Direct cell-to-cell exchange of matter in synthetic clostridium syntrophies enabling CO2 fixation and an expanded metabolic space.

In microbial fermentations to produce metabolites, at least 33% of the sugar-substrate carbon is lost as CO2 during pyruvate decarboxylation to acetyl-CoA. Previous attempts to reduce this carbon loss focused on engineering a single organism. In nature, microorganisms live in complex communities where syntrophic interactions result in superior resource utilization. Microbial communities are ubiquitous in nature and have a wide range of applications, including production of biofuels and chemicals. Syntrophic and other microbial co-cultures/consortia carry out efficient bio-transformations that are the result of multiple complementary metabolic systems working together. It is now well appreciated that the capabilities of multi-microorganism systems cannot be predicted by the sum of their parts. Rather, synergistic interactions at different levels often result in better overall performance of these systems. Importantly, integration of diverse metabolic systems through syntrophic dependencies make co-culture systems robust to environmental fluctuations. Clostridium organisms are of major importance for developing new technologies to produce biofuels and chemicals. Three major types of Clostridium organisms have been the focus of studies for the sustainable production of fuels and chemicals. Solventogenic clostridia utilize a large variety of biomass-derived carbohydrates (hexoses, pentoses, disaccharides, and hemicellulose), and can produce C2-C4 chemicals. Acetogenic clostridia can fix inorganic H2, CO2, and CO to generate C2 acids and alcohols. Other specialized clostridia possess diverse biosynthetic capabilities for production of a wide variety of metabolites including C4 – C8 carboxylic acids and alcohols, which could serve as commodity chemicals, biofuels, or biofuel precursors. Here, we first examined a synthetic syntrophy consisting of the solventogen Clostridium acetobutylicum, which converts simple and complex carbohydrates into a variety of chemicals, and the acetogen C. ljungdahlii, which fixes CO2. This synthetic co-culture achieved carbon recoveries into C2-C4 alcohols almost to the limit of substrate-electron availability, with minimal H2 and CO2 release. The syntrophic co-culture produced robust metabolic outcomes over a broad range of starting population ratios of the two organisms. Significantly, the co-culture exhibited unique direct cell-to-cell interactions and material exchange among the two microbes, which enabled unforeseen rearrangements in the metabolism of the individual species that resulted in the production of non-native metabolites, namely isopropanol and 2,3-butanediol. Next, we expanded this co-culture system to include C. kluyveri, which can metabolite ethanol and acetate to produce C6 and C8 carboxylic acids. Both C. acetobutylicum and C. ljungdahlii produce ethanol and acetate, which makes C. kluyveri and ideal partner for a triple synthetic co-culture system capable to converting biomass-derived carbohydrates to C6 and C8 chemicals. Supported by the National Science Foundation through the US Army Research Office (ARO; Award No. W911NF-17-1-0343) and the US Department of Energy (DOE; Award No. DE-SC0019155)

Metabolism and cell-to-cell interactions of anaerobic syntrophic Clostridia co-cultures

Papoutsakis, EleftheriosThe greenhouse gas, CO2, in Earth’s atmosphere is a threat to our planet and a cause of global warming. Utilizing CO2 industrial waste gases as fermentation feedstock for biofuel production is a promising technology for the reduction of industrial waste. One of the notable genera of bacteria that can accomplish this is the genus Clostridium. Clostridium spp. are Gram-positive, anaerobic Firmicutes that can metabolize a diverse amount of substrates, including sugars, acids, alcohols, and gases like CO2, H2, and CO. Engineering non-acetogenic clostridia to efficiently consume waste as feedstock has been the subject of many recent studies, but using multiple species of Clostridium in co-culture has been shown to be an achievable and economical option for optimizing fermentation carbon recoveries and production of non-natural products. ☐ Many different co-cultures between Clostridium spp. have been studied, but one pair that has not been investigated previously is between the chain elongating C. kluyveri, and the well-studied solventogen, C. acetobutylicum. Our lab has previously established a co-culture between C. acetobutylicum and the CO2 fixing acetogen, C. ljungdahlii, and this co-culture has been shown to improve carbon efficiency of clostridia fermentation when compared to mono-culture. The addition of a third organism, C. kluyveri, to the co-culture could potentially use the products of syntrophic interactions between C. ljungdahlii and C. acetobutylicum to produce medium-chain fatty acids that can be converted to biofuels. ☐ In this study, we examined the possibilities of C. acetobutylicum and C. kluyveri in co-culture as well as a triple organism co-culture between C. acetobutylicum, C. ljungdahlii, and C. kluyveri. This triple organism co-culture has the potential to use sugars for syntrophic production acids and alcohols, which would be further converted to of medium-chain fatty acids by C. kluyveri. C. acetobutylicum and C. kluyveri co-cultures were shown to be need further optimization due to pH discrepancies between species, but the addition of C. ljungdahlii to the culture was able to produce the medium-chain fatty acid, hexanoate from additional acetate and ethanol in co-culture. In order to further understand syntrophic interactions between Clostridium spp., we designed an anaerobic, highly-fluorescent reporter system using the fluorescence activating protein, FAST, and the fluorogenic ligand, HMBR. Commonly used fluorescent proteins reporters require oxygen for chromophore maturation, and anaerobic fluorescent proteins lack brightness comparable to aerobic fluorescent proteins. FAST does not require oxygen, and fluoresces instantaneously when the fluorogenic ligand, HMBR, is added. In addition to being a successful fluorescent reporter, FAST was also used to successfully tag and view protein localization of the cell division protein, ZapA, in live C. acetobutylicum cells. This fluorescent system opens the door for research on other Clostridium spp. and other anaerobes to study protein interactions with oxygen-independent fluorescence. FAST could be used in the future for further investigation of protein localization, particularly shedding light on localization of proteins that may be involved in syntrophic co-cultures.University of Delaware, Department of Biological SciencesM.S