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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.

Since sewage is a hotspot for antibiotic resistance genes (ARGs), the identification of ARGs in environmental waters impacted by sewage, and their correlation to fecal indicators, is necessary to implement management strategies. In this study, sewage treatment plant (STP) influent samples were collected and analyzed using quantitative polymerase chain reaction (qPCR) to investigate the abundance and correlations between sewage-associated markers (i.e., Bacteroides HF183, Lachnospiraceae Lachno3, crAssphage) and ARGs indicating resistance to nine antibiotics (belonging to aminoglycosides, beta-lactams, sulfonamides, macrolides, and tetracyclines). All ARGs, except blaVIM, and sewage-associated marker genes were always detected in untreated sewage, and ermF and sul1 were detected in the greatest abundances. intl1 was also highly abundant in untreated sewage samples. Significant correlations were identified between sewage-associated marker genes, ARGs and the intl1 in untreated sewage (τ = 0.488, p = 0.0125). Of the three sewage-associated marker genes, the BIO-ENV procedure identified that HF183 alone best maximized correlations to ARGs and intl1 (τ = 0.590). Additionally, grab samples were collected from peri-urban and urban sites along the Brisbane River system during base and stormflow conditions, and analyzed for Escherichia coli, ARGs, the intl1, and sewage-associated marker genes using quantitative polymerase chain reaction (qPCR). Significant correlations were identified between E. coli, ARGs, and intl1 (τ = 0.0893, p = 0.0032), as well as with sewage-associated marker genes in water samples from the Brisbane River system (τ = 0.3229, p = 0.0001). Of the sewage-associated marker genes and E. coli, the BIO-ENV procedure identified that crAssphage alone maximized correlations with ARGs and intl1 in river samples (τ = 0.4148). Significant differences in E. coli, ARGs, intl1, and sewage-associated marker genes, and by flow condition (i.e., base vs. storm), and site types (peri-urban vs. urban) combined were identified (R = 0.3668, p = 0.0001), where percent dissimilarities between the multi-factorial groups ranged between 20.8 and 11.2%. Results from this study suggest increased levels of certain ARGs and sewage-associated marker genes in stormflow river water samples compared to base flow conditions. E. coli, HF183 and crAssphage may serve as potential indicators of sewage-derived ARGs under stormflow conditions, and this merits further investigation. Data presented in this study will be valuable to water quality managers to understand the links between sewage pollution and ARGs in urban environments.