Artificial reef age reshapes benthic microbial communities and modulates the genetic potential for nitrogen and...

Artificial reefs (ARs) are widely used to restore coastal ecosystems; however, the impact of reef age on microbial communities and their biogeochemical functions remains unknown. This study integrated metagenomic sequencing with physicochemical analysis to examine successional changes in benthic nitrogen and sulfur cycling along a chronosequence spanning from non-artificial reefs (0 years) to 14-year-old ARs in the coastal waters of the Bohai Sea, China. Our analysis revealed a systematic, time-dependent reorganization of the benthic microbiome, characterized by significant enrichment of ammonia-oxidizing archaea (Nitrososphaerota) and bacteria (Nitrospirota) in reefs older than 6 years. Conversely, taxa involved in coupled nitrate reduction and sulfur oxidation (Sulfurovum) declined significantly. Functionally, this led to a shift in genetic potential: the abundance of nitrification genes (amoB and amoC) increased, while genes associated with dissimilatory nitrate reduction (nirB and nrfA), denitrification (nosZ and napB), thiosulfate reduction (phsC and ttrB), and sulfur oxidation (sqr and sox) decreased. Genome-resolved analysis further demonstrated that these functional shifts were driven by the proliferation of nitrifiers and concurrent decline of versatile bacterial lineages. Importantly, this genomic shift was corroborated by geochemical observations of decreased ammonium and increased nitrate concentrations in both bottom seawater and sediments of ARs compared to non-artificial reefs. These results indicate that reef age reshapes benthic microbial communities and functions, favoring aerobic nitrification over anaerobic or microaerophilic nitrate reduction and sulfur metabolism. This study provides a scientific basis for AR adaptive management, underscoring the necessity of integrating microbial functional metrics into the long-term impact assessment of marine infrastructures.