Chain elongation into medium-chain carboxylates, such as n-caproate and n-caprylate, with ethanol as an electron donor and with open cultures of microbial consortia (i.e., reactor microbiomes) under anaerobic conditions is being developed as a biotechnological production platform. The goal is to use the high thermodynamic efficiency of anaerobic fermentation to convert organic biomass or organic wastes into valuable biochemicals that can be extracted. Several liter-scale studies have been completed and a first pilot-plant study is underway. However, the underlying microbial pathways are not always well understood. In addition, an interdisciplinary approach with knowledge from fields ranging from microbiology and chemical separations to biochemistry and environmental engineering is required. To bring together research from different fields, we reviewed the literature starting with the microbiology and ending with the bioprocess engineering studies that already have been performed. Because understanding the microbial pathways is so important to predict and steer performance, we delved into a stoichiometric and thermodynamic model that sheds light on the effect of substrate ratios and environmental conditions on product formation. Finally, we ended with an outlook.

Geobacteracea are distinct for their ability to reduce insoluble oxidants including minerals and electrodes without apparent reliance on soluble extracellular electron transfer (ET) mediators. This property makes them important anode catalysts in new generation microbial fuel cells (MFCs) because it obviates the need to replenish ET mediators otherwise necessary to sustain power. Here we report cyclic voltammetry (CV) of biofilms of wild type (WT) and mutant G. sulfurreducens strains grown on graphite cloth anodes acting as electron acceptors with acetate as the electron donor. Our analysis indicates that WT biofilms contain a conductive network of bound ET mediators in which OmcZ (outer membrane c-type cytochrome Z) participates in homogeneous ET (through the biofilm bulk) while OmcB mediates heterogeneous ET (across the biofilm/electrode interface); that type IV pili are important in both reactions; that OmcS plays a secondary role in homogenous ET; that OmcE, important in Fe(III) oxide reduction, is not involved in either reaction; that catalytic current is limited overall by the rate of microbial uptake of acetate; that protons generated from acetate oxidation act as charge compensating ions in homogenous ET; and that homogenous ET, when accelerated by fast voltammetric scan rates, is limited by diffusion of protons within the biofilm. These results provide the first direct electrochemical evidence substantiating utilization of bound ET mediators by Geobacter biofilms and the distinct roles of OmcB and OmcZ in the extracellular ET properties of anode-reducing G. sulfurreducens.

It has been previously noted that mixed communities typically produce more power in microbial fuel cells than pure cultures. If true, this has important implications for the design of microbial fuel cells and for studying the process of electron transfer on anode biofilms. To further evaluate this, Geobacter sulfurreducens was grown with acetate as fuel in a continuous flow 'ministack' system in which the carbon cloth anode and cathode were positioned in close proximity, and the cation-selective membrane surface area was maximized in order to overcome some of the electrochemical limitations that were inherent in fuel cells previously employed for the study of pure cultures. Reducing the size of the anode in order to eliminate cathode limitation resulted in maximum current and power densities per m(2) of anode surface of 4.56 A m(-2) and 1.88 W m(-2) respectively. Electron recovery as current from acetate oxidation was c. 100% when oxygen diffusion into the system was minimized. This performance is comparable to the highest levels previously reported for mixed communities in similar microbial fuel cells and slightly higher than the power output of an anaerobic sludge inoculum in the same ministack system. Minimizing the volume of the anode chamber yielded a volumetric power density of 2.15 kW m(-3), which is the highest power density per volume yet reported for a microbial fuel cell. Geobacter sulfurreducens formed relatively uniform biofilms 3-18 mum thick on the carbon cloth anodes. When graphite sticks served as the anode, the current density (3.10 A m(-2)) was somewhat less than with the carbon cloth anodes, but the biofilms were thicker (c. 50 mum) with a more complex pillar and channel structure. These results suggest that the previously observed disparity in power production in pure and mixed culture microbial fuel cell systems can be attributed more to differences in the fuel cell designs than to any inherent superior capability of mixed cultures to produce more power than pure cultures.

The advent of rapid complete genome sequencing, and the potential to capture this information in genome-scale metabolic models, provide the possibility of comprehensively modeling microbial community interactions. For example, Rhodoferax and Geobacter species are acetate-oxidizing Fe(III)-reducers that compete in anoxic subsurface environments and this competition may have an influence on the in situ bioremediation of uranium-contaminated groundwater. Therefore, genomescale models of Geobacter sulfurreducens and Rhodoferax ferrireducens were used to evaluate how Geobacter and Rhodoferax species might compete under diverse conditions found in a uraniumcontaminated aquifer in Rifle, CO. The model predicted that at the low rates of acetate flux expected under natural conditions at the site, Rhodoferax will outcompete Geobacter as long as sufficient ammonium is available. The model also predicted that when high concentrations of acetate are added during in situ bioremediation, Geobacter species would predominate, consistent with fieldscale observations. This can be attributed to the higher expected growth yields of Rhodoferax and the ability of Geobacter to fix nitrogen. The modeling predicted relative proportions of Geobacter and Rhodoferax in geochemically distinct zones of the Rifle site that were comparable to those that were previously documented with molecular techniques. The model also predicted that under nitrogen fixation, higher carbon and electron fluxes would be diverted toward respiration rather than biomass formation in Geobacter, providing a potential explanation for enhanced in situ U(VI) reduction in low-ammonium zones. These results show that genome-scale modeling can be a useful tool for predicting microbial interactions in subsurface environments and shows promise for designing bioremediation strategies. The ISME Journal (2011) 5, 305-316; doi:10.1038/ismej.2010; published online 29 July 2010 Subject Category: integrated genomics and post-genomics approaches in microbial ecology Keywords: Geobacter; Rhodoferax; community modeling; bioremediation; systems microbiology Introduction A wide phylogenetic diversity of microorganisms that are capable of dissimilatory metal reduction has been recovered from subsurface environments Lovley, 2006). The factors controlling which species predominate in a given subsurface environment are poorly understood, but may have important environmental consequences. For example, some dissimilatory metal-reducing microorganisms, such as those from the Geobacteraceae family, are capable of reducing U(VI) to U(IV), which can impact the mobility of uranium in the subsurface (Lovley, 1991(Lovley, , 2001(Lovley, , 2006Lovley et al., 1993Lovley et al., , 2004Wall and Krumholz, 2006), whereas others do not reduce U(VI) Lovley, 2006). Stimulating dissimilatory metal reduction to promote the reductive precipitation of uranium shows promise as a bioremediation strategy for uranium-contaminated groundwater (Anderson et al., 2003;Vrionis et al., 2005), but relies on stimulating the ...

Different microbial pathways can elongate the carbon chains of molecules in open cultures of microbial populations (i.e. reactor microbiomes) under anaerobic conditions. Here, we discuss three such pathways: 1. homoacetogenesis to combine two carbon dioxide molecules into acetate; 2. succinate formation to elongate glycerol with one carbon from carbon dioxide; and 3. reverse β oxidation to elongate short-chain carboxylates with two carbons into medium-chain carboxylates, leading to more energy-dense and insoluble products (e.g. easier to separate from solution). The ability to use reactor microbiomes to treat complex substrates can simultaneously address two pressing issues: 1. providing proper waste management; and 2. producing renewable chemicals and fuels.