Albert D. Venosa

Biodegradation of Crude Oil Contaminating Marine Shorelines and Freshwater Wetlands

Abstract This paper is a summary of the various factors influencing weathering of oil after it has been released into the environment from a spill incident. Special emphasis has been placed on biodegradation processes. Results from two field studies conducted in 1994 and 1999 involving bioremediation of an experimental oil spill on a marine sandy shoreline in Delaware and a freshwater wetland on the St. Lawrence River in Quebec, Canada have been presented in the paper.

A numerical model for density-and-viscosity-dependent flows in two-dimensional variably saturated porous media

We present a formulation for water flow and solute transport in two-dimensional variably saturated media that accounts for the effects of the solute on water density and viscosity. The governing equations are cast in a dimensionless form that depends on six dimensionless groups of parameters. These equations are discretized in space using the Galerkin finite element formulation and integrated in time using the backward Euler scheme with mass lumping. The modified Picard method is used to linearize the water flow equation. The resulting numerical model, the MARUN model, is verified by comparison to published numerical results. It is then used to investigate beach hydraulics at seawater concentration (about 30 g l−1) in the context of nutrients delivery for bioremediation of oil spills on beaches. Numerical simulations that we conducted in a rectangular section of a hypothetical beach revealed that buoyancy in the unsaturated zone is significant in soils that are fine textured, with low anisotropy ratio, and/or exhibiting low physical dispersion. In such situations, application of dissolved nutrients to a contaminated beach in a freshwater solution is superior to their application in a seawater solution. Concentration-engendered viscosity effects were negligible with respect to concentration-engendered density effects for the cases that we considered.

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure.

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The methods accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

The dose-response relationship between No. 2 fuel oil and the growth of the salt marsh grass, Spartina alterniflora

The effect of No. 2 fuel oil on the biomass production of the salt marsh plant, Spartina alterniflora, was studied in a greenhouse dose-response experiment. S. alterniflora were transplanted into soil with 10 dosage levels of No. 2 fuel oil ranging from 0 to 456 mg g(-1) dry soil. Three months after transplantation, values for plant biomass, stem density, and shoot height decreased significantly with increasing fuel oil level in a dose-response fashion. Evapo-transpiration rates were correlated with the total biomass response. Relative to the control, a significant decrease in total (above- plus below-ground) plant biomass was observed at concentrations above 57 mg g(-1) dry soil. Within the 3-month experimental period, detrimental effects on below-ground biomass accumulation and bioluminescence of the marine bacterium Viberio fisheri in the Microtox Solid Phase Test were observed at oil concentrations >29 mg g(-1) dry soil, suggesting that biological effects of oil within the sediment matrix may be more pronounced than on above-ground biomass, requiring a dosage 228 mg g(-1) dry soil to elicit a significant detrimental effect. Hence, measurements of oil effects with biological end-points based solely on above-ground responses may underestimate the potential impacts of petroleum hydrocarbon spills, especially when the oil has penetrated the soil. While S. alterniflora was proved to be relatively tolerant to the No. 2 fuel oil spills, its effectiveness in phytoremediation operations may be limited at fuel oil levels 228 mg g(-1) dry soil, as both plant growth and microbial activity may be constrained.

Use of hopane as a conservative biomarker for monitoring the bioremediation effectiveness of crude oil contaminating a sandy beach

Much of the variability inherent in crude oil bioremediation field studies can be eliminated by normalizing analyte concentrations to the concentration of a nonbiodegradable biomarker such as hopane. This was demonstrated with data from a field study in which crude oil was intentionally released onto experimental plots on the Delaware shoreline. Five independent replicates of three treatments were examined: no nutrient addition, addition of inorganic mineral nutrients alone, and nutrient addition plus indigenous oil-degrading microorganisms from the site. Samples collected biweekly were analyzed for the Most Probable Numbers (MPNs) of alkane and aromatic degraders and oil component analysis by GC/MS. The data were normalized to either the mass of sand that was extracted or to the concentration of hopane that was measured. Hopane normalization enabled detection of significant treatment differences in hydrocarbon biodegradation that were not detected when the data were normalized to sand mass. First-order loss rates for the hopane-normalized data were lower than those for the sand-normalized data because hopane normalization accounts only for loss due to biodegradation whereas sand normalization includes all loss mechanisms. Plots amended with nutrients alone and nutrients plus the inoculum showed enhanced removal of hydrocarbons compared to unamended control plots. However, no differences were detected between the nutrient-amended plots and the nutrient/inoculum-amended plots.

Biodegradability of Corexit 9500 and dispersed South Louisiana crude oil at 5 and 25 °C.

