Expansive microbial metabolic versatility and spatial biodiversity of anammox coupling with denitrification granular sludge under different feeding strategies

\Anammox coupling with denitrification has attracted increasing attention in low-strength wastewater treatment. However, its wide application in continuous-flow process is seriously restricted by insufficient anammox capacity due to the failed biomass retention. The functional capacities of communities inhabiting the fluctuating habitat has been little understood. This study developed a novel granular sludge-based partial denitrification coupling with anammox (PD/A) process with the feeding strategy shifting from sequencing to continuous flow in a Continuous Stirred Tank Reactor (CSTR), treating low-strength wastewater containing ammonia (NH4+-N) of 46.1 mg/L and nitrate (NO3--N) of 60.7 mg/L. Significantly enhanced specific anammox activity was demonstrated, attributed to the robust nitrite (NO2--N) generation with NO3--N to NO2--N transformation ratio (NTR) averaged 84.1 %. Remarkable total nitrogen (TN) removal of 95.4 % was thus achieved with anammox pathway contribution as high as 88.3 %. Interestingly, a dynamic and stratified microbial structure was obtained in CSTR, in which anammox migrated to outer layer together with Thauera capable of partial denitrification (PD) in big granules, resulting in more efficient cross-feeding and strengthening the cooperation between anammox and heterotrophic PD bacteria in CSTR. Metagenomic-based metabolic inferences provide first insight into the versatile microbial metabolic potential of anammox bacteria for reducing NO3--N with acetate as electron donor in PD/A process. They were successfully enriched and retained with the relative abundance of 8.4 %, being attributed to the enhanced mass transfer efficiency. On the contrary, dominant denitrifier Thauera decreased sharply to 4.2 %. This study provides a new alternative of anammox coupling with heterotrophics granular sludge for efficient low-strength wastewater treatment under continuous feeding, and open up new perspectives for selecting distinctive communities that stand out by expansive metabolic versatility and excellent microbial collaboration.