Acid stress-driven microbial community succession and metabolic regulation in anaerobic hydrogen production

Self-acidification from volatile fatty acids (VFAs) accumulation limits microbial activity and process stability during anaerobic hydrogen production. However, microbial responses to acid stress remain insufficiently understood. Here, batch fermentation with xylose under seven initial pH conditions (4.0-5.5) was used to track acid-stress driven succession and metabolic regulation. Hydrogen content remained stable at initial pH 4.8-5.5 (49.4-51.8%) but declined markedly at pH 4.0-4.6. Hydrogen yield exhibited a similar trend, peaking at 0.99-1.10 mol H₂/mol xylose and declined as the pH decreased. Total VFAs concentration peaked at initial pH 5.5 (11.3 g/L) and declined progressively as initial pH decreased. Notably, increasing acid stress progressively inhibited the butyrate metabolic pathway while enhancing the acetate pathway. Community analysis revealed a dominance shift from hydrogen-producing Clostridium and Paraclostridium to acid-tolerant Limosilactobacillus. Mechanistically, acid stress aggravated oxidative stress, with reactive oxygen species peaking at 30,962.5 U at 12 h (initial pH 4.2). Within the adaptive pH range, extracellular polymeric substance (EPS) production increased (fluorescence intensity: 777.6 R.U.), likely enhancing cellular protection in conjunction with dynamic regulation of membrane permeability. Under sustained or more severe acidification, EPS production was constrained, compromising cellular protection and accelerating functional decline. ATP acts more on the regulation of inner membrane permeability rather than on hydrogen production. Overall, this study defines the pH tolerance boundaries of hydrogen-producing consortia and links pH-driven oxidative damage, EPS-membrane-ATP coupling, and pathway switching to community turnover. These mechanistic insights provide a foundation for enhancing the robustness and industrial reliability of biohydrogen production systems.