Advanced isolation strategies for mimicking the actual environment to reveal key biofilm-forming bacteria in an actual sewage-treating membrane bioreactor

Membrane fouling is the most critical challenge limiting the long-term performance of membrane bioreactors (MBRs) in wastewater treatment. This study aimed to isolate and characterize key biofilm-forming bacteria (BFB) responsible for membrane fouling by mimicking the low-nutrient conditions of actual sewage environments. We employed a combination of conventional plate culture (using diluted nutrient media) and an in situ iChip cultivation device to capture both culturable and previously unculturable bacterial strains from a fouled MBR membrane treating municipal sewage. Over 400 bacterial strains were isolated and classified into 15 genera, including dominant biofilm producers such as Pseudomonas, Flavobacterium, and members of the Enterobacteriaceae. Biofilm formation assays revealed that strains cultured under low-nutrient conditions or in activated sludge supernatant exhibited significantly enhanced biofilm production compared to those grown in standard rich media. Crossflow filtration tests confirmed that some strains produced soluble extracellular polymeric substances (EPS) that substantially increased membrane resistance. Notably, strain IK01 demonstrated unique biofilm formation under low-nutrient conditions, accompanied by upregulated expression of osmotic stress response ( envZ , ompR ) and EPS biosynthesis-related ( exoP , wza , gmd , fcl , and wcaI ) genes. These results highlight the utility of culturing strategies mimicking the actual conditions in identifying overlooked BFB and provide biological insights into membrane fouling. • The low-nutrient media that better replicate environmental conditions are effective in isolating even unculture bacteria. • Superior biofilm production is exhibited in low-nutrient or activated sludge supernatant conditions. • Soluble extracellular polymeric substances (EPSs) contribute to high filtration resistance. • The unique biofilm formation of strain IK01 is induced by low-nutrient conditions.