Impacts of organic loading rate fluctuations and division of labor on sugar-based chain elongation revealed through metatranscriptomics

• Organic loading rate fluctuations did not improve caproate production. • Glucose was preferentially consumed compared to xylose. • Caproiciproducens and Caproicibacter were abundant at higher organic loading rates. • Chain elongators and sugar fermenters divide labor. • Pantothenate, folate, biotin, CoA, and several amino acids are shared metabolites. Medium-chain carboxylates (MCCs) can be produced using open, mixed cultures of microorganisms in a process termed “chain elongation.” Chain elongating bacteria can increase ATP yield by producing six-carbon caproate rather than four-carbon butyrate. Therefore, requiring chain elongating bacteria to maximize ATP yield for cell synthesis may be a way to increase production of the more valuable caproate. To test this, duplicate bioreactors were operated, and the impact of organic loading rate (OLR) fluctuations were assessed with glucose and xylose as substrates in media that did not include amino acids, vitamins, or other growth factors. Increasing the OLR did not reliably improve caproate production despite several known caproate-producing bacteria being present. 16S rRNA gene amplicon sequencing and shotgun metagenomics revealed that the same chain-elongating and sugar-degrading species were enriched in both bioreactors, including members of the Caproiciproducens , Caproicibacter , Olegusella , and Tractidigestivibacter genera. Further, metatranscriptomic results suggest a distinct division of labor associated with critical growth factors, including amino acids, folate, and pantothenate. This division of labor, while potentially beneficial to the microbial community, may result in low caproate production.