Biophotolysis vs. Anaerobic Digestion—An Experimental Comparison of Two Pathways for Biohydrogen Production by Tetraselmis subcordiformis

Biohydrogen is considered to be one of the fuels of the future, so there is a justified need to find efficient and cost-effective technologies for its production. This study evaluated the efficiency of two biohydrogen production pathways, specifically biophotolysis and dark fermentation, using Tetraselmis subcordiformis biomass. Microalgae production was performed in three variants, where the separation criterion was the type of culture medium: a control sample (synthetic medium; V1–PCR), agricultural wastewater from hydroponic tomato cultivation (V2–SL-WW), and effluent from a microbial fuel cell (V3–MFC-WW). The highest increase in biomass of T. subcordiformis was obtained in V2–SL-WW—2730 ± 212 mg VS/L, which was also associated with the maximum chlorophyll a content (65.0 ± 5.1 mg Chl-a/L). In biophotolysis, the highest specific hydrogen yields were obtained in V1–PCR (55.3 ± 4.3 mL/g VS) and V2 (54.3 ± 3.7 mL/g VS). The total hydrogen production in these variants was 166 ± 13 mL (V1–PCR) and 163 ± 11 mL (V2–SL-WW), respectively. The average H2 production rate reached 4.70 ± 0.33 mL/h in V2–SL-WW, and the rate constant (k) was 0.030–0.031 h−1. In anaerobic fermentation, the highest total and specific H2 production was obtained in V1–PCR, 453 ± 31 mL and 45.3 ± 3.1 mL/g VS, respectively. The qualitative composition of the biogas confirmed a high hydrogen content: 61.4% (biophotolysis, V1) and 41.1% (dark fermentation, V2–SL-WW). The results obtained confirm that T. subcordiformis can be effectively cultivated on waste media and that the biohydrogen production maintains a high technological efficiency through both photolytic and fermentative mechanisms. The medium from hydroponic tomato cultivation (V2–SL-WW) proved to be particularly promising, as it combines high biomass productivity with a satisfactory biohydrogen production profile.