养基中以反丁烯二酸为电子接受体可在pH 5.0-9.0范围内生长,且在pH 5.5-8.0范围内生长的最大OD_600nm值高于1.0,说明在该pH范围内所富集混合产电菌利用葡萄糖生长时活性高于利用CMC。同时所富集混合产电菌以葡萄糖为底物在三电极体系中在pH 5.0及pH 9.0条件下均可以生长但电流密度均低于10μA/cm²,且在pH 5.5-8.0时产电性能的范围仅为45.6±3.27μA/cm²-185.42±21.00μA/cm²,可见所富集混合产电菌利用CMC为底物时的产电性能在pH 8.0时是以葡萄糖为底物时的4.6倍。当将底物CMC换为乙酸钠时,所富集混合产电菌在以反丁烯二酸为电子受体的生长培养基中可在pH 5.3-8.7范围内生长,相较于利用CMC时可生长pH范围缩窄,但在pH 6.0-8.0范围内利用乙酸钠时的最大OD_600nm值为0.526-0.712,高于在该pH范围内利用CMC时的生长活性。同时,所富集混合产电菌在三电极体系中在pH 5.3-8.7范围内以乙酸钠为底物有较好的产电能力,并在pH 8.0时产电性能最高为971.56±64.78μA/cm²,该值是以葡萄糖为底物时产电能力的5.2倍,也高于以CMC为底物时的850.03±17.22μA/cm²,然而在pH 5.3-7.2时的产电性能（346.35±16.96μA/cm²-572.20±8.27μA/cm²）却低于以CMC为底物时的（504.25±20.93μA/cm²-776.23±34.58μA/cm²）。（3）本研究在三电极体系中利用电化学交流阻抗法进一步对所富集混合产电菌利用不同底物在几个典型的pH值时产电性能的不同进行分析。研究发现,电荷转移电阻(R_ct)存在明显的差异,R_ct由小到大的顺序为122.6Ω（乙酸钠-pH 8.7）<314.6Ω（CMC-pH 4.8）<466.6Ω（乙酸钠-pH 5.3）<538.7Ω（CMC-pH 9.0）<604.7Ω（葡萄糖-pH 8.0）。电荷转移电阻越小,表明附着在电极表面的电化学活性微生物膜与电极间的电子转移速率越高,越有利于提高混合产电菌的产电性能,这与在相应底物和pH值时的产电性能的变化规律是一致的。此外,对该混合产电菌附着在电极表面的几个典型微生物膜的形貌进行了分析,发现相应底物和相应pH值时形成的生物膜的厚度从小到大的顺序为17.05μm（CMC-pH 9.0）<23.57μm（乙酸钠-pH 5.3）<26.16μm（CMC-pH 4.8）<30.27μm（乙酸钠-pH 8.7）。附着的微生物膜越厚,其具有的电化学活性微生物越大,越有利于提高混合产电菌的产电性能,这些结果再次与在相应底物和pH值时的产电性能的变化规律相一致的。（4）以该混合产电菌在pH 4.8和pH 9.0时利用CMC、pH 5.0和pH 9.0时利用葡萄糖以及pH 5.3和pH 8.7时利用乙酸钠为底物时为研究对象,对其群落结构进行分析。结果表明,构成混合产电菌中相对丰度排前10的物种相同,优势菌属均为Geobacter、Anaerospora、Lachnoclostridium和Proteiniclasticum,但所富集混合产电菌的微生物多样性、物种相对丰度和可能的代谢产物差异导致了其电化学性能不同。此外,经Beta多样性分析可知,在以CMC、葡萄糖和乙酸钠为底物时,pH 4.8、pH 5.0、pH 5.3环境更易使所富集混合产电菌群落结构产生差异。更多还原

【Abstract】 Under the background of shortage of fossil fuel reserves,environmental pollution and global warming,the development and application of new energy is a field that the world is actively investing in and researching.The ability of microorganisms to transfer intracellular electrons to the outside of the cell inspired the idea of using microorganisms to generate energy,that is,microbial fuel cells（MFCs）.At present,although MFCs cannot achieve large-scale practical application,it still has great development potential because it is a green and environmentally friendly new energy source.On the other hand,cellulose is the most abundant renewable polysaccharide in the world,and it is also one of the hot research objects of resource utilization.In this study,it was found that MFCs can use cellulose as a substrate to generate electricity,and the change of pH value had a great influence on the electricity generation performance of mixed bacteria using cellulose.At the same time,the comparison of the electricity-generating properties of the mixed culture using cellulose and other substrates could reveal the characteristics of the electricity-producing mixed bacteria using cellulose.In this paper,the self-enriched mixed bacteria grown on soluble starch in the laboratory were used as the inoculum source,and sodium carboxymethyl cellulose（CMC）was used instead of starch as the sole carbon source for re-acclimation,and studied its power generation performance at different pH values.Meanwhile,a comparative study was carried out on the pH range of growth,electricity generation performance,biofilm morphology and microbial community structure when glucose and sodium acetate were used as substrates for the mixed electrogenic culture.The main conclusions are as follows:（1）When CMC was used as the substrate,the maximum output current density of the microbial film formed by the mixed electrogenic culture pre-enriched with fumaric acid as the electron acceptor in the three-electrode system was 596.3μA/cm².After enrichment by the working electrode,the lag period was shortened from about 