In Situ Enhanced Natural Attenuation of Monoaromatic Compounds in Groundwater: Beyond Aerobic Biodegradation?

Background information BTEX compounds (benzene, toluene, ethylbenzene, and xylene isomers) are among the most widespread groundwater contaminants owing to their solubility and toxicity. Despite being biodegradable, their natural attenuation is often limited by environmental conditions, particularly electron acceptor availability, making engineered bioremediation necessary, typically by promoting aerobic biodegradation through air/oxygen sparging or injection of calcium peroxide-based oxygen-releasing compounds (ORCs). This study compared natural attenuation and different enhanced bioremediation approaches for the remediation of residual BTEX contamination in groundwater at an industrial site in northern Italy, where the aquifer is characterized by mild reducing conditions (low dissolved oxygen and slightly negative redox potential). Laboratory microcosms (1:3 wt. solid-liquid ratio) with soil and groundwater from the site were set up to simulate in situ conditions and enhanced BTEX removal through ORCs or nitrate dosing. Phosphate buffer was added to all microcosms to mitigate the pH variation caused by calcium peroxide. Main results Over a 60-day incubation period, periodic analysis of residual monoaromatic compounds, oxygen, and different nitrogen species (ammonium, nitrate, and nitrite), as well as microbial activity, revealed rapid degradation of BTEX in all microcosms. Notably, microcosms under denitrifying conditions (control or NO₃ supplementation) exhibited higher degradation rates, with a four-order-of-magnitude decrease in initial BTEX concentrations, compared with ORC-supplemented microcosms under aerobic conditions, which achieved a decrease of only two to three orders of magnitude. Molecular analyses, including 16S rRNA sequencing and qPCR, revealed a diverse microbial community in pre-test water and soil samples, including Comamonadaceae and Desulfuromonadaceae, known hydrocarbon-degraders under nitrate-reducing conditions, as well as Chitinophagaceae, Pseudomonadaceae, which are aerobic hydrocarbon-degraders. The faster biodegradation under denitrifying conditions suggests that these represent the predominant in situ conditions, to which the microbial populations are better adapted. As indicated by the time evolution of nitrogen species, in control and NO₃-supplemented microcosms, the accumulation of NO₂⁻ was observed depending on the relative abundance of BTEX and NO₃. ORC addition led to relevant shifts in the microbial community structure and functional gene expression as the increased oxygen availability introduced new selective pressures, requiring an adaptation phase. Conclusions This study highlights the interplay between aerobic and denitrifying BTEX degradation pathways and underscores the importance of considering multiple electron acceptor conditions in engineered remediation. Additionally, this suggests the potential benefits of nitrate amendments, possibly combined with oxygen release, to maximize contaminant removal while minimizing unintended geochemical changes. Further investigations are recommended to evaluate long-term field-scale applications and refine dosing strategies.