Single-stage microbial conversion of fish waste into linear and branched medium-chain fatty acids.

Anaerobic fermentation of biowaste into medium‑chain fatty acids (MCFAs) offers a scalable route for resource recovery within a circular bioeconomy framework, yet production from protein‑rich substrates such as fish waste remains underexplored. This study investigated the valorization of fish waste into MCFAs via mono- and co-fermentation in a single-stage chain elongation process. By leveraging endogenous electron donors and protein ammonification, the system maintained favorable pH conditions and sustained MCFA synthesis without external chemical inputs. Mono-fermentation favored isocaproate (4-methylvaleric acid) production, reaching a peak yield of 166.9 ± 11.6 mg COD/g VS (5.8 ± 0.4 g COD/L). Co-fermentation with carbohydrate-rich food waste in equal proportions shifted selectivity toward n-caproate (214.5 ± 18.8 mg COD/g VS) and n-heptanoate (145.8 ± 27.7 mg COD/g VS), achieving a maximum total MCFA yield of 366.3 ± 53.1 mg COD/g VS (12.8 ± 1.9 g COD/L). Bioaugmentation with Saccharomyces cerevisiae triggered solventogenesis and excessive ethanol oxidation (EEO) possibly due to elevated ammonium concentrations. Although sodium 2-bromoethanesulfonate (BES) addition transiently suppressed EEO, EEO resumed as H2 production declined, suggesting that promoting lactate-driven chain elongation via co‑fermentation is a more robust strategy under ammonia stress. Metagenomic analysis revealed that isocaproate formation during mono‑fermentation was associated with Stickland‑type amino‑acid fermentation, with Sporanaerobacter acetigenes as a potential key contributor. In contrast, co‑fermentation enriched genes associated with lactate metabolism, acetyl‑CoA generation, and linear MCFA synthesis, primarily linked to Clostridia and Betaproteobacteria. These results reveal tunable routes to straight‑ and branched‑chain MCFAs from protein‑rich waste, supporting green approaches to platform chemical generation.