Deciphering Microbe-Mediated Dissolved Organic Matter Reactome in Wastewater Treatment Plants Using Directed Paired Mass Distance

Understanding the reaction mechanism of dissolved organic matter (DOM) during wastewater biotreatment is crucial for optimal DOM control. Here, we develop a directed paired mass distance (dPMD) method that constructs a molecular network displaying the reaction pathways of DOM. It couples direction inference and PMD analysis to extract the substrate–product relationships and delta masses of potentially paired reactants directly from sequential mass spectrometry data without formula assignment. Using this method, we analyze the influent and effluent samples from the bioprocesses of 12 wastewater treatment plants (WWTPs) and build a dPMD network to characterize the core reactome of DOM. The network shows that the first step of the transformation triggers reaction cascades that diversify the DOM, but the highly overlapped subsequent reaction pathways result in similar effluent DOM compositions across WWTPs despite varied influents. Mass changes exhibit consistent gain/loss preferences (e.g., +3.995 and −16.031) but different occurrences across WWTPs. Combined with genome-centric metatranscriptomics, we reveal the associations among dPMDs, enzymes, and microbes. Most enzymes are involved in oxygenation, (de)hydrogenation, demethylation, and hydration-related reactions but with different target substrates and expressed by various taxa, as exemplified by Proteobacteria, Actinobacteria, and Nitrospirae. Therefore, a functionally diverse community is pivotal for advanced DOM degradation.