From substrate composition to microbial metabolism: performance, molecular, taxonomic and functional insights into sludge-derived hydrothermal carbon sources for denitrification

Liquid recovered from hydrothermal sludge treatment is an attractive denitrification carbon source, but it carries nitrogen that raises effluent total nitrogen and recalcitrant organics that leave residual chemical oxygen demand (COD). Thermal hydrolysis (TH), wet air oxidation (WAO) and two-stage WAO with catalytic WAO (WAO-CWAO) liquids were compared with sodium acetate in sequencing batch reactors, and the mechanisms were resolved by Fourier transform ion cyclotron resonance mass spectrometry, 16S rRNA gene sequencing and metagenomics. TH and WAO liquids failed as carbon sources, with effluent COD and nitrogen above the Chinese discharge standard, whereas the WAO-CWAO liquid converted the recalcitrant organics into volatile fatty acids (VFAs), removed ammonia, and approached, but did not match, the sodium acetate control. An apparent nitrogen balance showed that residual nitrogen reflected the load introduced with the carbon source, and residual COD was traced to a CHON-rich pool unique to these effluents. Community composition followed carbon source quality, shifting from a diverse consortium sustained by slowly biodegradable organics to a streamlined assemblage of VFAs-utilizing denitrifiers. Genome-resolved analysis of the recovered metagenome-assembled genomes indicated a substrate-based division of labor: the dominant Saprospiraceae carried an expanded hydrolytic arsenal but no nitrate reductase, whereas Thauera carried complete denitrification and VFA-utilization gene sets but no polysaccharide hydrolases. A second, catalytic stage at higher temperature and oxygen pressure therefore alleviates the trade-off between nitrogen release and incomplete oxidation, yielding a sludge-derived carbon source that approaches sodium acetate.