Metabolic responses of purple phototrophic bacteria to elevated hydrogen partial pressure: Metatranscriptomic insights into biohydrogen production and consumption.

During biohydrogen production, self-generated gas accumulation can lead to elevated hydrogen partial pressure (HPP) in the headspace. However, the effects of elevated HPP on hydrogen yield in photo fermentation systems and the metabolic responses of phototrophic bacteria (PPB) under such conditions remain poorly understood. This study investigated the effects of elevated HPP on PPB-based biohydrogen production and examined the underlying metabolic responses using metagenomic and metatranscriptomic analysis. PPB maintained efficient H2 production (0.67 ± 0.09 L H2·g COD-1; 0.09 ± 0.01 L H2·L-1·d-1) until HPP reached about 30 %, after which both H2 production and growth declined. Transcriptomic results showed reduced nitrogenase and increased hydrogenase expression, disturbing electron allocation and redox balance and suppressing the tricarboxylic acid (TCA) cycle, gluconeogenesis, and photosynthesis. When HPP increased to about 40 %, H2 production stopped and shifted to H2 consumption, while alternative electron sinks such as the reverse TCA cycle and branched-chain amino acid synthesis were activated to partially restore metabolism. Upon headspace flushing, H2 production rapidly resumed, demonstrating the robustness of the PPB system to short-term HPP inhibition. This study provides practical insight for PF system operation, highlighting the importance of maintaining HPP below inhibitory threshold to sustain efficient H2 production.