Formation mechanism and metabolic pathways of photogranule in batch and continuous-flow mode under high salinity stress

• Photogranules with excellent properties were obtained under high salinity stress. • Enriched SMP polysaccharides facilitated cyanobacteria gliding motility. • Aromatic protein-like SMP steered biomass to photogranules in continuous-flow mode. • Aromatic protein-like and humic acid-like EPS promoted photogranule formation. • Metabolism and genetic information processing ensured excellent photogranulation. Photogranules have considerable advantages for treating high saline wastewater economically and efficiently. However, photogranulation mechanism needs to be further investigated. In this study, a continuous-flow photogranular bioreactor (R1) and sequencing batch photogranular bioreactor (R2) were setup for exploring the formation mechanism of photogranules. Results showed that R1 achieved satisfying pollutant removal efficiencies with TOC of 85.36 ± 2.84%, NH 4 + of 93.30 ± 2.07% and PO 4 3- of 77.68 ± 5.81%. Microscopy and 16S/18S rRNA sequencing indicated that the dominate bacteria showed discrepancy with Phormidium sp. in R1 and Leptolyngbya sp . in R2, and a specific network structure was formed on photogranules in R1. Additionally, more abundant polysaccharides in soluble microbial products (SMP) were observed in R1, which facilitated cyanobacteria gliding motility, thereby accelerating the photogranulation. Meanwhile, three dimensional excitation-emission matrix (3D-EEM) analysis implied that the pivotal SMP components driving photogranulation were different, with aromatic protein-like substances accumulating in R1 and humic acid-like substances and soluble microbial by-product-like substances in R2. In contrast, R1 exhibited lower abundance of extracellular polymeric substances (EPS) but higher proteins to polysaccharides ratio (PN/PS) than those in R2. High PN/PS showed advantages for accelerating photogranule formation with a compact structure. Furthermore, more enriched aromatic protein-like and humic acid-like EPS were identified in R2 compared with R1, suggesting they were more significant contributors to granulation in R2. Finally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed that metabolism and genetic information processing were the most abundant metabolic pathways, which were responsible for excellent pollutants removal and stable granulation. Therefore, this study demonstrates the impacts of SMP and EPS on photogranulation and reveals the biological mechanism that drives granular structure and function.