Effect of Nitrosomonas europaea on Chlorella vulgaris in bio-hydrogels for startup of microalgal-bacterial granular sludge: Performance and microscopic mechanism.

Algal-bacterial granular sludge technology holds significant promise for treating municipal wastewater with carbon emission reduction. However, its practical application has been constrained by the long startup period. In this study, bio-hydrogels co-encapsulating autotrophic ammonia-oxidizing bacteria (Nitrosomonas europaea) and photomixotrophic microalgae (Chlorella vulgaris) were used in a microalgal-bacterial granules system (MBGS) for the treatment of low C/N wastewater. The operating conditions of the MBGS system included inoculation volume ratio of 1:6 (bio-hydrogel granules: wastewater), initial biomass of 0.33 g suspended solids (SS) /L, light intensity of 300 μmol/m2·s, and no aeration. The results showed that this strategy reduced the typical MBGS startup period to just 6 days, with biomass accumulation (1.53 g SS/L, >4 g SS/L after running 120 days) and efficient chemical oxygen demand (COD) and total nitrogen (TN) removal (effluent COD <20.00 mg/L and TN <2.50 mg/L). Transcriptomic analysis revealed that the symbiosis with Nitrosomonas significantly upregulated the expression of the PetH gene, which encodes a reductase (EC:1.18.1.2) in the photosynthetic system of Chlorella (log₂ fold change=13.63), thereby enhancing the supply of NADPH required for the Calvin cycle. Concurrently, the expression of the rbcL gene, encoding the large subunit of Rubisco in Chlorella, was upregulated by 1.4-fold, which promoted photosynthetic carbon fixation. The symbiosis also suppressed heterotrophic metabolism in Chlorella, as evidenced by downregulation of 72.5 % of genes in glycolysis and the tricarboxylic acid cycle, thereby mitigating the adverse effects of encapsulating material biodegradation on the bio-hydrogel structure. In addition, cell proliferation of Chlorella was stimulated (over 13-fold upregulation of DNA replication licensing factors MCM5 and MCM6), which increased biomass and nutrient removal efficiency. Furthermore, a higher light intensity of 400 μmol/m²/s promoted a significant increase in extracellular polymeric substances (EPS) content (106.76 mg/g SS), improved granule stability (integrity coefficient < 20 %), and enhanced biomass production (4.68 g SS/L after 120 days of operation). These findings highlight a promising strategy for sustainable wastewater treatment.