Achievement of prosperous nitrification cycle fosters diversity in freshwater microbiome within recirculating aquaculture systems after adjustment period

Abstract

In recirculating aquaculture systems (RAS), a significant challenge involves managing the biological cycling of nitrogenous waste. As a result, there is worldwide focus on the diversity of biological groups involved in nitrogen cycling and their impacts. However, there is still a lack of information concerning the microbial diversity, particularly in relation to biological nitrogen cycling. We hypothesized that biological nitrification in RAS becomes fully established only after a distinct microbial adjustment period following microbial inoculant (MI) treatment, and that this phase features distinct shifts in microbiome composition and nitrogen compound dynamics. To test this, we monitored water chemistry and microbial community structure over an 8-week pre-treatment maturation phase in freshwater RAS for Japanese eel (Anguilla japonica) cultivation. Using 16S rRNA gene amplicon sequencing and qPCR targeting ammonia- and nitrite-oxidizing bacteria, we found that nitrifier abundance increased sharply from week 3 post-treatment. Random forest analysis and structural equation modeling revealed that nitrate levels—serving as a proxy for complete nitrification—had the strongest direct influence on microbiome diversity. Diversity indices and beta-dispersion analyses confirmed that microbial communities during the adjustment period (weeks 0–3) were significantly different from those in the stabilization phase (weeks 4–8). These findings demonstrate the existence of a critical, temporally defined adjustment phase in RAS maturation, during which microbiome structure and nitrification processes co-develop. Our study provides mechanistic insights into microbiome assembly in RAS and highlights the importance of temporal monitoring in optimizing MI-based water treatment strategies.