Petroleum Hydrocarbon Concentration as the Primary Driver of Soil Bacterial Community Dynamics During Mycorrhizal Fungi and Rhizobacteria Assisted Phytoremediation

Petroleum hydrocarbon (PHC) contamination poses widespread environmental risks in Canada, where it is known to contribute to 60% of existing contaminated sites. It requires effective remediation strategies in the boreal ecozone due to the cold climate resulting in slow site weathering processes and soil nutrient cycling. This study utilized PHC-contaminated field soil (25,700 mg/kg total petroleum hydrocarbons - TPH) and background soil (<120 mg/kg TPH) collected from a Canadian boreal site where PHCs had weathered in place for 25+ years. Additionally, a 1:1 diluted soil was prepared (12,600 mg/kg TPH), and with the field contaminated soil, used to investigate the effects on rhizobacterial community composition. In two-year greenhouse experiments (2023 - 2025), the soils were planted with native or na turalized plant species and inoculated with i) arbuscular mycorrhizal fungi (AMF), ii) the biosurfactant-producing and plant-growth-promoting rhizobacterium (PGPR) - Bacillus subtilis ATCC 21332, or iii) both AMF and B. subtilis 21332. PHC contamination was determined to exert a dominant influence on rhizobacterial community composition. Alpha- and beta-diversity analyses revealed that neither plant species nor microbial inoculants significantly altered bacterial diversity or community structure beyond the overriding effect of PHC concentration. Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi dominated across all soils, with hydrocarbon-degrading genera such as KCM-B 112 and Sphingomonas significantly enriched in the 25,700 mg/kg TPH soil. Functional gene profiling identified widespread aerobic (e.g., alkB, CYP153, assA, nahAc, pheA, xylM, xylE, todC1, bphA1, and pahE) hydrocarbon-degradation pathways across phyla, suggesting extensive horizontal gene transfer and functional redundancy across treatments and plant species. Exogenously introduced B. subtilis ATCC 21132 established only in the background soil, suggesting competitive dominance of indigenous PHC-degraders. These findings underscore the magnitude of PHC concentration as the primary driver of rhizobacterial dynamics and indicate that augmenting native microbial capacity through soil carbon enhancement, rather than bioaugmentation, may significantly impact soil bacterial community composition during rhizodegradation of boreal soils.