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[ASAP] Manipulation of Unfrozen Water Retention for Enhancing Petroleum Hydrocarbon Biodegradation in Seasonally Freezing and Frozen Soil

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Manipulating the retention of unfrozen water in freezing contaminated soil to achieve prolonged bioremediation in cold climates remains unformulated. This freezing-induced biodegradation experiment shows how nutrient and zeolite amendments affect unfrozen water retention and hydrocarbon biodegradation in field-aged, petroleum-contaminated soils undergoing seasonal freezing. During soil freezing at a site-specific rate (4 to −10 °C and −0.2 °C/d), the effect of nutrients was predominant during early freezing (4 to −5 °C), alleviating the abrupt soil-freezing stress near the freezing-point depressions, elevating alkB1 gene-harboring populations, and enhancing hydrocarbon biodegradation. Subsequently, the effect of increased unfrozen water retention associated with added zeolite surface areas was critical in extending hydrocarbon biodegradation to the frozen phase (−5 to −10 °C). A series of soil-freezing characteristic curves with empirical α-values (soil-freezing index) were constructed for the tested soils and shown alongside representative curves for clays to sands, indicating correlations between α-values and nutrient concentrations (soil electrical conductivity), zeolite addition (surface area), and hydrocarbon biodegradation. Heavier hydrocarbons (F3: C16–C34) notably biodegraded in all treated soils (22–37% removal), as confirmed by biomarker-based analyses (17α(H),21β(H)-hopane), whereas lighter hydrocarbons were not biodegraded. Below 0 °C, finer-grained soils (high α-values) can be biostimulated more readily than coarser-grained soils (low α-values).