Enrichment and characterisation of ethanol chain elongating communities from natural and engineered environments

Abstract

chain elongation is a microbial process in which an electron donor, such as ethanol, is used to elongate short chain carboxylic acids, such as acetic acid, to medium chain carboxylic acids. this metabolism has been extensively investigated, but the spread and differentiation of chain elongators in the environment remains unexplored. Here, chain elongating communities were enriched from several inocula (3 anaerobic digesters, 2 animal faeces and 1 caproic acid producing environment) using ethanol and acetic acid as substrates at pH 7 and 5.5. This approach showed that (i) the inoculum's origin determines the pH where native chain elongators can grow; (ii) pH affects caproic acid production, with average caproic acid concentrations of 6.4 ± 1.6 g·L −1 at pH 7, versus 2.3 ± 1.8 g·L −1 at pH 5.5; however (iii) pH does not affect growth rates significantly; (iv) all communities contained a close relative of the known chain elongator Clostridium kluyveri; and (v) low pH selects for communities more enriched in this Clostridium kluyveri-relative (57.6 ± 23.2% at pH 7, 96.9 ± 1.2% at pH 5.5). These observations show that ethanol-consuming chain elongators can be found in several natural and engineered environments, but are not the same everywhere, emphasising the need for careful inoculum selection during process development.Carboxylic acids with carbon chains of six to twelve carbon atoms, also named medium chain carboxylic acids (MCCA), are a group of chemicals with a wide range of potential applications; as antimicrobial agents in feed and fodder these chemicals could replace conventional antibiotics, while they can also be used as a corrosion inhibitor 1 . Furthermore, chemical derivatization of MCCA creates additional product opportunities, e.g. esterification of MCCA can lead to a range of fragrance and flavour products for use in cosmetics and food 2 . On top of that, conversion of the carboxylic acids to their relative alcohols and alkanes, can supply solvents and fuels 3 , while Kolbe electrolysis of MCCA allows the production of long chain alkanes from MCCA, for instance decane, usable as jet-fuel 4 .Caproic acid, the shortest of the MCCA (six-carbon chain), is currently sourced from plant oils and animal fats 5 with a production capacity of 25,000 tonnes per year and a market value of approx. €2000/tonne 6 . It has generated great research interest because of the aforementioned application potential, in combination with the possibility of sustainable sourcing of caproic acid through bioproduction from waste streams, such as organic wastes and syngas 7-9 . This bioproduction occurs through the so-called reversed-β oxidation pathway, by which an electron donor, such as ethanol, is converted to acetyl-coA. This acetyl-CoA is then used in a cyclical process that converts acetic acid to butyric acid, which is in turn converted to caproic acid. This stepwise two-carbon increase of the carboxylic acid acyl-chain is why this metabolism is often named chain elongation. Clostridium kluyveri was the first isolated bacter...

Product Inhibition and pH Affect Stoichiometry and Kinetics of Chain Elongating Microbial Communities in Sequencing Batch Bioreactors

“…Possibly, a higher catabolic rate could not effectuate a higher growth rate at pH 7.0 because the culture was already growing at µ max . However, the average growth rate in our systems was 0.058 h −1 , which is significantly lower than µ max -values reported in literature (0.1-0.24 h −1 ) for C. kluyveri but comparable to growth rates (0.05-0.09 h −1 ) of communites enriched from anaerobic digester sludge in batch bottles (Weimer and Stevenson, 2012;Candry et al, 2018Candry et al, , 2020. Alternatively, faster growth might have been inhibited by relatively large amounts of fermentation products, leading to a trade-off between additional energy generation and maintaining endurable product concentrations.…”

“…This equaled an ethanol-to-acetate ratio of 6:1 in the bioreactor feed. A relatively high concentration of ethanol versus acetate was reported to positively influence caproate production (Liu et al, 2016) and a 6:1 ratio enabled both butyrate and caproate formation at pH 5.5 and pH 7.0 (Candry et al, 2020). Therefore, this feeding regime was chosen to allow for the characterization of the effect of both butyrate and caproate on chain elongating communities at different pH values.…”

“…Therefore, it is likely that the functional differences observed between the two enrichments were related to the inhibitory effects of the fermentation products. Two main mechanisms of toxicity of the elongated products can be distinguished (Candry et al, 2018(Candry et al, , 2020Roghair et al, 2018). First of all, protonated short-and medium chain carboxylic acids induce ATP consumption for the extrusion of protons and dissociated acids (Herrero, 1983).…”

“…Caproic acid is naturally produced in different types of microbial ecosystems (Barker and Taha, 1942;Weimer and Stevenson, 2012;Candry et al, 2020) by elongating shortchain carboxylic acids using ethanol or lactate as electron donor through reversed β-oxidation (Spirito et al, 2014). Chain elongating organisms compete for substrate with acetoclastic methanogens, syntrophic ethanol oxidizers and sulfate reducing organisms and provide substrate to, for example, homoacetogens, syntrophic butyrate oxidizing organisms and hydrogenotrophic methanogens (Muyzer and Stams, 2008;Cavalcante et al, 2017).…”

Recirculation of H2, CO2, and ethylene improves carbon fixation and carboxylate yields in anaerobic fermentation

“…However, the selectivity of 2-BES comes with caveats. Recent studies indicated collateral effects of 2-BES addition on carboxylate production, such as inhibition of i- butyrate formation 6, 8 and an increase of sulfate-reducing bacterial populations, suggesting 2-BES degradation in the long term 9–11 . Moreover, application of 2-BES at the high concentrations (50 mM 12 ) needed to inhibit hydrogenotrophic methanogenesis (Equation 4) might be economically unfeasible in industrial scale.…”