Eukaryotic algae represent a highly heterogeneous group in terms of organization, lifestyle, and metabolic capabilities. Unicellular green microalgae are capable of biohydrogen production through direct and indirect photolysis as well as dark fermentation. Most algae hydrogen studies focus on axenic algal cultures, although these are difficult and expensive to maintain for continuous operation. Moreover, the complex interplays and metabolic fluxes between algae and bacteria in natural ecosystems provide a number of clear biological and technological benefits to large-scale functional algae-based systems. Two green algae species from the Chlamydomonas and Chlorella genera were used to engineer stable synthetic communities by incorporating a starch-degrading bacterium from the Bacillus genus into the inter-kingdom consortium. Continuous photoheterotrophic biohydrogen production was achieved by elaborating an appropriate algal–bacterial ratio and fine-tuning the culture conditions for the synthetic consortia. Medium with starch as only carbon source served as a simple model of cheap substrate for algal hydrogen generation. The engineered pairwise algal–bacterial associations showed increased biomass and biohydrogen yield compared to the axenic control conditions. Chlorella sp. MACC-360 produced a significantly higher amount of hydrogen when both the bacterium partner and starch were added to the media compared to the axenic algae. Continuous, elevated algal hydrogen production was achieved in media supplemented with 8 g L⁻¹ starch as sole carbon source when carefully selected initial cell number values were used for the Chlorella sp. MACC-360–B. amlyloliquefaciens co-cultures.

Biohydrogen production from wastewater using eukaryotic green algae can be facilitated by appropriately selected bacterial partners and cultivation conditions. Two Chlorella algal species were chosen for these experiments, based on their robust growth ability in synthetic wastewater. The applied three Bacillus bacterial partners showed active respiration and efficient biomass production in the same synthetic wastewater. Bacillus amyloliquefaciens, Bacillus mycoides, and Bacillus cereus as bacterial partners were shown to specifically promote algal biomass yield. Various inter-kingdom co-culture combinations were investigated for algal–bacterial biomass generation, for co-culture-specific exopolysaccharide patterns, and, primarily, for algal biohydrogen evolution. Chlorella sp. MACC-38 mono- and co-cultures generated significantly higher biomass compared with that of Chlorella sp. MACC-360 mono- and co-cultures, while in terms of hydrogen production, Chlorella sp. MACC-360 co-cultures clearly surpassed their Chlorella sp. MACC-38 counterparts. Imaging studies revealed tight physical interactions between the algal and bacterial partners and revealed the formation of co-culture-specific exopolysaccharides. Efficient bacterial respiration was in clear correlation with algal hydrogen production. Stable and sustainable algal hydrogen production was observed in synthetic wastewater for Chlorella sp. MACC-360 green algae in co-cultures with either Bacillus amyloliquefaciens or Bacillus cereus. The highest algal hydrogen yields (30 mL H₂ L⁻¹ d⁻¹) were obtained when Chlorella sp. MACC-360 was co-cultured with Bacillus amyloliquefaciens. Further co-culture-specific algal biomolecules such as co-cultivation-specific exopolysaccharides increase the valorization potential of algal–bacterial co-cultures and might contribute to the feasibility of algal biohydrogen production technologies.