Exploring the Danish nitrifiers in MicroFlora Danica, Denmark’s microbial atlas

Flora Danica, an atlas of Denmark’s native plants compiled over 122 years, contains more than 3,000 botanical engravings. Initially inspired by this work, we introduce Microflora Danica, the first microbial atlas of Denmark, built from 10,689 samples spanning Denmark’s diverse habitats and dominant land uses. From these, we generated 10,683 short-read metagenomes, near full-length 16S rRNA gene datasets from 412 samples, 16S/18S rRNA operon sequences from 450 samples, and assembled 19,253 microbial genomes (MAGs). Agriculture covers 63% of Denmark, and while disturbed soils (e.g. farmland, roadsides, and urban parks) showed high within-sample richness (alpha diversity), their overall habitat diversity (gamma diversity) was lower than in natural areas, reflecting species homogenisation in managed landscapes. As agriculture depends on nitrogen cycling, we focused on the nitrifiers, characterising their distribution and diversity. We built and refined new HMM search models for the key nitrifier marker genes amoA and nxrA, adding sequences from MAGs we recovered, and improving resolution to separate canonical nitrifiers from homologous enzymes and putative new nitrifier candidates. MAG-based analyses, using single copy marker gene searches (SingleM) and genome quantification (sylph), confirmed and extended our gene-based findings. Linking to high-quality MAGs from long-read sequencing further allowed metabolic and functional inference through the analysis of gene synteny. These analyses uncovered novel taxa, including a widespread potential new nitrite oxidizer previously misclassified as Nitrobacter, and revealed that comammox Nitrospira clade B, currently lacking any cultured representative, is the most widely distributed and abundant nitrifier in natural Danish habitats. In contrast, poorly characterised archaeal ammonia oxidisers (e.g. TA-21 NS-δ) dominate disturbed soils. We reassembled genomes to represent the type material of these lineages, recovering a closed circular genome for “Ca. Nitronatura plena” (comammox clade B) and an eight contig MAG of “Ca. Nitrososappho danica” (TA-21). Altogether, Microflora Danica establishes a comprehensive national resource for investigating functional microbial groups from both gene- and genome-level perspectives. Linking the species-level resolution of our work to in situ activity and greenhouse gas emissions is the next step towards microbially informed agricultural management, ecosystem monitoring and restoration.