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

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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.