Jon E. Lindstrom

The Roles of Photooxidation and Biodegradation in Long-term Weathering of Crude and Heavy Fuel Oils

Abstract Although spilled oil is subject to a range of natural processes, only combustion, photooxidation and biodegradation destroy hydrocarbons and remove them from the biosphere. We present laboratory data that demonstrate the molecular preferences of these processes, and then examine some oil residues collected from previously documented releases to confirm the important roles that these processes play in removing spilled oil from both marine and terrestrial environments.

Biodegradation of petroleum hydrocarbons at low temperature in the presence of the dispersant Corexit 9500

Our study examined the effects of Corexit 9500 and sediment on microbial mineralization of specific aliphatic and aromatic hydrocarbons found in crude oil. We also measured gross mineralization of crude oil, dispersed crude oil and dispersant by a marine microbial consortium in the absence of sediment. When provided as carbon sources, our consortium mineralized Corexit 9500 the most rapidly, followed by fresh oil, and finally weathered oil or dispersed oil. However, mineralization in short term assays favored particular components of crude oil (2-methyl-naphthalene > dodecane > phenanthrene > hexadecane > pyrene) and was not affected by addition of nutrients or sediment (high sand, low organic carbon). Adding dispersant inhibited hexadecane and phenanthrene mineralization but did not affect dodecane and 2-methyl-naphthalene mineralization. Thus, the effect of dispersant on biodegradation of a specific hydrocarbon was not predictable by class. The results were consistent for both high and low oiling experiments and for both fresh and weathered oil. Overall, our results indicate that environmental use of Corexit 9500 could result in either increases or decreases in the toxicity of residual oil through selective microbial mineralization of hydrocarbons.

Microbial community analysis: a kinetic approach to constructing potential C source utilization patterns

Abstract The analysis of multiple substrate metabolism by assemblages of bacterial strains may be used to differentiate inocula from environmental samples. Biolog plates, 96-well microtiter plates containing nutrients, a single carbon test substrate in each well and a tetrazolium redox dye to monitor substrate oxidation, have been used for this purpose. One of the difficulties faced by users of this technique is determining which substrates have been metabolized. Reliance on single-time-point absorbance data for each well is problematic due to variably non-linear rates of color development for each well. Previous efforts to use color-normalized single plate readings have been successful in discriminating between environmental sample types, but substrate-use contributions to sample classifications vary depending on the duration of the plate incubation. We present a model based on the logistic equation for density-dependent population growth providing a good (low χ 2 ) fit to the sigmoidal kinetics of color development data. The kinetic parameters generated by the model can be used as surrogates for single-time-point data in constructing carbon source utilization patterns, and contribution of substrate use to sample classification does not depend on incubation time. This technique obviates the need to choose the time following inoculation to read the plate absorbance data and also provides two kinetic parameters that are invariant with respect to inoculum density. We provide a comparison of community potential substrate use analyses using single-time-point microplate data and parameters from our kinetic model.

Long-term effects on microbial communities after a subarctic oil spill

Abstract A combination of microbial assays was used to examine soil population structure and community-level metabolism at the site of a 1976 experimental crude oil spill conducted in Alaska. Estimates of total bacterial numbers and soil C mineralization potentials were not significantly different between pristine and hydrocarbon-affected soils. In contrast, net N mineralization potential was lower, metabolically active (FDA stain) bacteria were less abundant and hydrocarbon degrading microbes were more abundant in the oiled soils. Additionally, the effects of dilution on the kinetics of community-level substrate use were examined in multiple substrate microplates. Microplate kinetic patterns varied less with dilution and by season in oiled soils. In oiled soils, absence of seasonal variation in soil C mineralization potentials, coupled with the microplate data, indicated that population diversity (evenness, richness or both) was diminished compared to the pristine soils. Further analysis of microplate data suggested that the communities surviving in the oiled soils may be considered metabolic generalists. By using several independent microbial assays, differences in soil microbial community structure attributable to oiling could be seen decades after the spill event.

Distribution of hydrocarbon-degrading microorganisms in sediments from Prince William Sound, Alaska, following the Exxon Valdez oil spill

Abstract Biodegradation by naturally occurring populations of microorganisms is a major mechanism for the removal of petroleum from the environment. Therefore, measurements of microbial populations are an important component of contaminated site assessment studies. Over a 3 year period following the T V Exxon Valdez oil spill in Prince William Sound, Alaska, we counted numbers of hydrocarbon-degrading microorganisms in intertidal and subtidal sediments affected by the spill. We found significantly higher numbers of hydrocarbon-degrading microorganisms at sites within the path of the oil slick than at reference sites, indicating rapid acclimation of the resident microbial populations. In offshore surface sediments, we saw a temporal increase in numbers of hydrocarbon-degrading microorganisms. Our data suggest that microbial measurements are good indicators of exposure of sediments in Prince William Sound to hydrocarbons and of mobilization of oil to surface sediments offshore over time.

