Wetlands harbor lactic acid-driven chain elongators

ABSTRACT Wetlands are globally significant carbon storage hotspots. Recent research has suggested that microbially derived metabolites may contribute to soil organic matter formation. Identifying pathways driving the formation of such metabolites is critical to understand the global impact of wetland carbon cycling. Here, we evaluate the presence of chain-elongating organisms converting two to three carbon compounds (i.e., lactic and acetic acid) to medium-chain carboxylic acids (MCCA; i.e., six-carbon caproic acid) in wetland soils. We demonstrate the enrichment of a lactic acid-driven chain-elongating community from wetland soils producing a mixture of butyric and caproic acid. The enriched community was dominated by Clostridiaceae, Ruminococcaceae, and Lachnospiraceae, three families with known chain elongators. Amplicon sequencing identified three Ruminococcaceae and one Clostridiaceae zero-radius OTU (zOTU) that were (i) present in the soil, (ii) enriched over 1% relative abundance in the bioreactor, and (iii) were closely related to known chain elongators. Moreover, close relatives of the three Ruminococcaceae zOTU were also observed in several other wetland microbiomes. From this observation, we conclude that close relatives of known chain elongators, potentially capable of lactic acid-driven MCCA production themselves, are present in wetland soils. This observation may have implications for our understanding of carbon cycling and storage in wetland ecosystems. IMPORTANCE Wetlands are globally significant carbon cycling hotspots that both sequester large amounts of CO2 as soil carbon as well as emit a third of all CH4 globally. Their outsized role in the global carbon cycle makes it critical to understand microbial processes contributing to carbon breakdown and storage in these ecosystems. Here, we confirm the presence of chain-elongating organisms in freshwater wetland soils. These organisms take small carbon compounds formed during the breakdown of biomass and turn them into larger compounds (six to eight carbon organic acids) that may potentially contribute to the formation of soil organic matter and long-term carbon storage. Moreover, we find that these chain-elongating organisms may be widely distributed in wetlands globally. Future work should identify these organisms’ contribution to carbon cycling in wetlands and the potential role of the products they form in carbon sequestration in wetlands.

Microbial chain elongation anaerobically converts two to four carbon compounds (e.g., ethanol, acetic acid, lactic acid, etc.) into six to eight carbon medium-chain carboxylic acids (MCCA, e.g., six-carbon caproic acid) (6) whose aliphatic nature and potential mineral-organic interactions might contribute to SOM formation (7,8).While research on chain elongators has mostly focused on biotechnological applications, their presence in and contribution to environmental systems, specifically wetlands, remain understudied and poorly understood.
Chain elongators have been observed in environmental settings.For instance, Clostridium kluyveri-an ethanol-consuming chain elongator-was isolated from canal mud (9), while two recent studies enriched ethanol-elongating mixed communities from environmental samples including soils and animal feces (10,11), and another study incidentally observed chain elongation in H 2 -supplied soil incubations (12).Despite their potential significance, the presence of lactic acid-consuming chain elongators in wetlands-or other terrestrial environments-has not yet been investigated.
Amplicon sequencing was used to analyze community composition over the course of the enrichment.Broadly, three families accounted for over half of the community throughout the enrichment (Fig. 1B): (i) Clostridiaceae (20%-35% relative abundance), (ii) Ruminococcaceae (gradual decrease from 31.3% to 6.7% relative abundance), and (iii) Lachnospiraceae (gradual increase from 1.0% to 28.7% relative abundance).While all three families harbor known chain elongators (13), each of these families contains a wide range of functional diversity beyond chain elongation.
We evaluated the presence of these putative chain elongators in other environmental and engineered systems by searching the zOTU in public sequencing data (S.1.6.).All zOTU were found in other systems (Fig. S4 and S5; Table S3).The four zOTU were present in 33%-86% of all bioreactor or enrichment samples (n = 275, median abundances of 0.0022%-40.5209%),while detection in wetland sites was sparser with zOTU 3, 13, and 32 detected in 1.6%-7.1% of samples (n = 127, median abundances of 0%-0.0141%).zOTU 8 was not detected in any wetlands besides the site studied here.Notably, saline, sulfate-rich soils contained none of the four zOTU, potentially due to substrate competi tion between sulfate reducers and chain elongators in these settings (13).
The data presented here indicate lactic acid chain-elongating communities can be enriched from wetland soils, and putative chain elongators were present in a range of wetland soils.
The functionality of zOTU was inferred from abundances in the enrichment and their closest phylogenetic relatives.However, close relatives and even different strains within a single species may exhibit divergent properties.Moreover, predicting chain elongation potential from full genomes remains a challenge, which is further compli cated by some isolates producing MCCA only under certain conditions (15,19,20).Given these limitations, we conclude that close relatives of known chain elongators, potentially capable of lactic acid-driven MCCA production themselves, were found in wetland soils.It should be highlighted that this is the first report of chain elongators in wetland ecosystems as well as the first report of lactic acid chain elongators in natural environments.
The presence of lactic acid chain elongators in wetlands may have implications for our understanding of wetland carbon cycling.Short-chain carboxylic acids (e.g., acetic, propionic, and lactic acid) are typically converted to CH 4 under anaerobic conditions.If chain elongators are present and active in wetlands, they may divert carbon away from CH 4 toward MCCA, potentially sequestering carbon and altering the system's net greenhouse gas flux.While we show their presence and potential role, open questions remain on which environmental conditions favor chain elongators in wetlands and to what extent they alter the microbial cross-feeding network in the ecosystem (11,13).Chain elongators have a competitive advantage over methanogenic consortia in spatiotemporal niches with elevated H 2 partial pressures (21).Such temporal niches may occur during periods of flooding or high organic matter influx (22,23), or in spatial niches allowing longer-term proliferation of chain elongators.In parallel, interactions between MCCA and SOM (7, 8) could extend the retention time of carbon in the system, providing a potential mechanism for chain elongators to simultaneously reduce CH 4 flux by outcompeting methanogens as well as contribute to longer-term carbon storage.These hypotheses, however, remain to be confirmed with experimental data.
This study presents the enrichment of a lactic acid-driven chain elongation commun ity from wetland soils and identifies close relatives of known chain elongators that were present in the original soil as well as other wetland soils.This observation may have implications for our understanding of carbon cycling and storage in wetland ecosystems.

FIG 1
FIG 1 Enrichment of a lactic acid-driven chain elongation community from wetland soil.Panel (A) shows CSTR effluent product profiles with orange triangles indicating days sampled for community characterization.Panel (B) shows the relative abundance of community members classified at the family level.Panel (C) shows a phylogenetic tree of the V4-V5 region of (Continued on next page)

FIG 1 (
FIG1 (Continued)    the 16S rRNA gene for zOTU of interest along with relevant relatives.zOTU of interest (i) were detected in the wetland soil inoculum, (ii) had a relative abundance of at least 1% at any time during reactor operation, and (iii) are related to known chain elongators.Bootstrap values greater than or equal to 50% are shown as percentages at each node.Scale indicates substitutions per nucleotide position.