30 hours to about 5 hours,and the maximum output current density was increased to 774.2μA/cm².Compared with the maximum output current density when the inoculation source used soluble starch as the substrate（528.9μA/cm²）increased to 1.46 times.To further study the growth and electricity generation performance of enriched mixed electrogenic bacteria using CMC as substrate at different pH.The study found that the mixed culture enriched with CMC as carbon source can grow in the range of pH 4.8 to 9.0 when fumaric acid is used as electron acceptor,and its maximum OD_600nm value is 0.225 to 0.527.The OD_600nm value reaches a maximum value of0.527 at pH 6.0,and is 0.225 at pH 9.0 and 0.356 at pH 4.8.In the three-electrode system,a potential of+0.3 V was applied to the working electrode,and the maximum current density of the enriched mixed electrogenic culture gradually increased from 504.25±20.93μA/cm² to850.03±17.22μA/cm²when the pH value increased from 4.8 to 8.0,then the maximum current density decreased to 291.59±19.70μA/cm² when the pH value increased to 9.0.It can be seen that the mixed electricity-generating bacteria had the highest electricity-generating capacity at pH 8.0 when CMC was used as the substrate,and also had better electricity-generating capacity at pH 4.8 and 9.0.This performance is rarely reported.In addition,the electricity-generating performance of the enriched electricity-generating mixed bacteria was further studied using a dual-chamber microbial fuel cell system.When the pH value of the anodic chamber solution increased from 4.8 to 5.5,6.0,7.2 until 8.0,the maximum output power densities of the MFCs increased,which were 0.215±0.003 W/m²,0.467±0.038 W/m²,0.494±0.016 W/m²,0.849±0.038 W/m² and 1.17±0.016 W/m²,respectively,and when the pH increased to 9.0,the maximum power density of the maximum output of the battery decreased to 0.209±0.005 W/m².This was consistent with the hybrid electrogenic bacteria in the three-electrode system having the highest electrogenic capacity at pH 8.0.These results show that the optimal pH value of the enriched mixed electrogenic bacteria is 8.0.In addition,the mixed electricity-generating bacteria also had certain electricity-generating capacity at pH 4.8 and pH 9.0,which were 0.215±0.003 W/m² and 0.209±0.005 W/m²,respectively,especially at pH4.8.It has broken through the limitations reported in the literature that degraded cellulose can generate electricity in a neutral environment,which lays the foundation for the further application of MFCs in cellulose degradation and electricity generation under weakly acidic conditions.（2）When the CMC was changed to glucose as the substrate,the enriched mixed electrogenic culture can grow in the pH range of 5.0 to 9.0 with fumaric acid as the electron acceptor in the growth medium.And the maximum OD_600nm value grown in the range of pH5.5 to 8.0 was higher than 1.0,indicating that the enriched mixed electrogenic bacteria in this pH range were more active when growing on glucose than when using CMC.At the same time,the enriched mixed electrogenic culture can grow but the current density was less than10μA/cm² using glucose as the substrate in the three-electrode system at pH 5.0 and pH 9.0,and the range of electricity-generating performance at pH 5.5 to 8.0 is only 45.6±3.27μA/cm²to 185.42±21.00μA/cm²,it can be seen that the electricity production performance of the enriched mixed electrogenic culture using CMC as substrate is 4.6 times higher than that of glucose as substrate at pH 8.0.When the substrate CMC was changed to