Multi-omic investigation of hydrocarbon and nitrite bioremediation in Antarctic soils

Petroleum hydrocarbon (HC) contamination is a prominent anthropogenic contaminant in polar regions, arising from fuel spills linked to tourism, logistics and historical mismanagement. Hydrocarbon bioremediation is a common treatment option in polar locations due to low operational costs, on-site application and minor disturbances to local ecosystems. This thesis examines long-term effects of HC bioremediation on microbiomes and soil geochemistry in east Antarctica, focusing on mechanisms driving the accumulation and removal of nitrite, an emerging secondary contaminant. With preliminary evidence suggesting that aerobic biopiles favour the proliferation of ammonia oxidising bacteria, we hypothesized that microaerophilic soil conditions may lead to more efficient nitrite removal. Using a multi-omic approach across 211 soil samples from urea-amended Antarctic biopiles, Shirley Island and Station Nord (Greenland), this work addresses: (1) development of a metaproteomics pipeline to identify active xenobiotic-degrading and nutrient-cycling proteins in low-biomass polar soils; (2) uncovering microbial pathways and mechanisms driving nitrite accumulation in engineered biopiles and its removal from hydrocarbon-remediated soils; (3) construction of MAGs of major taxa to assess contributions to bioremediation; (4) evaluation, using a multi-omic framework, of soil washing as a nitrite-removal strategy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by reverse-phase chromatography (SDS-RP) was found to be the ideal metaproteomic extraction protocol, yielding a three-fold increase in protein identifications over the next best method. In total, 5,624 recovered proteins from Shirley Island soils displayed functions in carbon and nitrogen cycling and tolerance to cold and osmotic stress. Between two Station Nord sites, 1,030 proteins were significantly (p < 0.05) differentially enriched, many associated with legacy hydrocarbon and heavy-metal contamination. A longitudinal omics analysis of a Casey Station biopile (BP 7) showed total recoverable hydrocarbons (TRH) declined fourfold in the first 21 months, then plateaued. Nitrite was first detected at 36 months, peaked at 60 months and reached lowest levels by 109 months. Its emergence coincided with Nitrosospira rising to > 1.2% relative abundance in low-TRH samples, suggesting hydrocarbon toxicity may repress ammonia-oxidising organisms. Metagenomics and metaproteomics showed that ammonia monooxygenase genes (amoB, amoC) declined from time 0, with a significant decrease (p < 0.05) in glutamine synthetase expression observed between 21 and 36 months- indicating that reduced ammonia assimilation as bioremediation plateaued likely contributed to nitrite accumulation. Increases in nrfAH and fdrA gene copies at 109 months revealed a shift toward anaerobic respiration and heterotrophic denitrification. Soil washing was evaluated as a mechanism for nitrite removal in partially remediated biopiles (BP3 and BP4). Five wash cycles reduced combined nitrite and nitrate levels from 52.5 mg/kg to 0.5 mg/kg, yet within two years nitrite re-accumulated to 11.5–139.5 mg/kg, with lower concentrations found at the shallower depth. Amplicon sequencing and shotgun metagenomics indicated that Tomitella spp. may play an important role in hydrocarbon degradation and nitrogen cycling, with relative abundances between 26.1% and 16.9% observed throughout the study. In addition, a Tomitella biformata MAG contained a complete denitrification pathway and alkane-degradation genes (alkT, ALDH). Metaproteomics detected 1,870 proteins across biopile and Casey Utility Building (CUB) soil samples at 5 cm and 50 cm. Ammonia assimilation and primary production was enriched at shallow soils (5 cm) in the CUB slope, with NAD-specific glutamate dehydrogenase 2.3 log-fold higher and phycocyanin 1.7 log-fold higher than in the biopile soils. In this thesis, metaproteomics was successfully utilised in low biomass Antarctic soils to uncover geochemical cycling and xenobiotic proteins (Ch. 2). Additionally, our multi-omic analyses identified the mechanism of nitrite accumulation as a reduction in ammonia assimilation coupled with increased ammonia oxidation (Ch. 3-4). Two routes for nitrite removal were uncovered: (1) in biopile soils, microaerophilic conditions promoted heterotrophic denitrification; and (2) in CUB slope soils, which remained aerobic at all depths, elevated surface-layer primary production supplied carbon that facilitated glutamine synthetase activity, preventing rapid nitrite accumulation in washed soils. Together, these findings indicate that controlled oxygen gradients and carbon availability are actionable levers to steer nitrogen fluxes and alleviate nitrite accumulation during hydrocarbon bioremediation in polar soils.