The occurrence and ecology of microbial chain elongation of carboxylates in soils

Abstract

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.

Fig. 1. Microbial chain elongation activity in microcosms (25 g soil and 75 mL medium) with Tempe, Bozeman, Lucas, and Goodyear soils amended with 100 mM acetate and 100 mM ethanol or 100 mM ethanol.

The data are averages with standard deviation of duplicate microcosms.

Fig. 2. Metabolites during microbial chain elongation in soils initially fed with 100 mM acetate and 100 mM ethanol and subjected to semi-batch enrichment.

A semi-batch cycle consisted of removing one third of microcosm liquid (25 mL) and replacing with 25 mL medium containing 100 mM acetate and 100 mM ethanol. The incubation time for each cycle (between 6–14 days) is shown in Table 2. The plotted carboxylates and alcohols are final measured metabolite concentrations at the end of each cycle before liquid was removed and medium with substrates was readded. The data are averages with standard deviation of duplicate microcosms.

Fig. 3. Metabolites during microbial chain elongation in soils initially fed with 100 mM ethanol and subjected to semi-batch enrichment.

A semi-batch cycle consisted of removing one third of microcosm liquid (25 mL) and replacing with 25 mL medium containing 100 mM ethanol. The incubation time for each cycle (between 6–43 days) is shown in Table 2. The plotted carboxylates and alcohols are final measured metabolite concentrations at the end of each cycle before liquid was removed and medium with substrates was readded. The data are averages with standard deviation of duplicate microcosms.

Fig. 4. Microbial chain elongation of acetate and ethanol at lower substrate concentrations in Tempe and Lucas soil microcosms (25 g soil and 75 mL medium).

The data are averages with standard deviation of duplicate microcosms.

Fig. 5. The predominance of Firmicutes in chain-elongating soil microbial communities enriched on acetate and ethanol or ethanol.

The data are averages of sequences from duplicate microcosms before enrichment (at time zero) and after enrichment in 4 or 5 semi-batch cycles as specified in Table 2.

Fig. 6. Diversity of select enriched genera and species in (a) Tempe, (b) Bozeman, (c) Lucas, and (d) Goodyear soil chain-elongating microbial communities.

The plotted ASVs show relative abundance from the total sequences obtained in each condition and sampled according to Table 2. The data are averages of sequences from duplicate microcosms. Labels: t 0 = soil before enrichment; NS =  No Substrates, A = 140 mM Acetate; AE = 100 mM Acetate + 100 mM Ethanol; E = 100 mM Ethanol; AH = 100 mM Acetate + H₂; EH = 100 mM Ethanol + H₂.