Bioremediation of petroleum hydrocarbons: multi-scale investigation of effects of pore size and role of diurnal temperature changes

Indigenous, cold-adapted, hydrocarbon-degrading bacteria have been frequently detected in soils from cold region sites suggesting bioremediation, as a cost-effective remediation technology is potentially viable for cleanup of contaminated sites in cold regions. Previous studies on bioremediation of petroleum hydrocarbons in cold regions have been conducted with coarse textured soils. However, fine-grained or clayey soils are commonly found in several cold regions. The overall objective of this research was to investigate the factors controlling biodegradation of aged, clayey petroleum hydrocarbon contaminated soils from a sub-arctic site, under relevant temperature regimes. The biodegradation rates and extents of petroleum hydrocarbons were investigated for a clayey site contaminated soil in four pilot scale biopiles maintained at 15˚C. Non-volatile petroleum hydrocarbons (C16-C32) were biodegraded 24 to 38% under continuous aeration and with amendments of 17.5% or 23.5% moisture and also 17.5% moisture with 95 mg-N/ kg soil. In contrast, statistically significant biodegradation of the non-volatile hydrocarbon fraction was not observed in a biopile tank containing soils amended with 23.5% moisture content and 1340 mg-N/ kg soil. While a significant shift occurred in soil microbial community over 60 days in the biopile tank amended with 23.5% moisture only, there was no change in the microbial community of soil samples amended with a similar level of moisture and 1340 mg-N/ kg soil, indicating that high amounts of nitrogen amendment inhibited bioremediation.This study investigates the effect of diurnal temperature variations on the biodegradation of petroleum hydrocarbon contaminated soil typical for the site from northern Canada from which contaminated soils were obtained. The extents of biodegradation of petroleum hydrocarbons after 70 days, in systems incubated at temperatures varying between 5 ˚C to 15 ˚C daily (representative of daily high and low temperatures) were similar to systems incubated at constant temperature of 15 ˚C, and were significantly higher than systems incubated at 5 ˚C. The soil microbial community of systems with daily changes in temperatures between 5 ˚C to 15 ˚C was similar to those incubated at 15 ˚C, but significantly different than those incubated at 5 ˚C, based on both 16S rRNA and alkB gene analyses. The results suggest that the highest temperature (15 ˚C) to which the soil was frequently exposed to, determined the microbial community profile in the site soil, and periods of low temperatures did not reduce the overall biodegradation efficiency or the microbial community compared to that maintained solely at 15 ˚C.Experiments were conducted to investigate if hexadecane, a biodegradable but effectively insoluble hydrocarbon phase, was accessible to, and degraded by hydrocarbon degrading bacteria when the hexadecane-water interface was separated from the microbial culture by membranes with specific pore sizes. In these systems, bacteria were unable to degrade hexadecane when the membrane pore size was 0.4 µm or 3 µm, but was able to degrade hexadecane when separated by pore sizes of 12 µm. To investigate the implications of these results to aggregates from clayey and sandy soils, the in-situ aggregate micro-structure of representative soil aggregates from petroleum contaminated clayey soil and petroleum contaminated sandy soil were compared by micro-scale computed tomography (micro CT) imaging. The “bioaccessible porosity” defined as fraction of aggregate volume corresponding to pores larger than 4 µm was comparable in case of clayey (26-27%) and sandy (24%) aggregates.