Metaproteomics Reveals Functional Rewiring and Metabolic Limits in Synthetic Bacterial Consortia Degrading Complex Waste Oils

Abstract

Elucidating the metabolic mechanisms underpinning complex hydrocarbon biodegradation by microbial consortia benefits from integration of community-level genomic and proteomic data. Here, we deployed a combined metagenomics and metaproteomic analysis to characterize functional dynamics within bacterial consortia enriched from hydrocarbon-contaminated soil, using military waste oil as the sole carbon source. Metagenomic profiling revealed a consortium dominated by Stenotrophomonas maltophilia alongside established hydrocarbon-degrading taxa including Pseudomonas fluorescens, Pseudomonas putida, and Delftia acidovorans. Quantitative label-free metaproteomics uncovered dynamic taxonomic restructuring and metabolic reorientation, with Delftia and Achromobacter proteins enriched during active degradation, indicating niche advantages under chronic contamination. Enzymatic mapping resolved expression of degradation pathways targeting aliphatic and aromatic substrates, with one specific consortium, BPS4, exhibiting preferential catabolism of catechol intermediates channeled toward the tricarboxylic acid cycle, supported by elevated protein abundance of oxidoreductases, dehydrogenases, and {beta}-oxidation enzymes. Gene ontology enrichment revealed heightened nitrogen cycling capacity, including upregulation of nitric oxide biosynthesis and nitrate reductase, suggesting adaptive nitrogen metabolism coupled to hydrocarbon mineralization. In-vitro batch-fed adaptation studies demonstrated limited improvements in degradation of recalcitrant fractions, particularly branched aliphatics and organophosphates, revealing intrinsic metabolic constraints within the consortium. These findings underscore the complementary utility of metaproteomic interrogation for resolving both population-level dynamics and functional metabolic capabilities, while highlighting fundamental limitations in expanding the degradative spectrum of enriched consortia through conventional adaptation strategies. This work establishes a foundation for rational ecological engineering of hydrocarbon-remediating consortia informed by metaproteomics. ImportanceWaste oils from transport and industry are complex and toxic mixtures that are difficult and expensive to treat using conventional chemical methods. Microorganisms offer a sustainable alternative, but their performance depends on how different species work together and where their metabolic limits lie. In this study, we show how advanced protein-based analysis using quantitative metaproteomics, can reveal microbial community activities whilst breaking down real-world waste oils. We enriched bacteria from contaminated soils and then identified the enzymes driving effective degradation. We investigated how communities reorganise under chemical stress, and why certain oil components remain resistant even after long-term adaptation. By uncovering both strengths and limitations of naturally enriched microbial consortia, this work provides practical insights for designing more reliable biological treatments for oil-based wastes and even contaminated environments, supporting greener approaches to pollution management and industrial waste recycling.