Microbial Interactions: Nitrifiers and their Partners in Complex Wastewater Microbiome

Microbial interactions shape the structure, function, and resilience of microbiomes in wastewater treatment plants (WWTPs), directly impacting performance. Nitrifiers, as chemolithoautotrophs and primary producers, are particularly suitable for studying these interactions by assessing carbon flow through metabolite tracing with stable isotopes. This project aims to uncover the principles governing microbial interactions between nitrifiers and their partners through three work packages: identifying interaction partners, inferring their interactions by genome analysis, and verifying these interactions in situ. Potential partners will be identified combining co-occurrence network analyses, fluorescence-activated cell sorting of activated sludge microflocs containing nitrifiers, and amplicon sequencing to identify co-sorted microbes. Fluorescence in situ hybridization (FISH) will be used to confirm their spatial co-localization with nitrifiers. Next, deep long-read metagenomics will generate metagenome-assembled genomes (MAGs) of nitrifiers and their potential partners for genome analysis and metabolic modeling, identifying possible types of interactions. The hypothesized interactions will be verified using FISH and stable isotope probing (SIP) combined with chemical imaging such as mid-infrared photothermal (MIP) imaging and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to identify exchanged metabolites in situ. This project is among the first to integrate SIP, FISH, and chemical imaging (MIP and MALDI-MSI) to study microbe-microbe interactions directly in complex microbiomes at the single-cell or cell cluster level. The findings will provide new insights into interaction mechanisms within complex microbiome and help design strategies to promote stable nitrifying communities in WWTPs. The developed workflow will also be highly valuable for studying microbial interactions in other environmental and medical microbiomes.