Ecological indicators and biological resources for hydrocarbon rhizoremediation in a protected area

Spillage from oil refineries, pipelines, and service stations consistently leads to soil, food and groundwater contamination. Bacterial-assisted phytoremediation is a non-invasive and sustainable solution to eliminate or decrease the concentration of xenobiotic contaminants in the environment. In the present study, a protected area interested by a fuel discharge was considered to assess a bioremediation intervention. From the spill point, a plume of contamination flowed South-West into the aquifer, eventually reaching a wetland area. Soils, groundwaters and plants belonging to the species Scirpus sylvaticus (L.) were sampled. In the majority of the soil samples, concentrations of total petroleum hydrocarbons, both C ≤ 12 and C > 12, exceeded legal limits set forth in Directive 2000/60/EC. The analysis of diatom populations, used as ecological indicators, evidenced morphology alterations and the presence of Ulnaria ulna and Ulnaria biceps species, previously detected in hydrocarbon-polluted waters. Tests for phytotoxicity and phytodegradation, carried out in soil mesocosms, planted with Zea mays and Helianthus annuus, demonstrated that both species significantly contributed to the removal of total petroleum hydrocarbons. Removal of C ≤ 12 and C > 12 petroleum hydrocarbons was in the range of 80%-82% for Z. mays and 71%-72% for H. annuus. Microbial communities inhabiting high organic carbon and vegetated soils were more active in hydrocarbon degradation than those inhabiting subsoils, as evidenced by soil slurry experiments. The abundance of functional genes encoding toluene-benzene monooxygenase (tbmD) and alkane hydroxylase (alkB), quantified in environmental samples, confirmed that the plant rhizosphere recruited a microbial community with higher biodegradation capacity. Bacterial strains isolated from the sampling site were able to grow on model hydrocarbons (hexane, hexadecane and o-, m-, p-xylene) as sole carbon and energy sources, indicating that a natural bio-attenuation process was on-going at the site. The bacterial strains isolated from rhizosphere soil, rhizoplane and endosphere showed plant growth promoting traits according to in vitro and in vivo tests on Z. mays and Oryza sativa, allowing to forecast a possible application of bacterial assisted rhizoremediation to recover the protected area.