Bergaust, L. | Search bioRxiv

bioRxiv2025

Distinct denitrification phenotypes in closely related bacteria: clues to understanding variations in nitrite accumulation among Stutzerimonas strains

Nitrite (NO2-) is a key denitrification intermediate, formed from nitrate (NO3-). Transient NO2- accumulation varies among denitrifiers, yet the underlying causes remain poorly understood, despite its potential toxicity and role in NO and N2O emissions. We profiled eighteen related Stutzerimonas strains, including the model S. perfectomarina ZoBell, and identified three phenotypic clusters (full, partial and low nitrite accumulators; FNA, PNA and LNA) based on the fraction of NO3--N transiently accumulated as NO2-. LNA strains lack or express the membrane-bound nitrate reductase (NarG) late, relying on periplasmic NapA for NO3- reduction, possibly explaining their balanced NO2- production/reduction. FNA and PNA strains possess NapA and NarG but differ in their nitrite reductase (NirS) clades. Delayed nirS transcription as FNA strains transition to NO3- respiration likely accounts for some NO2- accumulation. However, addition of NO3- halted NO2- reduction in FNA strains, suggesting additional metabolic control. This may require the cytochromes NirTB, which are only found in FNA strains. The regulator DnrE was also unique to NO2--accumulators, likely having a role in finetuning NO2- regulation. Our findings reveal diverse NO2--handling phenotypes among denitrifiers and provide insights for optimizing wastewater nitrogen removal and soil bioaugmentation strategies to mitigate N2O emissions.

microbiology↗

bioRxiv2024

Microbial consortia driving lignocellulose transformation in agricultural woodchip bioreactors

Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen-gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well-described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors, and their active enzymes. Our findings highlight a microbial community enriched for lignocellulose-degrading denitrifiers with key players from Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota). A wide substrate specificity is observed among the many expressed carbohydrate active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, even including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions. ImportanceFreshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota) playing significant roles in lignocellulose transformation and showcases a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improving water quality, protecting aquatic ecosystems, and reducing greenhouse gas emissions from WBRs.

microbiology↗