Effects of Ryegrass and Exogenous Carbon Addition on Petroleum Hydrocarbon Removal and Soil Microbial Communities

Extensive oil contamination left by the Gulf War, together with arid and nutrient-poor conditions, poses persistent challenges to the bioremediation of petroleum-contaminated soils in Kuwait. This study examined the effects of ryegrass and different forms of exogenous carbon on petroleum hydrocarbon removal and soil microbial communities. Twelve pot treatments combined ryegrass planting with different doses of β-hydroxybutyric acid (HB) or polyhydroxybutyrate (PHB) in soil artificially contaminated with engine oil. Total petroleum hydrocarbons (TPH), organic acids, microbial communities, and predicted hydrocarbon-degradation functions were analyzed. At 150 d, ryegrass combined with PHB at 500 mg/pot produced the numerically highest mean TPH reduction (39.09%), exceeding that of the unplanted, unamended control by 18.91 percentage points. However, final TPH concentrations did not differ significantly from those with ryegrass alone or PHB alone at the same dose (500 mg/pot). Increasing the PHB dose reduced remediation performance. HB and PHB produced distinct patterns of organic acid change and significantly affected bacterial community composition. At 60 d, Massilia and Dyadobacter were relatively abundant in the ryegrass + PHB 500 mg/pot treatment. PICRUSt2 analysis of 16S rRNA gene sequences attributed predicted gene families associated with aromatic intermediate transformation to these genera. Gordonia was relatively abundant in the ryegrass + HB 550 mg/pot treatment at 150 d. These findings highlight taxa potentially relevant to hydrocarbon degradation, although their actual contributions require direct functional validation. The results indicate that the form and dose of exogenous carbon influence plant-assisted remediation and bacterial community responses under the tested conditions. Applicability to field-contaminated soils in Kuwait requires further validation.