Background/Objectives: Petroleum contamination in soil exerts toxic effects on earthworms (Eisenia fetida) through non-polar narcotic mechanisms. However, the specific toxicities of individual petroleum components remain insufficiently understood. Methods: This study investigates the effects of four petroleum components—saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes—on earthworms in artificially contaminated soil, utilizing a combination of biochemical biomarker analysis and metabolomics to uncover the underlying molecular mechanisms. Results: The results revealed that aromatic hydrocarbons are the most toxic fraction, with EC50 concentrations significantly lower than those of other petroleum fractions. All tested fractions triggered notable metabolic disturbances and immune responses in earthworms after 7 days of exposure, as evidenced by significant changes in metabolite abundance within critical pathways such as arginine synthesis, a-linolenic acid metabolism, and the pentose phosphate pathway. According to the KEGG pathway analysis, saturated hydrocarbon fractions induced marked changes in glycerophospholipid metabolism, and arginine and proline metabolism pathways, contributing to the stabilization of the protein structure and membrane integrity. Aromatic hydrocarbon fractions disrupted the arachidonic acid metabolic pathway, leading to increased myotube production and enhanced immune defense mechanisms. The TCA cycle and riboflavin metabolic pathway were significantly altered during exposure to the colloidal fraction, affecting energy production and cellular respiration. The asphaltene fraction significantly impacted glycolysis, accelerating energy cycling to meet stress-induced increases in energy demands. Conclusions: Aromatic hydrocarbons accounted for the highest level of toxicity among the four components in petroleum-contaminated soils. However, the contributions of other fractions to overall toxicity should not be ignored, as each fraction uniquely affects key metabolic pathways and biological functions. These findings emphasize the importance of monitoring metabolic perturbations caused by petroleum components in non-target organisms such as earthworms. They also reveal the specificity of the toxic metabolic effects of different petroleum components on earthworms.

Petroleum pollution can pose a serious threat to soil health and its ecological functions. This study investigated the efficacy of bacterial treatments for bioremediation of diesel-contaminated soil under outdoor conditions for a period of 4 months. Unlike most previous studies conducted under laboratory conditions, this study applied single and multi-bacterial consortia directly into diesel-contaminated soil under outdoor conditions, evaluating both hydrocarbon degradation and soil toxicity changes. Three treatments using a single strain, a 4-strain consortium, and an 8-strain consortium were applied to 2% (v/w) diesel-contaminated soil, and their performance was compared to uncontaminated and untreated controls. Total petroleum hydrocarbon (TPH) degradation was quantified using GC-FID, and the soil toxicity was assessed using Eisenia fetida toxicity test and higher plant germination assays. As the experiment demonstrated, the multi-strain bacterial consortium (BT3) achieved the highest TPH degradation (78.3%) and demonstrated significant reduction in long-chain hydrocarbon fractions (C₁₄-C₂₈). Toxicity measurements showed that all three bioremediation treatments, especially BT3, significantly increased earthworm survival, body weight change and plant germination rate after the bioremediation. Microbial community analysis based on 16S rRNA sequencing revealed significant shifts in the dominant bacterial genera over time, accompanied by a noticeable reduction in alpha diversity. In particular, BT3 showed a significant decrease in Shannon diversity index values from 9.4 at S₁ to 6.9 at S₃ (p < 0.01), whereas BT1 and BT2 remained relatively stable (p > 0.05). Overall, the results demonstrated that all three bacterial treatments significantly enhanced diesel degradation and reduced soil toxicity under outdoor conditions, highlighting their potential for future large-scale applications in sustainable soil remediation. Importantly, this study combines constructed microbial consortia with multi-level toxicity assessments, providing a comprehensive framework to guide future bioremediation strategies.

Petroleum-contaminated terrestrial ecosystems require effective bioremediation strategies. In this study, genomic analysis revealed key biodegradation genes on the Acinetobacter calcoaceticus 21# chromosome: alkane hydroxylases (alkB, almA, LadA) and aromatic ortho-cleavage pathway genes (catABC). Phylogenetic and multiple sequence alignment analyses of the almA gene in strain 21# revealed the presence of signature motifs characteristic of Baeyer–Villiger monooxygenase. Functional annotation analysis demonstrated stronger phylogenetic affinity of this protein to previously characterized BVMOs than to hydroxylases. Therefore, it is suggested that the AlmA protein in 21# exhibits BVMO activity and participates in the subterminal oxidation pathway of alkane degradation. Wild-type 21# degraded both n-Octacosane (24.47%) and pyrene (34.03%). Engineered 21#-A3 showed significantly enhanced n-Octacosane degradation (28.68%). To validate AlmA function and assess impacts of exogenous gene integration, we expressed the almA gene from A. vivianii KJ-1 via pET-28a(+)-av-almA-BH vector. Enzymatic assays demonstrated no activity toward long-chain alkanes but high activity for 2-decanone (0.39 U/mg) and 2-dodecanone (0.37 U/mg). Metabolite analysis confirmed recombinant AlmA functions through subterminal oxidation. This study establishes a foundational framework for advancing the optimization of petroleum-degrading bacteria. To engineer more efficient hydrocarbon-degrading strains, future research should integrate meta-cleavage pathways to expand their substrate utilization range for polycyclic aromatic hydrocarbons.