Evidence for Quinol Oxidation Activity of ImoA, a Novel NapC/NirT Family Protein from the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1.

Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol in S. lithotrophicus.

Sporomusa ovata as Catalyst for Bioelectrochemical Carbon Dioxide Reduction: A Review Across Disciplines From Microbiology to Process Engineering.

Sporomusa ovata is a bacterium that can accept electrons from cathodes to drive microbial electrosynthesis (MES) of acetate from carbon dioxide. It is the biocatalyst with the highest acetate production rate described. Here we review the research on S. ovata across different disciplines, including microbiology, biochemistry, engineering, and materials science, to summarize and assess the state-of-the-art. The improvement of the biocatalytic capacity of S. ovata in the last 10 years, using different optimization strategies is described and discussed. In addition, we propose possible electron uptake routes derived from genetic and experimental data described in the literature and point out the possibilities to understand and improve the performance of S. ovata through genetic engineering. Finally, we identify current knowledge gaps guiding further research efforts to explore this promising organism for the MES field.

Reactors for Microbial Electrobiotechnology.

From the first electromicrobial experiment to a sophisticated microbial electrochemical process - it all takes place in a reactor. Whereas the reactor design and materials used strongly influence the obtained results, there are no common platforms for MES reactors. This is a critical convention gap, as cross-comparison and benchmarking among MES as well as MES vs. conventional biotechnological processes is needed. Only knowledge driven engineering of MES reactors will pave the way to application and commercialization. In this chapter we first assess the requirements on reactors to be used for bioelectrochemical systems as well as potential losses caused by the reactor design. Subsequently, we compile the main types and designs of reactors used for MES so far, starting from simple H-cells to stirred tank reactors. We conclude with a discussion on the weaknesses and strengths of the existing types of reactors for bioelectrochemical systems that are scored on design criteria and draw conclusions for the future engineering of MES reactors.

Simultaneous use of a crossflow filtration membrane as microbial fuel cell anode - Permeate flow leads to 4-fold increased current densities.

A new concept for the combination of membrane bioreactors and microbial fuel cells is introduced, that aims at the production of electricity for reducing the overall energy consumption of wastewater treatment. In contrast to previous approaches, the anode is integrated as microfiltration membrane in sidestream crossflow configuration. Using a stainless steel filtration membrane with G. sulfurreducens and an acetate-based synthetic medium, up to 4-fold higher current densities are achieved. In a standard setup without filtration, a membrane of filter grade 1 µm shows current densities of 5.8 A m-2 ±â€¯0.5 A m-2 compared to >11 A m-2 when it is used simultaneously as membrane filter. With smaller pore sizes of filter grade 0.5 µm, 4.4 A m-2 ±â€¯0.5 A m-2 in a standard setup and >15 A m-2 in a filtration setup are achieved. The permeate flow was identified as the main parameter leading to increased current densities.

The performance of microbial anodes in municipal wastewater: Pre-grown multispecies biofilm vs. natural inocula.

In this study, different inoculation strategies for continuously operated microbial anodes are analyzed and compared. After 20daysof operation with municipal wastewater anodes pre-incubated with a biofilm of the exoelectrogenic species Geobacter and Shewanella showed current densities of (65±8) µA/cm2. This is comparable to the current densities of non-inoculated anodes and anodes inoculated with sewage sludge. Analysis of the barcoded pre-grown multispecies biofilms reveal that 99% of the original biofilm was detached after 20daysof operation with municipal wastewater. This is in contrast to previous experiments where a pre-grown biofilm of exoelectrogens was operated in batch mode. To implement pre-grown biofilms in continuous systems it will thus be necessary to reveal a window of process parameters in which typical exoelectrogenic microorganisms including model organisms can be kept and/or enriched on anodes.