Reduced Extracellular Electron Shuttles as Electron Donors for H2 Production in Fermentative Bacterial Metabolism

CBET-0756054Finneran生物制氢是氢燃料电池的一种选择，如果工艺能够优化以产生显著的产量，则代表着一种可持续的能源形式。贝耶林克梭菌是一种用于生物制氢的发酵微生物。迄今为止的结果表明，产量相当低，除非a)大量的碳被用作基质，b)反应器的体积足够大，可以产生大量的沼气。我们这项工作的目标是通过以还原的胞外对苯二酚的形式提供多余的还原当量来增加氢产率，这些对苯二酚直接转化为氢。胞外对苯二酚被称为电子穿梭。它们使电子在混合的生物和非生物相互作用之间循环，或者在耦合生物相互作用的微生物之间循环。初步数据表明，贝耶林克氏假单胞菌在产氢的同时，会将对苯二磺酸(AH2QDS)氧化成对苯二磺酸(AQDS)。AQDS分子不会被消耗，因此可用于再还原和氧化。如果维持这个循环，那么AH2 QDS的所有还原当量都将用于氢气生产，这提供了相对于标准发酵的多余电子。我们的目标是表征生理学，并开发一种连续的、二元培养，包括发酵培养和AQDS还原微生物，通过还原电子轨道产生氢气。智力价值拟议工作的目标是：a。确认电子穿梭(对苯二酚)在发酵代谢中被氧化，从而产生氢气，b。)量化还原电子穿梭产生氢气的动力学，c.)了解增加或减少这一途径的化学和生物因素，以及d。)发展连续培养(首先是单一培养，然后是最终的二元培养)，以了解如何将这一策略应用于氢气回收工程系统。我们将同时使用贝耶林克杆菌的单一培养物和贝耶林克氏杆菌和金属还原地质杆菌的双重培养物。金属还原菌可以氧化葡萄糖发酵过程中产生的醋酸盐和丁酸，并将这种氧化耦合到AQDS还原形成AH2 QDS。然后，细胞外的AH2 QDS可以被贝耶林克链霉菌在耦合的生物反应中氧化，再生AQDS，完成电子穿梭循环。醋酸盐和丁酸盐中的电子等价物本质上是？穿梭的？通过当提供给发酵培养物时特定地产生氢的载体提供给发酵细胞。我们将使用定量逆转录聚合酶链式反应(Q-RT-PCR)来量化与AH2QDS氧化相关的关键发酵氧化还原酶的上调。二元培养将首先在批量实验(静态瓶)中进行测试，然后在搅拌槽式反应器中进行连续培养。AQDS在反应器中的浓度将被调整以增加或减少氢产率，这样我们就可以预测相对于电子穿梭浓度的反应效率。最后，提纯底物(例如葡萄糖)的结果将适用于具有非提纯底物的连续系统，如植物废物中的复杂淀粉和乙醇生产废物。广泛影响拟议工作的更广泛影响将在从学术界到咨询业的所有环境工程部门感受到，因为可持续能源生产是所有这些领域中出现的一个关键主题。尽管这项工作还处于起步阶段，距离商业应用或工业使用还有很长一段路要走，但这些数据确实表明，生产生物氢的一种独特方法与标准发酵技术非常不同。被纳入这项工作的学生将主要来自工程学中代表性不足的群体。这些学生将参与工作和数据传播的所有方面，这样他们的研究经验将有助于传播这个项目的基本知识。这些数据将直接分发给已经与该部门合作的咨询和行业团体；其中许多私营企业具有可持续性？他们公司内部的团队或战略计划。从生物燃料回收的角度来看，他们可能会对这种方法感兴趣，但更有可能的是，他们将在短期内提供对项目目标的关键见解，以便未来的实验将反映行业的需求。

CBET-0756054FinneranBiologically-produced hydrogen is one option for hydrogen fuel cells, and represents a sustainable form of energy if the process can be optimized to generate a significant yield. Clostridium beijerinckii is one fermentative microorganism that has been used for bio-hydrogen production. Results to date suggest that yields are quite low unless a) large quantities of carbon are used as a substrate and b) reactor volumes are large enough to generate significant biogas. Our goal for this work is to increase the hydrogen yield by providing excess reducing equivalents in the form of reduced, extracellular hydroquinones, which are converted directly to hydrogen. Extracellular quinones are referred to as electron shuttles. They cycle electrons between mixed biological and abiotic interactions or between microorganisms in coupled biological interactions. Initial data demonstrate the C. beijerinckii will oxidize anthrahydroquinone disulfonate (AH2QDS) to anthraquinone disulfonate (AQDS) with concomitant hydrogen production. The AQDS molecule is not consumed, and it is therefore available for re-reduction and oxidation. If this cycle is maintained then all reducing equivalents from the AH2QDS will be used for hydrogen production, which provides excess electrons relative to standard fermentation. Our goal is to characterize the physiology and develop a continuous, binary culture with a fermentative culture plus an AQDS-reducing microorganism to generate hydrogen via reduced electron shuttles.Intellectual MeritThe objectives of the proposed work are to: a.) confirm that electron shuttles (hydroquinones) are oxidized in fermentative metabolism leading to H2 production, b.) quantify the kinetics of H2 generation from reduced electron shuttles, c.) understand the chemical and biological factors that increase or decrease this pathway, and d.) develop a continuous culture (singular culture first and then eventual binary culture) to understand how this strategy can be applied to engineered systems for hydrogen gas recovery. We will use both single cultures of C. beijerinckii and binary cultures of C. beijerinckii and Geobacter metallireducens. G. metallireducens can oxidize the acetate and butyrate produced during glucose fermentation, and couples this oxidation to AQDS reduction to form AH2QDS. The extracellular AH2QDS can then be oxidized by C. beijerinckii in a coupled biological reaction, regenerating the AQDS and completing the electron shuttling cycle. The electron equivalents in the acetate and butyrate are in essence ?shuttled? to the fermentative cells via a carrier that specifically generates hydrogen when provided to the fermentative culture. We will quantify the up regulation of key fermentative redox enzymes using quantitative reverse transcriptase PCR (Q-RT-PCR) related to AH2QDS oxidation. The binary culture will be tested first in batch experiments (static bottles) and then in continuous culture with a stirred tank reactor. The concentration of AQDS in the reactor will be adjusted to increase or decrease the hydrogen yield, such that we can predict the efficiency of the reaction relative to electron shuttle concentration. Finally, the results with purified substrates (e.g. glucose) will be adapted to continuous systems with non-purified substrates such as complex starch in plant waste and ethanol production waste.Broader ImpactsThe broader impacts of the proposed work will be felt in all environmental engineering sectors from academia to consulting, as sustainable energy production is a critical theme emerging within all of these arenas. Although the work is at it starting point and still quite far from commercial application or industrial use, the data do suggest a unique approach for producing biological hydrogen that is very different from standard fermentative technologies. The students incorporated into this work will be primarily from underrepresented groups in engineering. These students will be involved in all aspects of the work and data dissemination such that their research experiences will help distribute the underling knowledge of this project. The data will be directly distributed to consulting and industry groups that already collaborate with the department; many of these private businesses have ?sustainability? teams or strategic initiatives within their companies. It is possible that they will become interested in this approach from a biofuel recovery perspective, but it is more likely in the short term that they will provide critical insight to the project goals such that the future experiments will reflect the needs of industry.