Screening of Gas Substrate and Medium Effects on 2,3-Butanediol Production with C. ljungdahlii and C. autoethanogenum Aided by Improved Autotrophic Cultivation Technique

Gas fermentation by acetogens of the genus Clostridium is an attractive technology since it affords the production of biochemicals and biofuels from industrial waste gases while contributing to mitigate the carbon cycle alterations. The acetogenic model organisms C. ljungdahlii and C. autoethanogenum have already been used in large scale industrial fermentations. Among the natural products, ethanol production has already attained industrial scale. However, some acetogens are also natural producers of 2,3-butanediol (2,3-BDO), a platform chemical of relevant industrial interest. Here, we have developed a lab-scale screening campaign with the aim of enhancing 2,3-BDO production. Our study generated comparable data on growth and 2,3-BDO production of several batch gas fermentations using C. ljungdahlii and C. autoethanogenum grown on different gas substrates of primary applicative interest (CO2 · H2, CO · CO2, syngas) and on different media featuring different compositions as regards trace metals, mineral elements and vitamins. CO · CO2 fermentation was found to be preferable for the production of 2,3-BDO, and a fair comparison of the strains cultivated in comparable conditions revealed that C. ljungdahlii produced 3.43-fold higher titer of 2,3-BDO compared to C. autoethanogenum. Screening of different medium compositions revealed that mineral elements, Zinc and Iron exert a major positive influence on 2,3-BDO titer and productivity. Moreover, the CO2 influence on CO fermentation was explored by characterizing C. ljungdahlii response with respect to different gas ratios in the CO · CO2 gas mixtures. The screening strategies undertaken in this study led to the production of 2.03 ± 0.05 g/L of 2,3-BDO, which is unprecedented in serum bottle experiments.

Efficient Hydrogen Delivery for Microbial Electrosynthesis via 3D-Printed Cathodes

The efficient delivery of electrochemically in situ produced H2 can be a key advantage of microbial electrosynthesis over traditional gas fermentation. However, the technical details of how to supply large amounts of electric current per volume in a biocompatible manner remain unresolved. Here, we explored for the first time the flexibility of complex 3D-printed custom electrodes to fine tune H2 delivery during microbial electrosynthesis. Using a model system for H2-mediated electromethanogenesis comprised of 3D fabricated carbon aerogel cathodes plated with nickel-molybdenum and Methanococcus maripaludis, we showed that novel 3D-printed cathodes facilitated sustained and efficient electromethanogenesis from electricity and CO2 at an unprecedented volumetric production rate of 2.2 LCH4 /Lcatholyte/day and at a coulombic efficiency of 99%. Importantly, our experiments revealed that the efficiency of this process strongly depends on the current density. At identical total current supplied, larger surface area cathodes enabled higher methane production and minimized escape of H2. Specifically, low current density (<1 mA/cm2) enabled by high surface area cathodes was found to be critical for fast start-up times of the microbial culture, stable steady state performance, and high coulombic efficiencies. Our data demonstrate that 3D-printing of electrodes presents a promising design tool to mitigate effects of bubble formation and local pH gradients within the boundary layer and, thus, resolve key critical limitations for in situ electron delivery in microbial electrosynthesis.

GASEOUS FUEL OBTAINING VIA FERMENTATION OF ORGANIC LANDFILL WASTE

Fossil carbon-containing fuel is currently one of the most common in industry and economy. The rapid depletion of reserves of this fuel makes it necessary to search for the alternative one. Landfills are a place where methane is spontaneously synthesized due to the decay of organic waste. Controlled and regulated fermentation of the landfill organics can provide biomethane as well as environmental bioremediation. The aim of the work was to study the patterns of methane fermentation of multi component organic waste and optimize the process to increase the efficiency of biomethane synthesis and waste decomposition. Colorimetric and potentiometric methods were used for pH and Eh measurement. Volumetric and chromatographic methods were applied to control volume and composition of synthesized gas. Fermentation parameters were calculated with the use of mathematical and statistical ones. The achievement of high efficiency of methane fermentation of organic waste due to the process regulation was shown. The modeling of unregulated fermentation of organic waste in landfills showed low efficiency of the process. It took 69 days. Weight of waste decreased only 5 times. Hydrogen yield was 5 L/kg of waste. Methane was not synthesized. The regular mass transfer, regulation of the process and waste grinding showed the greatest efficiency. Weight of waste decreased 20 times during only 14 days. Hydrogen yield was 27 L/kg, methane yield was 12 L/kg of waste. Thus, the absence of regulation caused long term decay of waste. The high efficiency is achieved due to regulation of the fermentation process. The results will serve as a basis for the development of industrial biotechnology for the utilization of organic waste to reduce the volume of existing landfills and produce methane energy. This will further allow bioremediation of contaminated areas, obtaining an alternative to fossil fuel biomethane.

Propionate production from carbon monoxide by synthetic co-cultures of Acetobacterium wieringae spp. and propionigenic bacteria

Gas fermentation is a promising way for converting CO-rich gases to chemicals. We studied the use of synthetic co-cultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore this co-cultivation approach was investigated. Four distinct synthetic co-cultures were constructed, consisting of: Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T); Ac. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T); Ac. wieringae strain JM and P. propionicus (DSM 2379T); Ac. wieringae strain JM and An. neopropionicum (DSM 3847T). Propionate was produced by all the co-cultures, with the highest titer (∼24 mM) measured in the co-culture composed of Ac. wieringae strain JM + An. neopropionicum, which also produced isovalerate (∼4 mM), butyrate (∼1 mM), and isobutyrate (0.3 mM). This co-culture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during co-cultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during co-cultivation to fully understand co-cultures behavior. IMPORTANCE Syngas fermentation has great potential for the sustainable production of chemicals from wastes (via prior gasification) and flue gases containing CO/CO2. Research efforts need to be driven to expanding the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol. This study provides the basis for a microbial process to produce propionate from CO using synthetic co-cultures composed of acetogenic and propionigenic bacteria and elucidates the metabolic pathways involved. Furthermore, based on proteomics results, we hypothesize that the two bacterial species engage in an interaction that results in amino acid exchange, which subsequently promotes isovalerate and isobutyrate production. These findings provide a new understanding of gas fermentation and a co-culturing strategy for expanding the product spectrum of microbial conversion of CO/CO2.