@article { WOS:001695226100001,
	title = {Metagenomics and Metabolomics Reveal Intrinsic Drivers of Pyrite-Based Mixotrophic Denitrifying Biofilters: Microbial Spatial Stratification, Nitrogen Removal Pathways, and Key Metabolites},
	journal = {ENGINEERING},
	volume = {57},
	year = {2026},
	month = {FEB},
	pages = {236-249},
	publisher = {ELSEVIER},
	type = {Article},
	address = {RADARWEG 29, 1043 NX AMSTERDAM, NETHERLANDS},
	abstract = {Microbial functions and metabolism are intrinsic drivers of pollutant removal in mixotrophic denitrification systems. Four pyrite-based mixotrophic denitrifying biofilters were constructed and monitored for 304 days. Variations in pollutant characteristics indicated that the hot zones of heterotrophic denitrification, autotrophic denitrification, and sulfate reduction were located in the bottom, middle-lower, and upper parts of biofilters, respectively. These hot zones corresponded to preferential enrichment of heterotrophic denitrifying, S-based mixotrophic denitrifying, and sulfate-reducing bacteria, respectively, highlighting microbial spatial stratification. Differential functional gene analysis for S reduction revealed that only a dissimilated sulfate reduction process could consistently provide biogenic S0 as a new electron donor via the flavocytochrome c sulfide dehydrogenase (Fcc) enzyme and extracellular polymeric substance protection systems, enhancing the denitrification process. X-ray photoelectron spectroscopy confirmed the accumulation of biogenic S0 . Untargeted metabolomic analysis suggested that vitamin B12 and tryptophan might be the key metabolites for realizing synergistic promotion of autotrophic and heterotrophic denitrification. The microbe-metabolite network indicated that dominant bacteria (e.g., Thiothrix and unclassified\_f\_Rhodocyclaceae) were specialists with less cross-feeding metabolism, while rare species (e.g., Thiobacillus and Desulfobacter) were generalists with complex cross-feeding metabolism in the constructed mixotrophic denitrification systems. The electron transfer pattern indicated that most of the electrons released from S, C, and Fe oxidation were utilized in denitrification processes as the dominant nitrogen removal pathway, including S2- /S0-based autotrophic, fermentation acetic acid production-heterotrophic, and Fe(II)-based autotrophic denitrification. Some electrons were utilized for coupling dissimilatory nitrate reduction to ammonia (DNRA) and anammox processes as an auxiliary pathway for systemic nitrogen removal. The findings of this study advance our understanding of the deeper intrinsic drivers of nitrogen removal by pyrite-based mixotrophic denitrifying biofilters, facilitating their optimization. (c) 2025 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).},
	keywords = {Electron transfer, mixotrophic denitrification, Pyrite, Sulfate reduction, Vitamin B 12},
	issn = {2095-8099},
	doi = {10.1016/j.eng.2025.05.006},
	author = {Qi Zhou and Wu, Weizhong and Wang, Jianlong}
}
