Dynamic characteristics of attachment genes and their role in aerobic granular sludge development

Aerobic granular sludge (AGS) is a microbial aggregate with a biofilm structure. Understanding microbial attachment at the genetic level is crucial for elucidating the potential mechanisms underlying AGS biofilm formation. This study applied RT‒qPCR to assess the correlation between key attachment genes (rmlA, rpfF, and fliD) and granulation. Comparative analysis of different sludge structures revealed that the abundances of attachment genes were 1–2 orders of magnitude greater in mature AGS than in floccular sludge. This indicated that the degree of microbial aggregation increased in parallel with the abundance of these attachment genes. Furthermore, the presence and dynamic characteristics of three key attachment genes showed significant positive correlations with granulation, particularly granule size (rmlA: r = 0.91, p < 0.001; rpfF: r = 0.88, p < 0.01, fliD: r = 0.97, p < 0.001). Dynamic quantification of attachment gene expression during AGS cultivation revealed stage-specific dominance: rpfF facilitated early-stage aggregation via quorum sensing, whereas fliD exhibited a steep increase (from 1.6 × 10³ to 2.56 × 10⁵ copies·(g·SS)⁻¹) during AGS stabilization, surpassing rmlA (1.47 × 10⁴ copies·(g·SS)⁻¹ and rpfF (1.12 × 10⁵ copies·(g·SS)⁻¹) during maturation. Bioaugmentation with Stenotrophomonas AGS-1 increased attachment gene amplification by 2–3 log units while competitively excluding low-abundance microbial groups and selectively enriching functionally dominant consortia. High-attachment bacteria may mediate AGS formation via attachment gene expression. This study revealed aerobic granulation mechanisms as a gene-driven bacterial aggregation process for the first time, highlighting the role of attachment genes in AGS development and providing guidance on biofilm regulation in AGS technology.