Metabolomic-Metabolite Profiling: Progressive Insight and Biochemical Pathway in Crude Oil Waste Sludge Co-Composting...

Background: Crude oil refinery waste sludge (COWS) ranks among the most compositionally complex and ecotoxicologically hazardous industrial residues. Although bulk total petroleum hydrocarbon (TPH) and summed polycyclic aromatic hydrocarbon (PAH) removal are routinely reported, the metabolite-level biochemical fate of individual petrogenic compounds, spanning ring dihydroxylation, catechol cleavage, and entry into central carbon metabolism, remains largely unmapped under co-composting with diverse animal manures. Objectives: This study aimed to construct a metabolite-resolved, microbially anchored biochemical fate map of crude oil sludge during co-composting. Methods: Aerobic microcosms combining crude oil sludge, garden soil, and a wood-chip bulking agent were amended separately with poultry, horse, cow, or swine/pig manure alongside an unamended control and then incubated at 22 °C for 300 days. Analyses integrated untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics, targeted PAH quantification (EPA Methods 3541/8270), 16S rRNA gene amplicon sequencing (Illumina MiSeq, V1-V3, paired-end 300 bp), physicochemical monitoring, and culture-dependent isolation, with National Institute of Standards and Technology (NIST) Mass Spectral library annotation. Results: GC-MS resolved 1169 metabolite features across 17 samples, comprising 538 annotated compounds within 11 chemical classes and 631 unknowns, of which 151 recurred in at least 10 samples. Petrogenic markers (n-alkanes C₁₄-C₃₆, hopanoids, steranes, and alkylated dibenzothiophenes) and ring-cleavage intermediates (2-hydroxyfluorene, 1,4-naphthoquinone, phenanthrene-methanol, benzenediols, butanedioic acid, fatty alcohols C₁₆-C₂₀) elucidated a four-stage degradation cascade consistent with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways map01220 and map00624. PAH mean-removal ranked swine/pig (88.0%) > horse (87.0%) > poultry (80.5%) > cow (79.1%) > control (68.2%). Sequencing recovered 2969 operational taxonomic units (OTUs) enriched in Pseudomonas, Achromobacter, Stutzerimonas, Dietzia, Gordonia, and Mycobacterium, with Pseudomonas dominating high-removal systems; respiration peaked at 18.7 mg CO₂-C g^-1 in poultry treatments. Conclusions: This work establishes a metabolite-resolved map linking hydrocarbonoclastic taxa to separate degradation steps. The co-occurrence of oxygenated PAH intermediates with decreasing parent PAH concentrations serves as an indicator of transformation processes and may assist in identifying potential residual-risk signals, thereby supporting remediation evaluation and process optimization.