【作者】 杨洋；

【导师】 邢德峰；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2020， 博士

【摘要】 阳极生物膜和电极界面间的胞外电子传递(Extracellular electron transfer,EET)效率是影响生物电化学系统(Bioelectrochemical systems,BES)性能的关键因素之一。作为一个重要的生态因子,温度影响着电化学活性菌(Electroactive bacteria,EAB)的代谢和阳极生物膜的成熟。目前,嗜冷电化学活性生物膜和电极界面间的EET调控机制尚需进一步研究。因此,本课题探究了群体感应(Quorum sensing,QS)信号分子与EET之间的关系;基于宏转录组测序鉴定和分析了对温度变化响应的功能微生物和功能基因;结合宏基因组学,单细胞基因组学和宏蛋白组学方法,鉴定了低温下对EET应激响应的EAB和功能蛋白,并对嗜冷EAB的代谢途径进行了重建。探究了QS信号分子与EET之间的关系,发现BES启动期信号分子种类丰富,但随着运行周期的延长类型减少,仅检测到C6-HSL、3-oxo-C8-HSL和3-oxo-C12-HSL。QS系统受到温度的影响,其中3-oxo-C8-HSL的分泌在低温条件下影响较大,C10-HSL信号分子的浓度变化与温度呈现一定的相关性,而C8-HSL和3-oxo-C14-HSL两类信号分子具有底物依赖性。只有C6-HSL始终存在于不同条件下BES的各个运行周期中,表明该信号分子主要参与了微生物的基础代谢活动。在Pseudomonas aeruginosa的纯培养体系内发现las-QS系统没有直接参与到EET的调控中,rhl-QS系统促进了BES的EET过程,然而PQS(Pseudomonas Quinolones Signal)系统会抑制EET效率。其中,C4-HSL可以将基因缺陷型菌株的电流输出恢复到正常菌株的水平,而PQS信号分子对电流输出无明显贡献。与QS相比,阳极生物量对EET无明显影响。探究了阳极生物膜群落对温度变化的响应,发现低温扰动限制了功率输出,并导致生物膜发生功率过冲现象。低温扰动在一定程度上影响了微生物群落的多样性,导致可操作分类单元(Operational taxonomic units,OTUs),Shannon指数和Simpson指数降低。然而,低温对优势EAB的系统发育没有明显影响,Geobacter始终在群落中占主导地位。低温环境下与膜转运相关的基因丰度降低,而与翻译相关的基因丰度增加,许多上调表达的活跃基因参与了包括冷休克蛋白(csp A)诱导在内的冷休克反应,表明生物膜群落对低温扰动进行了相应的适应。低温扰动过程中,Acinetobacter是一个对温度变化高度响应的物种。更多的差异表达基因(Differentially expressed genes,DEGs)在最初的降温阶段上调表达,随着进一步降温,下调表达的基因在系统中占主导地位。有239个DEGs始终随温度降低而上调表达,另外有19个DEGs随温度下降而下调表达。低温扰动下对温度敏感而上调表达的DEGs中包括与IV型菌毛合成相关的fli C,pil C,pil Q和pil U基因,表明生物膜在低温下仍然具有合成纳米导线并保持EET活性的能力。低温下氧化磷酸化生成的ATP主要用于维持基础代谢和合成蛋白以适应低温扰动,导致较少的电子参与EET过程。许多未知功能的基因对群落适应低温和实现EET具有潜在的重要意义。分析了生物膜在低温下对EET速率变化的响应,发现反应器在4℃下可以富集成熟稳定的电化学活性生物膜,以饱和甘汞电极为参比电极在-300 m V时出现高电流,而在相对高电位(0 m V)时电流减弱。嗜冷生物膜群落以Geobacter为主,EET速率短时间的改变并没有明显影响群落结构。低温下阳极生物膜群落的主要代谢过程是氨基酸代谢、碳水化合物代谢和能量代谢。16个对阳极电位敏感的差异表达蛋白(Differentially expressed proteins,DEPs)主要来自Geobacter。呼吸代谢相关功能蛋白(磷酸化、丙酸代谢嘌呤、核糖核苷三磷酸结合和TCA循环)的表达表明生物膜的基础代谢在低温下仍然保持一定活性。最佳电位促进了呼吸代谢相关DEPs的上调表达,次优条件下某些氧化代谢反应受到抑制,同时一些氨基酸代谢影响了生物膜的EET过程。高EET速率时有更多的DEPs发生活跃的相互作用,最佳电位和次优电位下参与能量代谢的互作蛋白比较相似,而一些参与核酸或氨基酸代谢的互作DEPs有差别。构建了嗜冷EAB的基因组草图并对其代谢活性进行了分析,细胞色素c和菌毛相关功能基因和蛋白的鉴定显示其具有EET的活性,表明嗜冷EAB的EET主要通过纳米导线和细胞色素c介导的直接电子传递机制实现。本研究通过揭示嗜冷电化学活性生物膜与电极互作的机制,为BES系统的优化调控提供了理论依据。更多还原

