Achieving nitrate removal without sulfide accumulation in sulfate-rich mariculture wastewater: Operational performance, kinetics, and microbial ecology of a PHBV-driven biofilm process

Coastal pollution associated with mariculture nitrogen discharge necessitates efficient denitrification processes. However, the mariculture wastewater treatment is challenged by its high sulfate content, which poses a risk of toxic sulfide production during conventional heterotrophic denitrification. This study conducted a systematic investigation into a poly-3-hydroxybutyrate-co-hydroxyvalerate (PHBV)-driven heterotrophic denitrification (PHD) biofilm process treating mariculture wastewater over 195 days. The PHD bioreactor achieved high nitrate removal efficiencies (53.5%–99.4%) and rates (110.0–630.5 mg L⁻¹ d⁻¹), with minor accumulation of nitrite (< 5.5%) and ammonia (< 3.7%). However, phosphorus removal was limited (4.0%–20.0%). Bioreactor performance was significantly influenced by hydraulic retention time, influent nitrate concentration, and temperature, but was resilient to variations in influent dissolved oxygen levels. Kinetic analysis indicates a shift in the optimal kinetic model for nitrate removal from half-order to zero-order as the influent nitrate concentration increased, reflecting an alteration in the rate-limiting step. Metagenomic sequencing revealed a diverse microbial community dominated by Proteobacteria (68.78%), with key genera including Sedimenticola (10.02%), Marinobacter (6.79%), Shimia (3.70%), Azoarcus (2.13%), and Thauera (2.10%). Functional gene analysis confirmed the metabolic pathways for PHBV degradation and denitrification as the primary nitrate removal route. Crucially, despite the high sulfate load, no significant sulfide accumulation was observed in the effluent, which could be attributed to the co-existence of sulfur-oxidizing and reducing bacteria in the biofilm. This study demonstrates that the PHD biofilm process is a robust strategy for effective nitrate removal in sulfate-rich mariculture wastewater without causing sulfide-related secondary pollution. • PHD biofilm efficiently removed the NO 3 – -N in mariculture wastewater (up to 99.4%). • No significant sulfide accumulation occurred despite high influent sulfate level. • NO 3 – -N removal kinetics shifted from half- to zero-order with rising NO 3 – -N levels. • A diverse community and functional genes drove coupled C, N, and S metabolisms. • Coexisting sulfur oxidizing and reducing bacteria balanced sulfur redox processes.