Human activities have increased reactive nitrogen input into the biosphere, leading to escalated emissions of N₂O (a potent greenhouse gas), NO, and HONO. Agriculture, particularly excessive use of synthetic fertilizers, is a major contributor to N₂O release through denitrification and nitrification. While CO₂ emissions are predicted to decrease, anthropogenic N₂O emissions are expected to continue increasing unless mitigation strategies are developed. Improved understanding of the organisms involved in N₂O production and reduction, and the environmental factors controlling these emissions, is crucial for developing such strategies. Denitrification is the stepwise microbial reduction of nitrate (NO₃⁻) to N₂ via nitrite (NO₂⁻), NO, and N₂O. Each step is catalyzed by a specific reductase (NarG/NapA, Nirk/NirS, cNor/qNor, NosZ). The denitrification pathway is modular, and the absence of one or more steps is common. Gene regulation, interacting with environmental factors, plays a key role in determining the N₂O/N₂ product ratio. Soil pH is a well-characterized environmental factor that profoundly affects the accumulation of denitrification intermediates. While NO₂⁻ accumulation increases with soil pH, there is a negative correlation between pH and N₂O emissions. Previous studies suggested that this negative correlation might be due to a post-transcriptional phenomenon affecting NosZ maturation at low pH, but this has not been extensively explored across diverse soil communities. The general occurrence of low NO₂⁻ concentrations in acidic soils has often been attributed to abiotic degradation, but recent research suggests that microbial reduction may play a significant role. This study aimed to investigate the effects of soil pH on denitrifier community composition and activity, and the accumulation/release of denitrification intermediates. The researchers employed an integrated multi-omics approach (metagenomics and metatranscriptomics) to analyze two soils with significantly different pH values (3.8 and 6.8), tracking denitrification intermediate and end-product dynamics during anoxic incubation.

The introduction thoroughly reviews existing literature on the impacts of anthropogenic nitrogen on the environment, focusing on N₂O emissions from denitrification and nitrification. The role of soil pH in controlling denitrification and the accumulation of intermediates is discussed, highlighting the contrasting findings regarding the relationship between pH and N₂O emissions. Previous studies on the effects of pH on the regulation and enzymology of denitrification steps and the potential post-transcriptional impact on NosZ maturation are summarized. The debate on the relative importance of abiotic vs. biological factors in controlling NO₂⁻ concentrations in acidic soils is also presented, setting the stage for the current study's investigation into these complex interactions using an integrated, multi-omics approach.

Two peat soils with significantly different pH values (SoilA, pH 3.8; SoilN, pH 6.8) were sampled from a long-term liming experiment. Clover was added to both soils before anoxic incubation at 15°C to ensure detectable transcription. Nitrate was added to the soils, and the vials were made anoxic. Gas (CO₂, O₂, NO, N₂O, N₂) and NO₂⁻ concentrations were measured at intervals during a 45-hour incubation. Samples for metagenomic (MG) and metatranscriptomic (MT) analyses were taken at specific time points. DNA and RNA were extracted from frozen soil samples using an optimized protocol. MG and MT sequencing were performed using HiSeq 2500 technology, while 16S rRNA gene sequencing for community composition analysis used MiSeq technology. Amplicon sequence analysis of 16S rRNA genes was performed using Greenfield Hybrid Analysis Pipeline (GHAP), which combines amplicon clustering and classification tools. MG and MT sequences were quality-controlled and functionally annotated using DIAMOND. Reads from nitrogen metabolism genes were compared against a custom dataset. Statistical analyses and graphing were done using in-house R scripts. Quantitative amplification-based analysis (qPCR) of 16S rRNA and denitrification genes (*nirK*, *nirS*, *nosZ* clade I) was also performed.

The denitrification kinetics showed striking differences between the two soils. SoilA (acidic) accumulated N₂O with minimal N₂ production initially, while SoilN (neutral) produced both N₂O and N₂ from the start. SoilA had severely delayed N₂O reduction despite early *nosZ* transcription, suggesting post-transcriptional limitations on NosZ maturation at low pH. This effect seemed to be common across diverse denitrifiers. Nitrite accumulation was high in SoilN and low in SoilA; the low levels in SoilA were attributed primarily to biological reduction, not abiotic decomposition. Metagenomic analysis revealed that *nirk* genes were much more abundant than *nirs* in both soils, contradicting results from standard PCR-based quantifications that showed the opposite. This highlighted significant primer bias in PCR-based studies. The acidic soil showed high *qnor* expression, indicating its importance in controlling NO levels. HONO production was estimated to be ten times higher in SoilA than SoilN, despite higher total nitrite-N in SoilN, suggesting other factors impact HONO emission. Analysis of accessory *nos* genes showed that the lack of N₂O reduction in SoilA wasn't due to defects in transcriptional control mechanisms. The microbial community composition differed between the two soils, but remained stable during incubation in SoilN. Taxonomic analysis of denitrification genes and transcripts showed that Proteobacteria, Actinobacteria, and Bacteroidetes were dominant in both soils, but the specific taxonomic distribution varied among individual genes and transcripts. The qPCR analysis consistently underestimated the abundance of *nirk* and *nosZ* clade I genes and transcripts compared to metagenomic and metatranscriptomic results.

The findings show that soil pH significantly impacts denitrification, causing a pH-dependent delay in N₂O reduction in acidic soil due to post-transcriptional limitations on NosZ activity. The study refutes the idea that abiotic decomposition is the primary cause of low nitrite concentrations in acidic soils. It also highlights that the abundance of denitrification genes and transcripts are poor predictors of metabolic activities, challenging the reliance on PCR-based quantification for studying these processes. The dominance of *qnor* in acidic soil suggests a functional adaptation to low pH, possibly due to its electrogenic nature and ease of horizontal gene transfer. The unexpected abundance of DNRA-related genes despite the lack of DNRA activity merits further investigation. The higher HONO emission from acidic soil, despite lower total nitrite-N, warrants more research to understand its controlling factors. The significant discrepancies between qPCR and multi-omics based quantifications illustrate the need to use more comprehensive techniques in soil microbial ecology studies.

This study significantly advances our understanding of soil denitrification by integrating meta-omics with detailed kinetic analysis. The pH-dependent limitations on N₂O reduction, the importance of biological nitrite reduction in acidic soils, and the dominance of *qnor* in controlling NO highlight the complexity of denitrification processes. The substantial primer bias evident in standard PCR approaches underscores the need for more comprehensive methods. Future research should focus on further elucidating the mechanisms behind post-transcriptional regulation of NosZ at low pH, the role of DNRA in these soils, and the environmental conditions affecting HONO emissions.

The study focused on two soils with distinct pH values; further studies with broader pH ranges and soil types are needed to generalize the findings. The metatranscriptomic analysis in the acidic soil was limited to the first 3 hours of incubation, potentially missing later transcriptional changes. While the study examined several accessory *nos* genes, incomplete datasets may limit the interpretation of their role in NosZ function. The limited taxonomic resolution in some parts of the analysis could affect the precision of interpretations.