We report the closed genome sequences of the acetogen Sporomusa sphaeroides ET (DSM 2875T) and of Sporomusa ovata H1T (DSM 2662T). The S. sphaeroides ET genome harbors a chromosome (4,956,256 bp) and a plasmid (59,087 bp). The genome of S. ovata H1T harbors one chromosome (5,433,971 bp).

Sporomusa ovata is a Gram-negative acetogen of the Sporomusaceae family with a unique physiology. This anerobic bacterium is a core microbial catalyst for advanced CO2-based biotechnologies including gas fermentation, microbial electrosynthesis, and hybrid photosystem. Until now, no genetic tools exist for S. ovata, which is a critical obstacle to its optimization as an autotrophic chassis and the acquisition of knowledge about its metabolic capacities. Here, we developed an electroporation protocol for S. ovata. With this procedure, it became possible to introduce replicative plasmids such as pJIR751 and its derivatives into the acetogen. This system was then employed to demonstrate the feasibility of heterologous expression by introducing a functional β-glucuronidase enzyme under the promoters of different strengths in S. ovata. Next, a recombinant S. ovata strain producing the non-native product acetone both from an organic carbon substrate and from CO2 was constructed. Finally, a replicative plasmid capable of integrating itself on the chromosome of the acetogen was developed as a tool for genome editing, and gene deletion was demonstrated. These results indicate that S. ovata can be engineered and provides a first-generation genetic toolbox for the optimization of this biotechnological workhorse.IMPORTANCES. ovata harbors unique features that make it outperform most microbes for autotrophic biotechnologies such as a capacity to acquire electrons from different solid donors, a low H2 threshold, and efficient energy conservation mechanisms. The development of the first-generation genetic instruments described in this study is a key step toward understanding the molecular mechanisms involved in these outstanding metabolic and physiological characteristics. In addition, these tools enable the construction of recombinant S. ovata strains that can synthesize a wider range of products in an efficient manner.

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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.

The biological conversion of carbon monoxide (CO) has been highlighted for the development of a C1 gas biorefinery process. Despite this, the toxicity and low reducing equivalent of CO uptake make biological conversion difficult. The use of synthetic co-cultures is an alternative way of enhancing the performance of CO bioconversion. This study evaluated a synthetic co-culture consisting of Citrobacter amalonaticus Y19 and Sporomusa ovata for acetate production from CO. In this consortium, the CO2 and H2 produced by the water-gas shift reaction of C. amalonaticus Y19, were utilized further by S. ovata. Higher acetate production was achieved in the co-culture system compared to the monoculture counterparts. Furthermore, syntrophic cooperation via various reducing equivalent carriers provided new insights into the synergistic metabolic benefits with a toxic and refractory substrate, such as CO. This study also suggests an appropriate model for examining the syntrophic interaction between microbial species in a mixed community.

BACKGROUND: Microbial electrosynthesis (MES) and gas fermentation are bioenergy technologies in which a microbial catalyst reduces CO2 into organic carbon molecules with electrons from the cathode of a bioelectrochemical system or from gases such as H2. The acetogen Sporomusa ovata has the capacity of reducing CO2 into commodity chemicals by both gas fermentation and MES. Acetate is often the only product generated by S. ovata during autotrophic growth.
RESULTS: In this study, trace elements in S. ovata growth medium were optimized to improve MES and gas fermentation productivity. Augmenting tungstate concentration resulted in a 2.9-fold increase in ethanol production by S. ovata during H2:CO2-dependent growth. It also promoted electrosynthesis of ethanol in a S. ovata-driven MES reactor and increased acetate production 4.4-fold compared to unmodified medium. Furthermore, fatty acids propionate and butyrate were successfully converted to their corresponding alcohols 1-propanol and 1-butanol by S. ovata during gas fermentation. Increasing tungstate concentration enhanced conversion efficiency for both propionate and butyrate. Gene expression analysis suggested that tungsten-containing aldehyde ferredoxin oxidoreductases (AORs) and a tungsten-containing formate dehydrogenase (FDH) were involved in the improved biosynthesis of acetate, ethanol, 1-propanol, and 1-butanol. AORs and FDH contribute to the fatty acids re-assimilation pathway and the Wood-Ljungdahl pathway, respectively.
CONCLUSIONS: This study presented here shows that optimization of microbial catalyst growth medium can improve productivity and lead to the biosynthesis of different products by gas fermentation and MES. It also provides insights on the metabolism of biofuels production in acetogens and demonstrates that S. ovata has an important untapped metabolic potential for the production of other chemicals than acetate via CO2-converting bioprocesses including MES.

A co-culture system comprising an acetogenic bacterium, Sporomusa ovata DSMZ2662, and a denitrifying bacterium, Pseudomonas stutzeri JCM20778, enabled denitrification using H2 as the sole external electron donor and CO2 as the sole external carbon source. Acetate produced by S. ovata supported the heterotrophic denitrification of P. stutzeri. A nitrogen balance study showed the reduction of nitrate to nitrogen gas without the accumulation of nitrite and nitrous oxide in the co-culture system. S. ovata did not show nitrate reduction to ammonium in the co-culture system. Significant proportions of the consumed H2 were utilized for denitrification: 79.9 ± 4.6% in the co-culture system containing solid-phase humin and 62.9±11.1% in the humin-free co-culture system. The higher utilization efficiency of hydrogen in the humin-containing system was attributed to the higher denitrification activity of P. stutzeri under the acetate deficient conditions. The nitrogen removal rate of the humin-containing co-culture system reached 0.19 kg NO3(-)-N·m(-3)·d(-1). Stable denitrification activity for 61 days of successive sub-culturing suggested the robustness of this co-culture system. This study provides a novel strategy for the in situ enhancement of microbial denitrification.

Microbial electrosynthesis, an artificial form of photosynthesis, can efficiently convert carbon dioxide into organic commodities; however, this process has only previously been demonstrated in reactors that have features likely to be a barrier to scale-up. Therefore, the possibility of simplifying reactor design by both eliminating potentiostatic control of the cathode and removing the membrane separating the anode and cathode was investigated with biofilms of Sporomusa ovata. S. ovata reduces carbon dioxide to acetate and acts as the microbial catalyst for plain graphite stick cathodes as the electron donor. In traditional \'H-cell\' reactors, where the anode and cathode chambers were separated with a proton-selective membrane, the rates and columbic efficiencies of microbial electrosynthesis remained high when electron delivery at the cathode was powered with a direct current power source rather than with a potentiostat-poised cathode utilized in previous studies. A membrane-less reactor with a direct-current power source with the cathode and anode positioned to avoid oxygen exposure at the cathode, retained high rates of acetate production as well as high columbic and energetic efficiencies. The finding that microbial electrosynthesis is feasible without a membrane separating the anode from the cathode, coupled with a direct current power source supplying the energy for electron delivery, is expected to greatly simplify future reactor design and lower construction costs.