New insights into microalgal photobiological hydrogen production: Potential role of aggregation forms in modulating the hydrogenase activity and metabolic properties of microalgae during hydrogen production

Photobiological hydrogen production of microalgae is highly influenced by environmental conditions, and the appropriate microalgal aggregation form is conducive to microalgae overcome environmental limitations and increase their hydrogen production capacity. In this study, the effects of three aggregation methods including self-flocculation, biofilm attachment, and gel immobilization on the photobiological hydrogen production of both prokaryotic Synechocystis sp. and eukaryotic Chlamydomonas reinhardtii were investigated. The results indicated that microalgal aggregates formed via gel immobilization exhibited superior physicochemical properties such as porosity, integrity, and oxygen diffusion coefficients, creating favorable conditions for enhanced hydrogenase activity. Upon gel-immobilization, hydrogen production of Synechocystis sp. and C. reinhardtii exhibited an increase of 1.89 and 2.02-folds, respectively, compared with suspended microalgae. Concurrently, hydrogenase activities increased to 2.07 and 32.9 H₂/(mg chlorophyll·h), respectively. The gel-immobilized aggregate form prompted the microalgae to maintain a relatively high photosynthetic activity, with the electron transfer rate reaching more than 1.58 folds of the control. Notably, three aggregated forms also accelerated oxygen consumption, and intracellular organic matter degradation, thereby optimizing electron utilization by microalgal hydrogenase. Furthermore, aggregation up-regulated hydrogenase gene expression in both species, particularly in gel-immobilized groups (2.34- and 4.14-folds increase compared with the control for Synechocystis sp. and C. reinhardtii, respectively). Subsequently, significantly upregulated gene expression related to oxidative phosphorylation and antioxidant systems was observed in C. reinhardtii aggregates, correlating with its relatively higher increase in hydrogen production compared with Synechocystis sp. aggregates. This study elucidated the mechanism by which aggregation strengthens microalgal hydrogen production and offers insights crucial for its industrialization.