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bioRxiv · 10.1101/2025.08.22.671748

Phytoremediation of 25+ Years Weathered Petroleum Contamination with Symbiotic Arbuscular Mycorrhizal Fungi and Bacillus subtilis ATCC 21332

Roy, P.
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Abstract

Petroleum hydrocarbon (PHC) contamination affects approximately 60% of Canadian contaminated sites, with the boreal ecozone facing acute exposure from industrial activities. This study evaluated the long-term effectiveness of plant-microbe enhanced phytoremediation of 25+ years weathered PHC-contaminated field soil over a 24-month period. Five native or naturalized plant species (three tufted grasses, a forb, and a tree) were tested across two contaminated soils and a control to compare remediation efficacy and plant health: 25,700 mg/kg total petroleum hydrocarbons (TPH) and a 1:1 contaminated and background site soil mixture (12,600 mg/kg TPH). Soil treatments included a Plants-Only Control, Arbuscular Mycorrhizal Fungi (AMF), plant-growth-promoting-rhizobacterium - Bacillus subtilis, and AMF+B. subtilis. Plant inoculation significantly improved remediation compared to natural attenuation (No Plants Control) in both PHC-contaminated soils. AMF-based treatments significantly remediated both soils: AMF in the 25,700 mg/kg TPH soil (74% mean TPH remediation) and AMF+B. subtilis in the 12,600 mg/kg TPH soil (96% remediation). In the 12,600 mg/kg TPH soil, all treatments successfully reduced PHC levels to below federal and provincial thresholds ([≥]95% removal) within 17 months. Picea mariana (black spruce) and Andropogon gerardii (big bluestem) above- and below-ground biomasses were not significantly impaired by the 25,700 and 12,600 mg/kg TPH soils, indicating their suitability for site remediation. B. subtilis significantly improved above-ground biomass and plant height and is therefore recommended for co-inoculation with AMF. These findings demonstrate that AMF-enhanced phytoremediation represents a highly effective, sustainable approach for long-term PHC remediation in Canadian boreal ecosystems, offering significant advantages over natural attenuation processes alone. HighlightsO_LIAMF-based treatments achieved highest PHC removal in 25+ year weathered boreal soils C_LIO_LIPrairie grasses remediated 12,600 mg/kg TPH soil by >95% in AMF+B. subtilis treatment C_LIO_LIEnhanced Picea mariana (black spruce) biomass and moisture in 25,700 mg/kg TPH soil C_LIO_LIBacillus subtilis significantly enhanced plant above-ground biomass and height C_LIO_LIAMF helped maximize PHC remediation, while B. subtilis promoted plant growth C_LI

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Roy, P.. 2025-08-27. Phytoremediation of 25+ Years Weathered Petroleum Contamination with Symbiotic Arbuscular Mycorrhizal Fungi and Bacillus subtilis ATCC 21332. https://doi.org/10.1101/2025.08.22.671748

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