Environ. Sci. Technol. 2006, 40, 2011-2017
Fertilization Stimulates Anaerobic Fuel Degradation of Antarctic Soils by Denitrifying Microorganisms S H A N E M . P O W E L L , * ,† SUSAN H. FERGUSON,‡ IAN SNAPE,‡ AND STEVEN D. SICILIANO§ School of Agricultural Science, University of Tasmania, Hobart 7001, Australia, Department of Heritage and Environment, Australian Antarctic Division, Kingston 7050, Australia, and University of Saskatchewan, Saskatoon, Canada S7N 5A8
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.
Introduction Polar soils have been contaminated by petroleum hydrocarbons as the result of accidental spillage during fuel transport and transfer, and from leaking vehicles and storage tanks (1). There are no reliable estimates of the amount of contaminated soil in the Antarctic, but it is possibly in the order of ∼1 million m3 (2). The most common management response to this problem has been to rely on natural attenuation or, occasionally, to excavate and ship the soil to * Corresponding author phone: +61 03 6226 2776; fax: +61 03 6226 2642; e-mail:
[email protected]. † University of Tasmania. ‡ Australian Antarctic Division. § University of Saskatchewan. 10.1021/es051818t CCC: $33.50 Published on Web 02/14/2006
2006 American Chemical Society
a temperate region for remediation (2). Excavation is expensive and can have severe environmental impacts. This combination of cost and environmental risk has promoted research into in situ alternatives such as bioremediation. In laboratory studies, Antarctic soil organisms degrade hydrocarbons such as n-alkanes, monoaromatics, and naphthalenes under aerobic conditions (3, 4), and field results indicate that a range of catabolic genes exist in Antarctic soils (5). Unfortunately, degradation rates are limited due to nutrient deficiencies, low temperatures, poor water holding capacities, and frozen water (6-8), but these limitations can be overcome by techniques such as fertilization, temperature control, and irrigation (7, 8). It is commonly assumed that there is sufficient aeration of Antarctic soils to prevent oxygen limitation (1). This assumption is based on gravimetric water contents between 3% and 11% and bulk densities between 1 and 1.2 g cm-3 (2) with a corresponding air volume fraction between 40% and 50%. The relatively low oxygen demand (∼600 µg O2 g-1 soil day-1) characteristic of many Antarctic soils suggests that there will be sufficient oxygen to prevent hypoxia (9). However, some Antarctic soils display gleying, an indication of oxygen limitation (10-12), and even in aerobic soils anaerobic conditions exist in anoxic microsites (13). Under intensive bioremediation where there is a carbon source (hydrocarbons) and nutrients (fertilizer) present in excess, it is probable that certain soil regions will become hypoxic or anoxic. Degradation of hydrocarbons by anaerobic organisms is different from aerobic degradation, as they do not use the oxygenases used by aerobic organisms (14, 15). Anerobic alkane degradation is poorly understood, but appears to be initiated by a fumarate addition via a radical reaction, eventually forming an alkyl-CoA compound later oxidized by β-oxidation (16-18). Degradation of alkyl-substituted aromatic hydrocarbons commences with the addition of fumarate to alkyl groups that is then oxidized to produce benzoyl coenzyme A (CoA). Benzoyl-CoA is reduced by benzoyl-CoA reductase and used for energy and carbon by the cell (19). Recently, two research groups developed conserved primers to amplify portions of the genes responsible for benzoyl-CoA reductase (14, 20). These primers are useful but are limited to the detection of the genes for alkylsubstituted aromatic degradation by some anaerobic organisms. Denitrifiers are common anaerobic soil organisms (21), and several isolates can metabolize hydrocarbons (16, 22, 23) using benzoyl-CoA reductase and the fumarate addition pathway. Denitrification is a respiratory process; however, denitrifying bacteria can either be obligately or facultatively anaerobic. Denitrification, the sequential reduction of nitrate to di-nitrogen gas, involves the genes nar/nap, nir, nor, and nos encoding for nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively (21). The nir and nosZ genes are considered indicative of denitrifying bacteria (24). There are two distinct forms of nitrite reductase, one with a cytochrome center (nirS) and one with a copper center (nirK); thus a quantitative assessment of denitrifiers in soil is more easily accomplished using primers specific for nosZ (25). However, in situ confirmation of denitrification activity is technically challenging. As nitrate reduction will not occur until oxygen levels have become depleted and iron reduction will not occur until nitrate has been consumed, the sequential use of electron acceptors is an accepted method of determining the dominant biogeochemical process (26). VOL. 40, NO. 6, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Denitrification is thought to be a minor process in polar environments because of nitrate limitations and oxygen surpluses. However, in the case of bioremediation, substantial nitrate is usually added and metabolic activity is stimulated. Thus, the nitrate limitation is removed and partial oxygen pressures reduced. In Antarctica, little is known about denitrifiers in soil with all previous work investigating denitrifiers in aquatic systems (27-29) and marine sediments (30). These investigators all found that denitrification does occur in Antarctica but to a limited extent. In contrast, denitrification is an important biogeochemical process in the Arctic (31, 32). Under nitrate-reducing conditions, hydrocarbon degradation by Arctic soil isolates was not limited by low temperatures with little difference seen in hydrocarbon degradation at 20 °C as compared to that at 7 °C (6). We hypothesized that, in fertilized soils, denitrifiers would contribute significantly to hydrocarbon degradation. If so, it may not be necessary to aerate contaminated soil sites. It is even possible that denitrification-based hydrocarbon biodegradation may prove to be more active at lower temperatures than alternative treatments involving expensive soil heating. This would allow low cost in situ treatment to proceed at a sufficient rate to achieve remediation goals in a reasonable time, thus reducing operational costs and the environmental risk. To investigate these possibilities, we examined the differences in hydrocarbon biodegradation and the structure of denitrifying communities in nutrient-treated, hydrocarbon contaminated soils in Antarctica.
Materials and Methods Field Site and Experiment. The study site is located in East Antarctica near the Old Casey Research Station and has been described elsewhere (2, 33). Approximately 1000 m3 of soil is contaminated from historical fuel spills that occurred over 20 years ago. Total petroleum hydrocarbons (TPH) concentrations at the site range between 10 000 and 47 000 mg kg-1. The main contaminant is Special Antarctic Blend (SAB) diesel, which contains predominantly n-alkanes. A landfarming trial was initiated in 1998 and has been previously described (2). Briefly, contaminated soil was sieved (
