Insights of light spectra on biohydrogen production by photo-fermentation

Biohydrogen production by purple non-sulfur bacteria typically relies on infrared light to excite bacteriochlorophyll a. However, the roles of visible wavelengths, especially red and blue, and the contribution of carotenoids remain poorly defined. In the current study, it was performed the first unified comparison of four LED spectra in a co-culture of Rhodobacter capsulatus and Rhodospirillum rubrum using lactose from milk whey permeate. Small‐scale tests (50 mL) under Cool White, Infrared, and Targeted-Spectrum LEDs revealed that Cool White LEDs achieved the highest H₂ productivity (8.23 mmol H₂ (L.day)). Remarkably, the Targeted Spectrum LED alone yielded significant H₂ (5.76 ± 0.14 mol H₂/mol lactose), demonstrating that pronounced carotenoid excitation can drive production with minimal emission for BChl ɑ excitation. The visible wavelengths can scatter more uniformly, benefiting non‐stirred systems, while Infrared alone underperforms when cells settle. Scaling to a 2.1 L stirred flat‐plate reactor increased productivity to 18.64 mmol H₂ (L.day) (3.26 % LCE), applying the Cool white LED as light source. Raising light intensity to 5,000 lx and renewing half the medium further boosted productivity to 26.93 mmol H₂ (L.day) (4.49 % LCE) while maintaining an optimal SOLR (∼4.8 g lactose/ g_VS.h). Finally, bi‐logistic and modified Gompertz models accurately described multiphasic H₂ accumulation, offering a robust framework for optimizing complex photofermentative systems. This work highlights the need to tailor light spectra, reactor design, and process control to maximize biohydrogen yields.