Integral biopile components for successful bioremediation in the Arctic

Timely bioremediation of petroleum-contaminated soils in the Arctic is possible with innovative engineering and environmental manipulation to enhance microbial activity beyond the natural effective season. Key parameters in extending the period of beneficial microbial activity in Arctic biopiles are temperature and substrate availability. A multidisciplinary team of engineers, microbiologists and electricians has designed and installed a thermally enhanced biopile at a diesel-contaminated gravel pad in Prudhoe Bay, AK. The combination of bioventing with active warming, fertilization and power cycling is working toward timely remediation at this site. Primary components for success are the (1) thermal insulation system (TIS) design, (2) microbiological monitoring plan, and (3) power optimization. (Alternate power sources are considered for use at this and future remote bioremediation sites.) This paper discusses the TIS design and extension of the effective treatment season, fertilization and the results of a treatability study that compared simple fertilization with application of commercially available bioproducts under simulated site conditions, and adjusting power utilization to prevent permafrost thaw. Through an integrated approach to bioremediation, we are treating diesel-contaminated soils at an Arctic site.

Weathering of a subarctic oil spill over 25 years: the Caribou-Poker Creeks Research Watershed experiment

A small experimental oil spill conducted in an open black spruce forest within the Caribou-Poker Creeks Research Watershed (CPCRW), 48 km north of Fairbanks, AK, in the winter of 1976 was designed to examine the effects of crude oil spills in permafrost terrain. No clean-up was attempted, and the site now provides an opportunity to follow the natural weathering of spilled oil under these conditions. In summer 2001, more than 25 years after the spill, we sampled soils from the spill plot and a nearby reference plot to determine how the oil had weathered, and to assess microbial populations and activity. All samples collected from the oiled plot contained substantial amounts of methylene chloride extractable oil, between 4% and 66% by weight. Using 17α(H)21β(H)hopane as a conserved internal marker within the oil, we determined that while some heavily oiled samples were almost unchanged since the spill, others had lost more than 80% of their initial hydrocarbon. Evaporation, biodegradation and photooxidation all seem to have played important roles in this process, but to varying degrees in different samples. Assays of culturable populations of total heterotrophs and crude oil emulsifiers, and mineralization potentials for hexadecane and phenanthrene, indicate that the microbial population in the oiled soils has remained acclimated to degrade hydrocarbons. We conclude that natural weathering processes will eventually lead to the removal of much of the hydrocarbon from these heavily oiled subarctic soils; however, the combination of low rates of nutrient turnover, a short thaw season, and high hydrocarbon concentrations will result in the persistence of oil residue for many more decades. Finding an environmentally appropriate cleanup technology for sites like this remains an important challenge for future research.

UAF radiorespirometric protocol for assessing hydrocarbon mineralization potential in environmental samples

Following the EXXOn Valdez oil spill, a radiorespirometric protocol was developed at the University of Alaska Fairbanks (UAF) to assess the potential for microorganisms in coastal waters and sediments to degrade hydrocarbons. The use of bioremediation to assist in oil spill cleanup operations required microbial bioassays to establish that addition of nitrogen and phosphorus would enhance biodegradation. A technique assessing 1-14C-n-hexadecane mineralization in seawater or nutrient rich sediment suspensions was used for both of these measurements. Hydrocarbon-degradation potentials were determined by measuring mineralization associated with sediment microorganisms in sediment suspended in sterilized seawater and/or marine Bushnell-Haas broth. Production of 14CO2 and CO2 was easily detectable during the first 48 hours with added hexadecane levels ranging from 10 to 500 mg/l of suspension and dependent on the biomass of hydrocarbon degraders, the hydrocarbon-oxidation potential of the biomass and nutrient availability. In addition to assessment of the hydrocarbon-degrading potential of environmental samples, the radiorespirometric procedure, and concomitant measurement of microbial biomass, has utility as an indicator of hydrocarbon contamination of soils, aqueous sediments and water, and can also be used to evaluate the effectiveness of bioremediation treatments.

Effects of chitin on microbial emulsification, mineralization potential, and toxicity of bunker C fuel oil.

Bunker C, one of the most frequently spilled petroleum products in the US, is difficult to remove from oiled surfaces and is relatively recalcitrant to biodegradation; therefore, emulsification and biodegradability must be optimized before bioremediation can be considered a viable treatment option. Sand from a freshly oiled beach near Dutch Harbor, Alaska, was incubated at 10 degrees C with nutrients (Bushnell-Haas (BH)) or nutrients with crab shell chitin (BH-C). BH-C amendment resulted in greater numbers of bunker C emulsifiers and greater mineralization potentials for hexadecane, phenanthrene, and fluorene than with BH only. Compared to BH alone, mineralization potentials for bunker C also were higher in BH-C, with an estimated 8% of fuel oil mineralized after 6 weeks. Microbially emulsified oil was more toxic than in uninoculated controls (p < 0.05) as measured by Microtox assays. However, toxicity was significantly lower in BH-C than BH after 4 and 6 weeks incubation (p < 0.05).

Toxicity of Dispersants and Dispersed Oil To An Alaskan Marine Organism

ABSTRACT The University of Alaska Fairbanks (UAF) conducted toxicity assays on Alaskan tanner crab larvae (Chionoecetes bairdi) using the oil dispersant Corexit 9500, Alaska North Slope (ANS) crude...