Mechanistic investigations of the syntrophy between Pseudomonas aeruginosa and 2,3-butanediol fermenters within the context of optimized phenazine-based current generation in bioelectrochemical systems

过去，利用微生物反应进行生物技术应用主要局限于纯培养。然而，在自然界中，微生物通常协同行动，因为协同合作能够克服代谢和能量挑战。不幸的是，我们对特定微生物相互作用的了解仍然很少。生物电化学系统（BESs）可以利用协同微生物将有机物厌氧转化（氧化）为二氧化碳和电流。因此，已经确定了具有特定代谢功能的关键微生物，如有机物水解，发酵，氧化还原介质穿梭或直接胞外电子转移。成功应用BES的一个主要挑战是开发优化的微生物BES催化剂。实现这一目标的明确途径是了解这些关键参与者如何相互作用，并设计社区来利用这些相互作用。先前的研究表明，2,3-丁二醇发酵剂产气肠杆菌和电子介质生产者铜绿假单胞菌之间具有重要的协同电流产生作用。在这种共培养中，发酵罐将糖主要消化为2,3-丁二醇，而后者又被铜绿假单胞菌吸收。P. aeruginosa使用2,3-丁二醇来产生更多的毒力因子pyocyanin，这是一种氧化还原介质，可以增加BES阳极的电流产生。本提案力求对这种协同作用的生态和分子理解，这似乎是由发酵产物2,3-丁二醇介导的。此外，这两种生物（以及具有类似发酵剂的铜绿假单胞菌）在土壤环境和肺部感染期间会相遇，这使得这种协同作用的含义远不止BES应用。在我们的工作中，我们将对铜绿假单胞菌和各种2,3-丁二醇发酵剂之间的协同作用进行全面的生态学评估：我们将研究两种伙伴的种间交流程度和生理反应，以及对铜绿假单胞菌群体感应和毒力因子形成的影响，其中BES将作为原位分析工具来量化pyocyanin。我们将进一步确定理想的生理条件，使铜绿假单胞菌：在BES发酵共培养的最佳增效行为，即我们将学会控制二元共培养。为了全面了解这种协同作用，我们将对铜绿假单胞菌的相关生理过程进行研究，以回答以下问题：2,3-丁二醇是如何被感知的？群体感应调节网络是如何受到影响的？除了信号之外，还有什么代谢作用吗？最后，我们将把所有观察到的表型和生理结果整合到一个共培养协同作用的生理生态模型中，这将为未来更复杂的BES微生物网络奠定基础。

In the past, exploiting microbial reactions for biotechnological applications was mainly limited to pure cultures. In nature, however, microorganisms typically act in concert because synergistic collaborations are able to overcome metabolic and energetic challenges. Unfortunately, our knowledge on specific microbial interactions is still scarce. Bioelectrochemical systems (BESs) can utilize collaborative microorganisms for the anaerobic conversion (oxidation) of organic matter to carbon dioxide and electric current. Thereby, key microbial players with specific metabolic functions like organics hydrolysis, fermentation, redox mediator shuttling or direct extracellular electron transfer have already been identified. One major challenge for the successful application of BES is the development of optimized microbial BES catalysts. A clear path to achieve this goal is to understand how these key players interact together and to design communities to exploit these interactions.Previous work has shown an important synergistic current production between the 2,3-butanediol fermenter Enterobacter aerogenes and the electron-mediator producer Pseudomonas aeruginosa. In this co-culture, the fermenter digests sugars to mainly 2,3-butanediol, which in turn, is taken up by P. aeruginosa. P. aeruginosa uses 2,3-butanediol to produce increased amounts of the virulence factor pyocyanin, a redox mediator, which enables increased current production at a BES anode. This proposal strives for an ecological and molecular understanding of this synergistic interaction, which seems to be mediated by the fermentation product 2,3-butanediol. Further, these two organisms (but also P. aeruginosa with similar fermenters) encounter one another in soil environments and during lung infections, making implications of this synergism much broader than just BES applications.In the proposed work, we will conduct a thorough ecological evaluation of the synergism between P. aeruginosa and various 2,3-butanediol fermenters: we will study the extent of interspecies communication and physiological responses in both partners and the effect on P. aeruginosa quorum sensing and virulence factor formation, whereby BES will be used as an in-situ analytical tool to quantify pyocyanin. We will further determine ideal physiological conditions for optimum synergistic behavior of P. aeruginosa : fermenter co-cultures in BES, i.e. we will learn to control the binary co-culture. For a comprehensive understanding of the synergism, the related physiological processes in P. aeruginosa will be investigated to answer the following questions: How is 2,3-butanediol sensed?; How is the quorum sensing regulatory network influenced?; Is there a metabolic effect beyond just signaling? Finally, we will integrate all observed phenotypic and physiological results in an physio-ecological model of the co-culture synergism, which will serve as a foundation for future, more complex microbial networks in BES.