Oriented accumulation of propionate during anaerobic digestion and its impact on bacterial communities

Our previous work have demonstrated that the metabolic and microbial profiles of the acidogenic food waste fermentation can be changed depending on the operating conditions in batch mode. In particular, initial pH, temperature and initial food waste concentration influenced the total metabolite production, the food waste conversion yield, and the propionate content among metabolites. Propionate reached its higher production at pH 9, T=35°C and FW= 7.8 gVS/L, reaching ~1.7 g/L and representing ~30% of the metabolites. This concentration remained low for furter valorisation of this molecule. To further increase propionate accumulation during the anaerobic digestion of food waste, an alternative method was studied. In fact, in conventional AD systems designed for methane production, propionate accumulation is generally considered detrimental to biogas production because propionate plays a central role in the stability and performance of the anaerobic processes. Its accumulation is widely recognised as an indicator of process imbalance or failure, often associated with a decrease in pH and methane yield (Capson-Tojo et al., 2017; Zhao, Westerholm, et al., 2018). The oxidation of propionate to acetate, hydrogen and carbon dioxide (Reaction 3.1) is thermodynamically unfavourable (ΔG°′ ≈ +76 kJ·mol⁻¹) and can proceed efficiently only when dissolved hydrogen is continuously consumed by hydrogenotrophic methanogens to produce methane (Reaction 3.2). This syntrophic cooperation between propionate-oxidising bacteria (POB) and hydrogenotrophic methanogens maintains low dissolved hydrogen concentration, thereby enabling propionate degradation and stable methane production (Kim et al., 2022; Mu et al., 2023). In parallel, acetoclastic methanogens convert acetate into methane, further pulling the oxidation of propionate forward. When this syntrophic equilibrium is disturbed due to factors such as organic overloading, ammonia inhibition, pH shifts, or other environmental stresses, propionate oxidation becomes energetically constrained, leading to its accumulation and subsequent inhibition of methanogenesis (Capson-Tojo et al., 2017). Reaction 3.1: CH3CH2COO− + 3 H2O → CH3COO− + HCO3− + H+ + 3 H2 Reaction 3.2: 4 H2 + HCO3− + H+ → CH4 + 3 H2O Propionate accumulation during AD has been observed at concentrations up to 21.6 g·L-1 (Capson-Tojo et al., 2017), under conditions such as high organic loading rates, elevated ammonia concentrations, high substrate-to-inoculum (S/X) ratios, and increased hydrogen partial pressures, all of which disturb syntrophic interactions between propionate oxidisers and methanogens (Capson-Tojo et al., 2017; Mu et al., 2023). The extent of propionate accumulation depends on multiple, often interrelated factors, including substrate characteristics, inoculum adaptation, and operating parameters. For instance, Li et al. (2017) reported that total ammonia nitrogen (TAN) concentrations above 2.5 g·L-1 inhibit propionate degradation, while Capson-Tojo et al. (2017) still observed methane production at TAN concentrations as high as 10 g·L-1. Similarly, propionate oxidation is thermodynamically inhibited when hydrogen partial pressure exceeds 10-3 atm (Harirchi, Wainaina, et al., 2022). The substrate-to-inoculum ratio (S/X) strongly influences microbial kinetics: low ratios (e.g., 0.5) favour VFA degradation, while higher ratios (≥ 1) promote accumulation. Consequently, parameters such as organic loading rate (OLR) and hydraulic retention time (HRT) are key operational levers for steering AD toward either biogas production or VFA accumulation (Alavi-Borazjani et al., 2020). Despite extensive literature describing propionate accumulation as a stress indicator, relatively little attention has been paid to the intentional control of AD conditions to selectively promote propionate accumulation. Understanding the interplay between substrate concentration and microbial adaptation could provide levers to drive AD toward propionate-rich end-products. Therefore, this study aims to elucidate the influence of substrate-to-inoculum ratio and food waste concentration on propionate accumulation and selectivity in successive batch systems, while providing insights into the microbial dynamics underpinning this phenomenon for the development of a propionate-oriented anaerobic process.