A recent study published in Engineering has shed new light on the mechanisms behind the efficient nitrogen removal capabilities of pyrite-based mixotrophic denitrifying biofilters. The research, conducted by Qi Zhou, Weizhong Wu, and Jianlong Wang, provides detailed insights into the microbial spatial stratification, nitrogen removal pathways, and key metabolites involved in these advanced biofilters.

The study investigates the intrinsic drivers of nitrogen removal in pyrite-based mixotrophic denitrifying biofilters, which have shown significant promise in wastewater treatment. These biofilters leverage a combination of pyrite and solid-phase carbon sources to facilitate the removal of nitrate nitrogen (NO₃⁻-N) and phosphorus, addressing the challenges posed by low carbon-to-nitrogen ratios in secondary effluents from wastewater treatment plants (WWTPs). The research involved the construction and monitoring of four up-flow biofilters over a period of 304 days, revealing detailed spatial variations in pollutant characteristics and microbial community dynamics.

The findings indicate that the biofilters exhibit distinct spatial stratification of microbial communities, with different zones corresponding to specific metabolic processes. The bottom of the biofilters is dominated by heterotrophic denitrifying bacteria, which utilize organic carbon sources for nitrate reduction. The middle-lower sections are enriched with sulfur-based mixotrophic denitrifying bacteria, while the upper parts are characterized by sulfate-reducing bacteria. This spatial distribution aligns with the hot zones for heterotrophic denitrification, autotrophic denitrification, and sulfate reduction, respectively.

A key discovery is the role of dissimilatory sulfate reduction in enhancing the denitrification process. The study confirms that the dissimilatory sulfate reduction process consistently provides biogenic elemental sulfur (S⁰) as a new electron donor, facilitating the denitrification process. X-ray photoelectron spectroscopy (XPS) analysis further verified the accumulation of biogenic S⁰ on the biofilms, highlighting the importance of this metabolic pathway.

Metabolomic analysis revealed that vitamin B₁₂ and tryptophan might be key metabolites involved in the synergistic promotion of autotrophic and heterotrophic denitrification. The study suggests that these metabolites play a crucial role in the metabolic exchanges between different microbial groups, contributing to the overall efficiency of the biofilters.

The research also highlights the coupling of dissimilatory nitrate reduction to ammonia (DNRA) and anammox processes as an auxiliary pathway for nitrogen removal. While the primary nitrogen removal pathway involves S²⁻/S⁰-based autotrophic, fermentation acetic acid production-heterotrophic, and Fe(II)-based autotrophic denitrification, the DNRA and anammox processes provide additional support for systemic nitrogen removal.

The study’s comprehensive evaluation of microbial functions and metabolites in pyrite–sawdust composite-based biofilters underscores the importance of understanding the deeper intrinsic drivers of nitrogen removal. The findings advance the field’s understanding of the complex interactions between microbial communities and their metabolic activities, providing valuable insights for the optimization of pyrite-based mixotrophic denitrifying biofilters in wastewater treatment applications.

Future research is expected to focus on applying these composites to real-world wastewater treatment scenarios, further exploring the parameters and intrinsic mechanisms through metatranscriptomics and metaproteomics. This will help elucidate the activities of important genes and enzymes at the expression level, ultimately revealing their contributions to nitrogen species removal.

The paper “Metagenomics and Metabolomics Reveal Intrinsic Drivers of Pyrite-based Mixotrophic Denitrifying Biofilters: Microbial Spatial Stratification, Nitrogen Removal Pathways, and Key Metabolites,” is authored by Qi Zhou, Weizhong Wu, Jianlong Wang. Full text of the open access paper: https://doi.org/10.1016/j.eng.2025.05.006. For more information about Engineering, visit the website at https://www.sciencedirect.com/journal/engineering.