Microscopic life from different angles: Insights into microbes and phages using metagenomics

Microorganisms are ubiquitous in nature. They inhabit soil, water, air, and even the human body. They are fundamental to life on Earth, driving processes such as nutrient cycling, waste degradation, and the production of bioactive compounds. Despite their importance, the majority of microbes remain poorly understood. To study these uncultured organisms, modern microbiology relies on metagenomics, a technique that enables the analysis of all genetic material present in an environmental sample and does not require growing microorganisms in the lab. This approach provides insight into which organisms are present and the potential functions they perform. Conventional methods for analyzing metagenomes often struggle to reconstruct and identify microorganisms in microbial communities, as they either disregard large portions of the genetic material or produce noisy results. In this thesis, I address part of these limitations by introducing a tool called the Read Annotation Tool (RAT), which integrates information across multiple levels of genome reconstruction. By combining these data sources and prioritizing them based on reliability, RAT provides an accurate and comprehensive representation of microbial community composition. Using this approach, I studied a groundwater bioremediation system in Utrecht, located at a former industrial site contaminated with hydrocarbons and other pollutants. Groundwater was sampled along a pipeline connecting the contaminated site to a treatment plant, and metagenomic analysis was used to reconstruct microbial genomes. The results showed a shift from anaerobic, sulfur-oxidizing bacteria in the park to aerobic, pollutant-degrading bacteria in the treatment plant. Genes involved in breaking down aromatic hydrocarbons were more abundant in the oxygen-rich treatment environment compared to the park. These findings show how oxygen availability and long-term operation shape microbial communities in engineered bioremediation systems and identify key microorganisms involved in the clean-up of polluted groundwater. Metagenomics can also be used to identify viruses, e.g. phages. Phages are the most abundant biological entities on the planet and play a central role in regulating microbial populations and facilitating gene exchange. However, their extreme genetic diversity and rapid evolution make them difficult to study. For this research, I analyzed DNA phages across 47,726 public metagenomes spanning 107 distinct ecosystems. I identified 39 million potential viral DNA sequences. Most sequences belonged to Caudoviricetes, while a substantial fraction remained unidentified, representing uncharacterized viral diversity and potentailly other genetic elements. Biome-level comparisons showed clear ecological patterns: human-associated phages were more frequently shared across environments, whereas marine and other environmental phages were more specific. This work provides an overview of phage diversity and distribution across global ecosystems and contributes a new, large dataset to explore the global DNA phageome. Finally, this thesis explores the emerging concept of phage bioaugmentation, where phages have the potential to enhance the degradation of pollutants in soil. Bioremediation often struggles with slow rates and environmental challenges, but phages may help overcome some of these limitations by delivering beneficial genes to native bacteria. These genes can improve host survival and pollutant breakdown, potentially transforming soil clean-up strategies. I review how soil complexity and microbial ecology affect pollutant bioavailability, outline evidence for phages carrying bioremediation-relevant AMGs in contaminated environments, and propose a framework for using phages in bioaugmentation. While many challenges remain, including understanding gene expression and ensuring ecological safety, phage bioaugmentation represents a promising next step toward more efficient, sustainable, and targeted soil remediation. Together, the studies presented in this thesis explore how the knowledge gained from metagenomics and improving bioinformatic methods can be used to better understand microbial communities. The research further shows how learning more about phages and their interaction with microorganisms in e.g. polluted environments could be used in the future to develop new techniques for environmental restoration.