sodium acetate,the enriched mixed electrogenic culture can grow in the range of pH 5.3 to 8.7 in the growth medium with fumaric acid as the electron acceptor.Compared with the use of CMC,the growth pH range was narrowed,but the maximum OD_600nm value was in the range of 0.526 to0.712 with sodium acetate in the pH 6.0 to 8.0 range,which was higher than the growth activity with CMC in this pH range.At the same time,the enriched mixed electrogenic culture had good electricity-generating capacity in the range of pH 5.3 to 8.7 in the three-electrode system with sodium acetate as the substrate,and the highest electricity-generating performance was971.56±64.78μA/cm² at pH 8.0.This value was 5.2 times higher than that of glucose as substrate,and also higher than that of 850.03±17.22μA/cm² when CMC was used as substrate.However,the electricity generation performance of sodium acetate as substrate at pH 5.3 to7.2（346.35±16.96μA/cm² to 572.20±8.27μA/cm²）was lower than that of CMC as substrate（504.25±20.93μA/cm² to 776.23±34.58μA/cm²）.（3）In this study,electrochemical impedance spectroscopy method performed in a three-electrode system was used to further analyze the difference in electricity production performance of the mixed electrogenic culture using different substrates at several typical pH values.The study found that there were obvious differences in charge transfer resistance(R_ct),the order of R_ct from small to large was 122.6Ω（sodium acetate-pH 8.7）<314.6Ω（CMC-pH 4.8）<466.6Ω（sodium acetate-pH 5.3）<538.7Ω（CMC-pH 9.0）<604.7Ω（Glucose-pH8.0）.The smaller the charge transfer resistance,the higher the electron transfer rate between the microbial film attached to the electrode surface and the electrode,which is more conducive to improving the electricity generation performance of the enriched mixed electrogenic culture.This was consistent with the change rule of the electricity generation performance under the corresponding substrate and pH value.In addition,the morphology of several typical microbial films attached to the electrode surface by the enriched mixed electrogenic culture was analyzed.It was observed that the thickness of the biofilm formed at the corresponding substrate and corresponding pH value was in descending order of 17.05μm（CMC-pH 9.0）<23.57μm（sodium acetate-pH 5.3）<26.16μm（CMC-pH 4.8）<30.27μm（sodium acetate-pH 8.7）.The thicker the attached microbial film,the more electrochemically active microorganisms it has,which is more conducive to improving the electricity generation performance of the enriched mixed electrogenic culture.These results are again consistent with the regularity of the electricity-generating properties at corresponding substrates and pH values.（4）Taking the enriched mixed electrogenic culture using CMC at pH 4.8 and pH 9.0,glucose at pH 5.0 and pH 9.0,and sodium acetate at pH 5.3 and pH 8.7 as the research objects,the community structures were analyzed.The results showed that the top 10 microorganisms in the mixed electrogenic bacteria were the same in relative abundance,and the dominant genera were Geobacter,Anaerospora,Lachnoclostridium and Proteiniclasticum.However,the microbial diversity,relative abundance of species and possible differences in metabolites of the enriched mixed electrogenic culture led to different electrochemical performances.In addition,the Beta diversity analysis showed that when CMC,glucose and sodium acetate were used as substrates,pH 4.8,pH 5.0,and pH 5.3 were more likely to cause differences in the enriched mixed electrogenic culture community structure.更多还原