The reported persistence of the dioctyl sodium sulfosuccinate (DOSS) surfactant in Corexit 9500 in the oil plumes formed during the Deepwater Horizon oil spill has contributed to concerns regarding the biodegradability and bioavailability of dispersed oil and dispersants used as an oil spill countermeasure in the Gulf of Mexico. We studied the biodegradation of DOSS and dispersed South Louisiana crude oil (SLC) in laboratory microcosms. Two oil-degrading cultures from the Gulf of Mexico were isolated, one from the surface (meso) and one from close to the area of the Macondo well (cryo). Each was enriched on SLC, the former at 25 °C, the latter at 5 °C. Results indicated that the meso culture rapidly and completely degraded DOSS, alkanes, and aromatics. The cryo culture metabolized the same compounds but with a lag of 28 d and a remaining residual of iso-alkanes, n-C(30-35), and the 4-ring PAHs.

The Baffled Flask Test for Dispersant Effectiveness: A Round Robin Evaluation of Reproducibility and Repeatability

Abstract After two previous investigations demonstrated that the baffled flask test was an effective and reproducible method for screening the effectiveness of dispersant products in the laboratory, the US Environmental Protection Agency decided that before the new protocol could be considered for replacement of the swirling flask test, it would have to be subjected to an interlaboratory round robin investigation. This paper describes how the round robin evaluation was carried out and presents repeatability and reproducibility calculations that demonstrate the superiority of the new method over previous methods. Probabilities for passing various threshold levels of effectiveness values were computed, and the results will be used by the Agency to develop final pass–fail decision rules for dispersant manufacturers wishing to have their products listed on the national contingency plan product schedule.

Optimal Nitrate Concentration for the Biodegradation of n-Heptadecane in a Variably-Saturated Sand Column

Bioremediation of oil spills on beaches commonly involves the addition of nutrients (especially nitrogen and phosphorus) to stimulate the growth of indigenous oil-degrading bacteria. Very little information is available regarding the relationship between nutrient concentration and the rate of oil biodegradation. This information is necessary to design an appropriate nutrient delivery technology. We used continuous-flow beach microcosms containing heptadecane-coated sand (2.0 g per kg of dry sand) to evaluate the effect of nitrate concentration on the hydrocarbon biodegradation rate. Heptadecane biodegradation was determined by monitoring oxygen consumption and carbon dioxide production in the microcosms. The maximum biodegradation occurred at 2.5 mg nitrate-N l−1. Nitrogen recycling by the biomass was evidenced by the presence of microbial activity at zero influent nitrate concentration.

Nutrient transport during bioremediation of contaminated beaches: Evaluation with lithium as a conservative tracer

Bioremediation of oil-contaminated beaches typically involves fertilization with nutrients that are thought to limit the growth rate of hydrocarbon-degrading bacteria. Much of the available technology involves application of fertilizers that release nutrients in a water-soluble form prior to bacterial uptake. Oil contamination of coastal areas from offshore spills usually occurs in the intertidal zone. This area is subjected to periodic flooding by a combination of tides and waves, which can affect the washout rate of water-soluble nutrients from the contaminated area. We used lithium nitrate as a conservative tracer to study the rate of nutrient transport in a low-energy, sandy beach on the southwestern shore of Delaware Bay. The rate of tracer washout from the bioremediation zone (i.e. the upper 25 cm below the beach surface) was more rapid when the tracer was applied at spring tide (when the tidal amplitude is largest) than at neap tide, but the physical path taken by the tracer plume was not affected. In both cases, the tracer plume moved vertically into the beach subsurface and horizontally through the beach in a seaward direction. The vertical transport was probably driven by waves infiltrating through the unsaturated zone. Hydraulic gradients that were established by differences between the rate at which the elevation of the water table in the beach changed and the rate at which the tide rose and fell contributed to horizontal movement of the plume.

Effects of nitrogen source on crude oil biodegradation

SummaryThe effects of NH4Cl and KNO3 on biodegradation of light Arabian crude oil by an oil-degrading enrichment culture were studied in respirometers. In poorly buffered sea salts medium, the pH decreased dramatically in cultures that contained NH4Cl, but not in those supplied with KNO3. The ammonia-associated pH decline was severe enough to completely stop oil biodegradation as measured by oxygen uptake. Regular adjustment of the culture pH allowed oil biodegradation to proceed normally. A small amount of nitrate accumulated in all cultures that contained ammonia, but nitrification accounted for less than 5% of the acid that was observed. The nitrification inhibitor, nitrapyrin, had no effect on the production of nitrate or acid in ammonia-containing cultures. When the culture pH was controlled, either by regular adjustment of the culture pH or by supplying adequate buffering capacity in the growth medium, the rate and extent of oil biodegradation were similar in NH4Cl- and KNO3-containing cultures. the lag time was shorter in pH-controlled cultures supplied with ammonia than in nitrate-containing cultures.