Microbial pre-colonization as a carrier modification strategy to enhance anammox granulation: Performance and...

The slow initial adhesion of autotrophic anammox bacteria to inert nuclei hinders flocculants granulation. Herein, a novel carrier modification strategy using microbial pre-colonization was proposed to promote anammox flocs granulation, where anaerobic heterotrophic bacteria were pre-colonized onto biochar, transforming it from a passive carrier into an active biofilm-primed nucleus. After 60 days of operation, the reactor supplemented with microbially pre-colonized biochar (R_MB) yielded the highest proportion of anammox granules with the biggest average size of 487 ± 12 μm, surpassing reactors with original biochar (R_B) and no biochar (R_C) by 67.3% and 62.3%, respectively. Enhanced granulation was attributed to increased specific surface area (126.5 ± 1.0 m²/g), polarity, and hydrophobicity of microbially pre-colonized biochar, which facilitated anammox bacterial adhesion and reduced extracellular polymeric substance (EPS) requirements, redirecting metabolic resources toward microbial growth and granulation. Consequently, R_MB exhibited the highest specific anammox activity (175.5 ± 13.4 mg N/g VSS/d) and anammox bacterial abundance (1.6 ± 0.3%), and the most stable nitrogen removal performance (87.2 ± 1.8%) among the three reactors. BugBase analysis further confirmed enriched biofilm-forming genes in R_MB, aligning with its robust granulation performance. Overall, this work establishes microbial pre-colonization as a viable carrier modification strategy for enhancing anammox granulation and system stability.