Determining the extent of biodegradation of fuels using the diastereomers of acyclic isoprenoids.

Improved testing and remediation procedures for sites contaminated with petroleum hydrocarbons are a priority in remote cold regions such as Antarctica, where costs are higher and remediation times are longer. Isoprenoid/n-alkane ratios are commonly used to determine the extent of biodegradation at low levels but are not useful once the n-alkanes have been removed. This study demonstrates how the diastereomers of the acyclic isoprenoids can be used to determine the extent of biodegradation in moderately biodegraded fuel in soils from a bioremediation trial at Casey Station, Antarctica. The biological diastereomers of pristane (meso; RS = SR) are depleted more rapidly during moderate biodegradation than the geological or mature diastereomers (RR and SS), and thus, the ratio of pristane diastereomers can determine the level of biodegradation. The statistical difference among mean diastereomer ratios for samples grouped according to the biodegradation scale and pristane/phytane ratios was highly significant. The ratios of norpristane and phytane diastereomers also change with biodegradation in a similar fashion, and different levels of sensitivity exist for each. Additional benefits are that the method can be performed on conventional gas chromatographs by non-specialist chemists and that the ratios are independent of evaporation and do not necessarily require a non-biodegraded reference (T0) sample. This study details a simple alternative method for determining the extent of biodegradation of fuels at moderate levels that can be applied to a wide range of petroleum products.

Using real-time PCR to assess changes in the hydrocarbon-degrading microbial community in Antarctic soil during bioremediation.

A real-time polymerase chain reaction (PCR) method to quantify the proportion of microorganisms containing alkane monooxygenase was developed and used to follow changes in the microbial community in hydrocarbon-contaminated Antarctic soil during a bioremediation field trial. Assays for the alkB and rpoB genes were validated and found to be both sensitive and reproducible (less than 2% intrarun variation and 25-38% interrun variation). Results from the real-time PCR analysis were compared to analysis of the microbial population by a culture-based technique [most probable number (MPN) counts]. Both types of analysis indicated that fertilizer addition to hydrocarbon-contaminated soil stimulated the indigenous bacterial population within 1 year. The proportion of alkB containing microorganisms was positively correlated to the concentration of n-alkanes in the soil. After the concentration of n-alkanes in the soil decreased, the proportion of alkane-degrading microorganisms decreased, but the proportion of total hydrocarbon-degrading microorganisms increased, indicating another shift in the microbial community structure and ongoing biodegradation.

Fertilization stimulates anaerobic fuel degradation of antarctic soils by denitrifying microorganisms.

Human activities in the Antarctic have resulted in hydrocarbon contamination of these fragile polar soils. Bioremediation is one of the options for remediation of these sites. However, little is known about anaerobic hydrocarbon degradation in polar soils and the influence of bioremediation practices on these processes. Using a field trial at Old Casey Station, Antarctica, we assessed the influence of fertilization on the anaerobic degradation of a 20-year old fuel spill. Fertilization increased hydrocarbon degradation in both anaerobic and aerobic soils when compared to controls, but was of most benefit for anaerobic soils where evaporation was negligible. This increased biodegradation in the anaerobic soils corresponded with a shift in the denitrifier community composition and an increased abundance of denitrifiers and benzoyl-CoA reductase. A microcosm study using toluene and hexadecane confirmed the degradative capacity within these soils under anaerobic conditions. It was observed that fertilized anaerobic soil degraded more of this hydrocarbon spike when incubated anaerobically than when incubated aerobically. We conclude that denitrifiers are actively involved in hydrocarbon degradation in Antarctic soils and that fertilization is an effective means of stimulating their activity. Further, when communities stimulated to degrade hydrocarbons under anaerobic conditions are exposed to oxygen, hydrocarbon degradation is suppressed. The commonly accepted belief that remediation of polar soils requires aeration needs to be reevaluated in light of this new data.

Investigation of evaporation and biodegradation of fuel spills in Antarctica: II-extent of natural attenuation at Casey Station.

In many temperate regions, fuel and oil spills are sometimes managed simply by allowing natural degradation to occur, while monitoring soils and groundwater to ensure that there is no off-site migration or on-site impact. To critically assess whether this approach is suitable for coastal Antarctic sites, we investigated the extent of evaporation and biodegradation at three old fuel spills at Casey Station. Where the contaminants migrated across frozen ground, probably beneath snow, approximately half the fuel evaporated in the first few months prior to infiltration at the beginning of summer. Once in the ground, however, evaporation rates were negligible. In contrast, minor spills from fuel drums buried in an abandoned waste disposal site did not evaporate to the same extent. Biodegradation within all three spill sites is generally very minor. We conclude that natural attenuation is not a suitable management strategy for fuel-contaminated soils in Antarctic coastal regions.

Investigation of evaporation and biodegradation of fuel spills in Antarctica. I. A chemical approach using GC-FID.

Little effort has been devoted to differentiating between hydrocarbon losses through evaporation and biodegradation in treatability studies of fuel-contaminated Antarctic soils. When natural attenuation is being considered as a treatment option, it is important to be able to identify the mechanism of hydrocarbon loss and demonstrate that rates of degradation are sufficient to prevent off-site migration. Similarly, where complex thermally enhanced bioremediation schemes involve nutrient addition, water management, air stripping and active heating, it is important to appreciate the relative roles of these mechanisms for cost minimisation. Following the loss of hydrocarbons by documenting changes in total petroleum hydrocarbons offers little insight into the relative contribution of evaporation and biodegradation. We present a methodology here that allows identification and quantification of evaporative losses of diesel range organics at a range of temperatures using successively less volatile compounds as fractionation markers. We also present data that supports the general utility of so-called biodegradation indices for tracking biodegradation progress. We are also able to show that at 4 degrees C indigenous Antarctic soil bacteria degrade Special Antarctic Blend fuel components in the following order: naphthalene and methyl-napthalenes, light n-alkanes, then progressively heavier n-alkanes; whereas isoprenoids and the unresolved complex mixture are relatively recalcitrant.

Effects of temperature on mineralisation of petroleum in contaminated Antarctic terrestrial sediments.

Although petroleum contamination has been identified at many Antarctic research stations, and is recognized as posing a significant threat to the Antarctic environment, full-scale in situ remediation has not yet been used in Antarctica. This is partly because it has been assumed that temperatures are too low for effective biodegradation. To test this, the effects of temperature on the hydrocarbon mineralisation rate in Antarctic terrestrial sediments were quantified. 14C-labelled octadecane was added to nutrient amended microcosms that were incubated over a range of temperatures between -2 and 42 degrees C. We found a positive correlation between temperature and mineralisation rate, with the fastest rates occurring in samples incubated at the highest temperatures. At temperatures below or near the freezing point of water there was a virtual absence of mineralisation. High temperatures (37 and 42 degrees C) and the temperatures just above the freezing point of water (4 degrees C) showed an initial mineralisation lag period, then a sharp increase in the mineralisation rate before a protracted plateau phase. Mineralisation at temperatures between 10 and 28 degrees C had no initial lag phase. The high rate of mineralisation at 37 and 42 degrees C was surprising, as most continental Antarctic microorganisms described thus far have an optimal temperature for growth of between 20 and 30 degrees C and a maximal growth temperature <37 degrees C. The main implications for bioremediation in Antarctica from this study are that a high-temperature treatment would yield the most rapid biodegradation of the contaminant. However, in situ biodegradation using nutrients and other amendments is still possible at soil temperatures that occur naturally in summer at the Antarctic site we studies (Casey Station 66 degrees 17(') S, 110 degrees 32(') E), although treatment times could be excessively long.