Native microbial community outperforms inoculated systems with Clostridium and Megasphaera during lactate-driven hydrogen fermentation

Hydrogen (H 2 ) production using lactic acid as a precursor has been studied under various operational conditions, showing competitive productivity. Although this process has mainly been attributed to lactic acid bacteria and lactic acid-consuming bacteria involved in cross-feeding, the overall microbial interactions remain poorly understood. This study evaluated three inoculation strategies for lactate-driven H 2 production in sequencing batch reactors using Megasphaera elsdenii, Clostridium beijerinckii, and pretreated sludge: (A) native community, (B) inoculation at start-up, and (C) periodic inoculation every third cycle. Microbial dynamics were analyzed through co-occurrence network analysis to elucidate potential microbial interactions during dark fermentation. Lactic acid from the fermentation of food waste served as substrate, and thermally pretreated sludge (5.0 gVS/L) was used as inoculum, along with 10.0 % of a strain mixture (68.0 % M. elsdenii , 32.0 % C. beijerinckii ). Strategy A exhibited the highest H 2 production (1039.7 mLH 2 /L reactor ). In contrast, strategies B and C showed declining H 2 over time, leading to CH 4 formation in later cycles. The three strategies showed a similar microbial community composition dominated by M. elsdenii , yet their dynamics varied across conditions. Network analysis suggested associations between M. elsdenii and Olsenella umbonata linked to H 2 production in strategy A. Conversely, the M. elsdenii – Methanobrevibacter boviskoreani association appeared to be related to hydrogenotrophic CH 4 production in strategies B and C. Negative associations, predominantly involving bacteria from the pretreated sludge, were also observed in these strategies. These results suggest that the native community of the substrate can establish more effective interactions for H 2 production than externally introduced strains.