It takes one to capture one: scientific and microbial community
Nature is the original engineer, and with a body of work billions of years long we know we are barely scratching the surface of discovery when it comes to her creations including the estimated millions of species of environmental microbes. Through Microflora Danica, our goal was to build a resource documenting the microbes shaping Danish ecosystems, and by charting this diversity we hoped to create the initial microbial baseline of a country. This baseline could act as a point of reference for future work revisiting sites of interest or exploring new hypotheses, including critical issues such as climate change and biodiversity loss. As the microbial life of Denmark is not well suited to illustration on copper plates (scale is an issue), we documented them by capturing their DNA “fingerprints” through near full-length 16S rRNA gene sequencing, rRNA operon sequencing (bigger fingerprints) and metagenome (environmental DNA) sequencing. Our ambitious sampling goals depended on our own team at Aalborg University (~1000 samples), but primarily our network of skilled and enthusiastic collaborators in the Microflora Danica consortium (~9000 samples). Together, we took 10,689 samples across the country, covering the majority of Danish habitat types. Due to the expertise of our collaborators, we could connect these samples to a detailed habitat classification system, or ontology, ensuring we could “box” our microbes into their expert-defined ecosystems at resolution. This was important, even though we were to find that many of these boxes have holes in their walls.
Scaling up quality and quantity
After sampling, our second challenge was processing so many samples in <122 (or 5) years. In our previous work, we spearheaded methods for both high quality and high-throughput sequence recovery. We initially used short-read platforms to capture the diversity of microbes in wastewater treatment plants (Karst & Dueholm et al., 2018), and then extended to long-read platforms with their advantages in contiguity and taxonomic resolution (Karst et al., 2021), with applications to global wastewater treatment and anaerobic digester microbial communities (Dueholm et al., 2022). Through these works, we greatly expanded the taxonomic reference databases for 16S rRNA genes, and contributed our methods to samples from the Earth Microbiome Project (Shaffer et al., 2022). Moving from “who is there” to “what might they be doing”, we focused on high-quality, contiguous genome recovery (Singleton et al., 2021). Expanding these initial field changing successes was the next logical step: why not capture the microbial citizens of a whole country? Microflora Danica was initially envisioned as a 16S rRNA gene atlas, but this ambition changed to metagenomes, enabling us to uncover functional potential. By adapting our protocols for metagenome generation (Jensen et al., 2024), we could achieve cost-effective short-read sequencing at a national scale. With the methods ready, we were able to capture the diversity, distribution and even functional potential of microbes at scale.
The virus we weren’t planning for
One year in, our project met its third challenge and the world met the coronavirus. Although it was one virus compared to the hundreds of thousands we were trying to capture in our samples, we soon realised it was our national duty to pivot our high-throughput operations to Corona Danica, sequencing and tracking Denmark’s coronavirus variants. The Poul Due Jensen foundation allowed us to divert our Microflora Danica laboratory resources to the rapid upscaling of virus sequencing, and the Villum Foundation allowed us to pause projects and divert our people to the task. We established the Danish Covid-19 Genome Consortium together with The Danish Statens Serum Institut (SSI, Denmark’s public health institute for disease control and research) and all major Danish hospitals. During the peak of the pandemic we sequenced more than 1000 SARS-CoV-2 positive samples a day and delivered data in near real-time to SSI and the Danish government. Despite lockdown and the change of focus, the sampling teams continued collecting samples in the background, ready for our return to business as usual.
A first country-wide microbial atlas
Denmark presented the perfect place for a country-wide microbiome campaign. It is small in size, but exemplified by its community mindedness, priding itself on flat hierarchies, consensus decision making and equal opportunities. Per H Nielsen and Mads Albertsen drew on their extensive networks amongst scientists, companies and funding foundations. This produced enthusiastic collaborations and varied metadata collected from the Microflora Danica consortium members belonging to different scientific fields, much of which is still being gathered and processed. Due to Denmark’s long history of environmental surveys, monitoring, and openness to data sharing, the potential for future detailed metadata is extensive. Through our collaborations, we have expanded into country-wide community science efforts, like the “Masseeksperiment 2025”. Here, 30,000 students from 500 schools around the country assess the health of Danish soils by examining soils and sending samples for additional physical and chemical property processing (Aarhus University and Technical University of Denmark), and analysis of the microbial community (us at Aalborg University). In this way, our work contributes not only to advances in our field, but also helps develop the environmental and scientific mindset of the next generation.
Nature, nurture and nitrification: microbial responses to human impacts
Over the course of this initial Microflora Danica campaign, clear ecological patterns emerged. We identified high local microbial diversity in the human-disturbed soils of road sides, parks and agriculture. But the same species consistently reoccurred, revealing striking homogeneity, especially compared to the overall diversity observed in the more natural equivalents. We added significant insight at the species level, identifying core populations associated with disturbance. Essentially, we were consistently identifying the microbial “weeds” or potential “pioneer” species that adapt fast to our disturbances. From the functional perspective, as Denmark is intensively agricultural, we focused on one of its most important microbial functional guilds, nitrifiers, responsible for nitrogen transformations leading to greenhouse gas emissions and environmental nitrogen pollution. Nitrifiers were among the core populations associated with the level of human disturbance, particularly one species of an uncharacterised archaeal ammonia oxidiser. Additionally, using the functional information afforded by metagenomes and metagenome-assembled genomes, we identified new potential nitrifiers, previously missed or misclassified, and determined the nitrifier profile of natural habitats. Identifying, at the species level, the functional groups inhabiting disturbed and natural terrestrial systems take us a step closer to improving land management practices as soil microbial communities are inextricably tied to soil health. For example, combined with additional data from field studies, knowledge of the microbial community could be used to encourage more natural distributions of nitrifiers associated with less nitrous oxide emissions, or to limit pollution, perhaps through selective fertiliser applications or crop regimes.
The microbial bridge to greener pastures
Microflora Danica extends current 16S rRNA gene databases by an order of magnitude, and combined with long-read based metagenome-assembled genome recovery from soils (totalling 34,567 genomes), extends database representation of species in soils an additional 8% in phylogenetic gain (Sereika et al., 2025). There is room for improvement, as only one time point is sampled, but we have provided the microbial map, and sites with indications of specific populations can be revisited for deeper analyses. We hope our approach will be extended by other countries, beyond northern temperate environments, to continue to capture our planet's microbial and functional diversity.
While we were hesitant to speculate too far beyond what we achieved in the study, our reviewers were cognizant of the wider relevance and encouraged us to be bold in the study’s implications for climate change and land use monitoring. Additionally, Microflora Danica has timely relevance following recent calls to include the microbial world in biodiversity assessments and conservation efforts. At home, it will no doubt contribute to the environmental leaps Denmark is preparing for with the initiation of the Green Tripartite. This historic agreement between government, farmers and industry sets concrete goals to restore nature, cut agricultural emissions and promote sustainable land use. Microflora Danica is the microbial lens needed to help track and guide this national transition.
This post was written by Mads Albertsen, Per Halkjær Nielsen, Thomas Bygh Nymann Jensen and Caitlin Singleton
Poster image credit: Søren Emil Søe Degn, Aalborg University