【Abstract】 The extracellular electron transfer efficiency between the anode biofilm and the electrode interface is one of the important factors affecting the performance of bioelectrochemical system(BES).As an important ecological factor,temperature affects the metabolism of electroactive bacteria(EAB)and the maturation of anode biofilm.At present,the regulation mechanism of EET between psychrophilic electroactive biofilm and electrode interface needs to be further studied.Therefore,the relationship between the QS signal molecules and the EET is explored.Based on the metatranscriptomics sequencing,the functional microbes and genes responding to temperature change were identified and analyzed.Combined with metagenomics,single cell genomics and metaproteomics methods,EAB and functional proteins responding to EET stress at low temperature were identified,and the metabolic pathway of psychrophilic EAB was reconstructed.The relationship between the QS signal molecules and EET was explored.It was found that there were rich types of signal molecules in the start-up period,but with the extension of operation cycle,only C6-HSL,3-oxo-C8-HSL and 3-oxoC12-HSL were detected.The QS system was affected by temperature.The secretion of 3-oxo-C8-HSL was affected by low temperature,while the concentration of C10-HSL was related to temperature.C8-HSL and 3-oxo-C14-HSL were substrate dependent.Only C6-HSL always existed in different BES operation cycles under different conditions,which indicated that C6-HSL was mainly involved in basic metabolic activities of microorganisms.It was found that las-QS system was not directly involved in the regulation of EET,and rhl-QS played a key role in the EET base on the Pseudomonas aeruginosa pure-cultured system.While,PQS(Pseudomonas quinolones signal)system inhibited the EET efficiency.Among them,C4-HSL could restore the current output of the gene deficient strain to the level of the normal strain,while PQS signal molecule has no significant contribution to the current output.Compared with QS,anode biomass had no significant effect on EET.The response of anodic biofilm community to temperature shifts was investigated.It was found that the power output was limited by low temperature disturbance,which led to power overshoot of biofilm.Low temperature disturbance affected the diversity of microbial community,resulting in the decrease of operational taxonomic units(OTUs),Shannon index and Simpson index.However,low temperature had no obvious effect on the phylogeny of dominant EAB,and Geobacter always dominated the community.Under low temperature,the abundance of genes related to membrane transport decreased,while the abundance of genes related to translation increased.Many up-regulated genes participated in the cold shock response including cold shock protein(csp A),which indicated that the biofilm community adapted to the low temperature disturbance.In the process of low temperature disturbance,Acinetobacter was a highly responsive species to temperature changes.More differentially expressed genes(DEGs)were up-regulated in the initial cooling stage.With further cooling down,the down regulated genes were dominant in the system.239 DEGs were continuously up-regulated with decreasing temperature,while 19 DEGs were continuously down-regulated.The up-regulated DEGs,which were sensitive to temperature under low temperature disturbance,included fli C,pil C,pil Q and pil U genes related to the synthesis of type IV pili,indicating that the biofilm still had the ability to synthesize nanowires and maintained EET activity at low temperature.At low temperature,most of the ATP generated in oxidative phosphorylation was used to maintain basic metabolic activities and protein synthesis for cold adaption,resulting in less electrons participating in EET.Many genes with unknown functions were potentially important for the community to adapt to low temperature and realize EET.The response of biofilm to EET rate shifts under low temperature was analyzed.It was found that the mature and stable electrochemical active biofilm could be enriched in reactor at 4 ℃.Using saturated calomel electrode as reference electrode,high current appeared at-300 m V,but decreased at relatively high potential(0 m V).The psychrophilic biofilm community was dominated by Geobacter,and the short-term change of EET rate did not obviously affect the community structure.The main metabolic processes of anodic biofilm community at low temperature were amino acid metabolism,carbohydrate metabolism and energy metabolism.16 of differentially expressed proteins(DEPs)sensitive to anodic potential mainly came from Geobacter.The expression of respiratory metabolism related functional proteins(phosphorylation,propionic acid metabolism purine,ribonucleoside triphosphate binding and TCA cycle)indicated that the basic metabolism of biofilm remained active at low temperature.The optimal potential promoted the up regulation of respiratory metabolism related DEPs,and some oxidative metabolism reactions were inhibited under suboptimal conditions.While,some amino acid metabolism affected the EET of biofilm.At high EET rate,more DEPs interacted with each other actively.The proteins involved in energy metabolism were similar under optimal potential and suboptimal potential,while some interacting DEPs involved in nucleic acid or amino acid metabolism were different.The draft genome of psychrophilic EAB was reconstructed and its metabolic activity was analyzed.The identification of cytochrome c and pilus related functional genes and proteins showed the activity of EET,indicating that EET of psychrophilic EAB at low temperature can be realized mainly through the directly transfer mechanism of nanowires and cytochrome c.In this study,the interaction mechanism between psychrophilic electroactive biofilm and electrode were revealed,which laid a theoretical basis for the optimal regulation of BES.更多还原