Higher-order interaction effects among operating conditions and feedstocks shape reactor microbiomes and fatty acid production profiles.

Arrested anaerobic digestion (AAD) offers a promising route for producing fatty acids (FAs) from organic residues, yet optimal conditions for selectively generating medium-chain fatty acids (MCFAs) remain poorly defined. Here, we systematically evaluated the main and interaction effects of pH (5, 7, 9), feedstock (food waste, manure), temperature (35 and 45 °C), and inoculum source on microbiome composition and FA production. Anaerobic digester sludge and a novel bison rumen inoculum were compared. Significant higher-order interactions among operating parameters governed FA profiles and microbiome structure. Butyric acid production was driven by a three-way interaction among pH, feedstock, and temperature (p < 0.001), with maximum concentrations achieved in food waste reactors at pH 5.0 and 35 °C (1.2 ± 0.1 g L-1 with sludge and 1.1 ± 0.3 g L-1 with rumen). MCFA production exhibited significant four-way interactions (p < 0.1 to p < 0.001). At 45 °C and pH 5.0, inoculum source tuned MCFA selectivity: sludge favored pentanoic acid (0.4 ± 0.1 g L-1), whereas rumen favored hexanoic and heptanoic acids (up to 0.4 ± 0.2 g L-1). Manure reactors produced < 0.2 g L-1 MCFAs under all conditions. Genera, including Megasphaera, Prevotella, and Lactobacillus, were associated with production of specific MCFAs. PICRUSt2-based pathway predictions were consistent with MCFA production patterns and suggested a potential role for lactic acid-driven chain elongation pathways. This study provides insights into how interacting operating conditions shape AAD microbiomes, their FA profiles, and advances the trajectory of research aimed at engineering robust and controllable microbiomes for waste valorization.