1. Kelly McFarlin, Mary Beth Leigh and Robert Perkins University of Alaska Fairbanks Indigenous Microorganisms Degrade Oil in Arctic Seawater Introduction Spill prevention techniques and response options are important considerations of exploration and production operations. After 40 years of research, many international organizations have documented evidence that the use of dispersants as an oil spill response can result in a lower overall environmental impact than otherwise may be caused by untreated oil. The use of dispersants rapidly moves oil from the air/water interface into the water column reducing the environmental impact of spilled oil on organisms that reside or move through this layer (e.g., marine birds and mammals, surface dwelling eggs, larvae and juveniles) and by preventing oil from impacting shorelines (Nordtug and Johansen, 2007). As oil is dispersed into the water column the small oil droplets rapidly dilute to very low concentrations. Dispersing the oil enhances natural biodegradation because of the increased surface area resulting from the small droplets (Lessard and Demarco, 2000); (George-Ares and Clark, 2000). The biodegradation of chemically dispersed oil and non- chemically dispersed oil is well documented in temperate conditions, but little is understood concerning the biodegradation potential of arctic marine microorganisms. A laboratory was assembled in Barrow, Alaska, to allow direct access to test the biodegradation potential of indigenous microorganisms. Discussion Arctic microorganisms degraded more chemically dispersed fresh crude than non-chemically dispersed fresh crude at 2°C. Arctic microorganisms degraded more chemically dispersed fresh crude with a low-level nutrient addition (0.1 mM nitrate, 0.15 mM phosphate) than chemically dispersed fresh crude without nutrients at 2°C. Microbial degradation and mineralization occurred with and without Corexit 9500 © at winter temperatures (-1°C). As expected, the initial modification of the hydrocarbons detectable via GC-MS, termed primary biodegradation surpassed mineralization measured via respirometry. The natural consortia of microorganisms degraded a higher percentage of fresh ANS crude oil than the weathered ANS crude. Test 1: Biodegradation in Seawater Collected in October at 2°C Figure 1. Percent mineralization calculated from the theoretical oxygen demand (ThOD) in a sealed respirometer experiment containing 10 ppm of fresh crude oil treatment in the presence and absence of Corexit 9500 and 1% Bushnell Haas growth medium at 2 ⁰ C, (1% nutrients represents 1% of the recommended volume of Bushnell Haas). Objective To determine the ability of indigenous microbes to biodegrade chemically dispersed and non –chemically dispersed crude oil in Arctic marine open water environments of the Beaufort and Chukchi Seas. Test 2: Biodegradation in Seawater Collected in March at -1°C Figure 2. Initial and final GC-MS of sealed respirometer experiment recovered from a 10 ppm loading of fresh ANS crude oil in the presence and absence of Corexit 9500 (1:20) and 0.5% Bushnell Haas at 2°C. Methods Two methods were used to address the objective: (1)manometric respirometry (2)gas chromatography - mass spectrometry (GC-MS) analysis of residual total petroleum hydrocarbons (TPH). Manometric respirometry measures oxygen consumption based upon a theoretical oxygen demand. Microorganisms in the sealed sample flasks consume O 2 and emit CO 2 in a sealed flasks. The CO 2 is absorbed by an alkali CO 2 trap which initiates an oxygen generating process to restore a pressure equilibrium in the sample flask. The measurable amount of oxygen generated is proportional to the ultimate breakdown of the sample to CO 2 and H 2 O; termed ultimate biodegradation or mineralization. GC-MS analysis of sample extracts is used to determine primary biodegradation; the loss or disappearance of specific compounds. Each sample was extracted three times by a liquid-liquid methylene chloride extraction technique and analyzed by GC-MS. Initial and final petroleum hydrocarbons were measured using 17α(H),21β(H)-hopane as a conserved internal marker, (Prince et al, 1994). Table 1. Comparison of Biodegradation as measured with GC-MS (primary biodegradation) and respirometry (mineralization) containing 10 ppm fresh crude oil, Corexit 9500 (1:20) and 1% of recommended Bushnell Haas. Table 2. Comparison of Biodegradation as measured with GC-MS (primary biodegradation) and respirometry (mineralization) containing 12 mg/L of 20% weather crude, 0.5% of recommended Bushnell Haas, with and without Corexit 9500 (1:20). Conclusion Biodegradation and mineralization occurred in fresh and 20% weathered ANS crude at both 2°C and -1°C with indigenous Arctic microorganisms. The presence of Corexit 9500 © was not inhibitory to natural microbial degradation. The addition of Corexit 9500 © enhanced the degradation of both fresh and weathered oil both in the presence and absence of low-level nutrients. Future Work An additional winter study is underway using chemically dispersed and non-chemically dispersed fresh crude oil with an amended chemical dispersion method. References 1. George-Ares, A. and J. R. Clark, "Aquatic Toxicity of Two Corexit Dispersants", Chemosphere, 40:897-906, 2000. 2. Lessard, R. R. and G. Demarco, "The Significance of Oil Spill Dispersants", Spill Science & Technology Bulletin, 6(1):59-68, 2000. 3. Nordtug, T. and Ø. Johansen, “Effect of Dispersed Oil – Status of Knowledge, Criteria for Establishment of Threshold Values and Recommendations for Further Experimental Studies” Report SINTEF A3036, 2007. 4. Prince, R. C., Elmendorf, D. L., Lute, J. R., Hsu, C. S., Haith, C. E., Senius, J. D., Dechert, G. J., Douglas, G. S. and Butler, E. L. “17a(H),21b(H)-hopane as a conserved internal marker for estimating the biodegradation of crude oil”, Environ. Sci. Technology, 28:142-145, 1994. kmmcfarlin@alaska.edu raperkins@alaska.edu Acknowledgements: Supported by a Joint Industry Project; Exxon-Mobil, Statoil, Shell, and Conoco-Philips. Project Manger: NewFields Northwest.