Multimodal, quantitative analysis enables process management of productive, mixotrophic biofilms.

Biofilms offer attractive opportunities for continuous biotechnological production processes; however, understanding biomass dynamics and electron fluxes within such communities remains a major challenge for process optimization and scale-up. In this study, a segmented-flow tubular capillary biofilm reactor was investigated using a dual-species consortium of Pseudomonas taiwanensis and Rhodopseudomonas palustris for nitrogenase-driven hydrogen production. Biofilm development and biomass dispersion were monitored using a newly established flow cytometry-based analysis pipeline capable of quantifying the abundance of the different organisms in the reactor outflow. Mature biofilms operated under N2-limited conditions exhibited stable species composition and cell numbers over a 72 h monitoring period, indicating semi-steady-state conditions characterized by slow growth and continuous erosive dispersion. Hydrogen production was strongly influenced by reactor gas composition. Unexpectedly, strictly anaerobic operation did not maximize hydrogen evolution. Instead, the addition of small amounts of O2 (≤1 %) significantly enhanced hydrogen production, likely by supporting the growth of the aerobic partner Pseudonomas taiwanensis, which stabilizes the biofilm structure and maintains favorable microoxic conditions. Hydrogen production declined in mature biofilms with high biomass accumulation, consistent with growth-coupled nitrogenase activity. To address this limitation, controlled biofilm dispersion was implemented by temporarily interrupting nutrient supply, inducing biofilm sloughing followed by rapid regrowth. This strategy restored high hydrogen production rates and enabled cyclical production phases while maintaining continuous reactor operation. Electron balance analysis further revealed substantial unaccounted electron fluxes, suggesting significant biofilm-associated maintenance energy demands. These results highlight the importance of biofilm management strategies for improving productivity and process control in biofilm-based production systems.