Functional insights into inhibition by steam-exploded brewery spent grain hydrolysate in anaerobic digestion: roles of ammonia, volatile fatty acids, and phenolics

Brewery spent grain (BSG) is a promising substrate for anaerobic digestion (AD). However, its lignocellulosic structure limits biodegradability. Steam explosion (SE) pretreatment enhances solubilization but may also generate inhibitory compounds, including free ammonia nitrogen (FAN), volatile fatty acids (VFAs), and p-cresol. This study evaluated the AD of BSG mixtures containing 0-100% SE hydrolysate in 85-day batch assays at 37 °C, assessing methane (CH4) production, inhibition dynamics, microbial community shifts, and predicted functional responses inferred from taxonomic profiles. The mixture containing 50% hydrolysate (EXP-0.5) exhibited the least compromised performance, with a CH4 yield of 101.0 ± 4.2 mL CH4/gVSadded and a reduced lag phase (λ = 1.7 d). Increasing hydrolysate proportions intensified inhibitory conditions, with FAN concentrations exceeding 500 mg/L, acetic acid reaching 2,448 mg/L, and p-cresol concentrations surpassing 280 mg/L. These conditions may have constrained key oxidation steps and, consequently, contributed to reduced CH4 production. Despite these stresses, Methanothrix remained the dominant methanogen, suggesting a persistent predominance of acetoclastic methanogenesis, albeit with reduced functional efficiency. Functional predictions for EXP-0.5 suggested a higher representation of pathways related to the citrate cycle, carbohydrate, and amino acid metabolism, implying a potential for sustained energy flux and adaptive responses to ammonia stress. Overall, the results infer that moderate hydrolysate incorporation can attenuate, but not eliminate, inhibition of SE-treated BSG during AD. Therefore, integrated strategies in lignocellulosic biorefinery schemes using SE hydrolysates are required to balance the benefits of enhanced solubilization with microbial metabolic resilience and long-term process stability in AD systems.