Brian A. Wrenn

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.

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.

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.

Nutrient and Oxygen Concentrations within the Sediments of an Alaskan Beach Polluted with the Exxon Valdez Oil Spill

Measurements of the background concentrations of nutrients, dissolved oxygen (DO), and salinity were obtained from a beach that has oil from the Exxon Valdez oil spill in 1989. Two transects were set across the beach, one passed through an oil patch while the other transect was clean. Three pits were dug in each transect, and they ranged in depth from 0.9 to 1.5 m. The DO was around 1.0 mg L(-1) at oiled pits and larger than 5 mg L(-1) at clean pits. The average nutrient concentrations in the beach were 0.39 mg-N L(-1) and 0.020 mg-P L(-1). Both concentrations are lower than optimal values for oil biodegradation (2 to 10 mg-N L(-1) and 0.40 to 2.0 mg-P L(-1)), which suggests that they are both limiting factors for biodegradation. The lowest nitrate and DO values were found in the oiled pits, leading to the conclusion that microbial oil consumption was probably occurring under anoxic conditions and was associated to denitrification. We present evidence that the oxygen level may be a major factor limiting oil biodegradation in the beaches.

A model for the effects of primary substrates on the kinetics of reductive dehalogenation

A kinetic model that describes substrate interactions during reductive dehalogenation reactions is developed. This model describes how the concentrations of primary electron-donor and -acceptor substrates affect the rates of reductive dehalogenation reactions. A basic model, which considers only exogenous electron-donor and -acceptor substrates, illustrates the fundamental interactions that affect reductive dehalogenation reaction kinetics. Because this basic model cannot accurately describe important phenomena, such as reductive dehalogenation that occurs in the absence of exogenous electron donors, it is expanded to include an endogenous electron donor and additional electron acceptor reactions. This general model more accurately reflects the behavior that has been observed for reductive dehalogenation reactions. Under most conditions, primary electron-donor substrates stimulate the reductive dehalogenation rate, while primary electron acceptors reduce the reaction rate. The effects of primary substrates are incorporated into the kinetic parameters for a Monod-like rate expression. The apparent maximum rate of reductive dehalogenation (qm, ap) and the apparent half-saturation concentration (Kap) increase as the electron donor concentration increases. The electron-acceptor concentration does not affect qm, ap, but Kap is directly proportional to its concentration.

TESTING THE EFFICACY OF OIL SPILL BIOREMEDIATION PRODUCTS

ABSTRACT Ten bioremediation products were tested in laboratory respirometers for their ability to enhance the biodegradation of artificially weathered Alaska North Slope crude oil compared to natur...

Evaluation of a model for the effects of substrate interactions on the kinetics of reductive dehalogenation

The effects of primary electron-donor and electron-acceptor substrates on the kinetics of TCA biodegradation in sulfate-reducing and methanogenic biofilm reactors are presented. Of the common anaerobic electron-donor substrates that were tested, only formate stimulated the TCA biodegradation rate in both reactors. In the sulfate-reducing reactor, glucose also stimulated the reaction rate. The effects of formate and sulfate on TCA biodegradation kinetics were analyzed using a model for primary substrate effects on reductive dehalogenation. Although some differences between the model and the data are evident, the observed responses of the TCA degradation rate to formate and sulfate were consistent with the model. Formate stimulated the TCA degradation rate in both reactors over the entire range of TCA concentrations that were studied (from 50 μg TCA/L to 100 mg TCA/L). The largest effects occurred at high TCA concentrations, where the dehalogenation kinetics were zero order. Sulfate inhibited the first-order TCA degradation rate in the sulfate-reducing reactor, but not in the methanogenic reactor. Molybdate, which is a selective inhibitor of sulfate reduction, stimulated the TCA removal rate in the sulfate-reducing reactor, but had no effect in the methanogenic reactor.

Influence of tide and waves on washout of dissolved nutrients from the bioremediation zone of a coarse-sand beach: Application in oil-spill bioremediation

Abstract Successful bioremediation of oil-contaminated beaches requires maintenance of a sufficient quantity of growth-limiting nutrients in contact with the oiled beach material. A conservative tracer study was conducted on a moderate-energy, sandy beach on Delaware Bay to estimate the washout rate for dissolved nutrients from the bioremediation zone at different stages during the lunar tidal cycle. When an aqueous solution of the conservative tracer (LiNO3) was applied to the beach surface in the upper intertidal zone at the full moon spring tide, it was completely removed within one day. When it was applied at neap tide, however, the tracer persisted in the bioremediation zone for several days. The amount of nutrient remaining in the bioremediation zone was highly correlated with the maximum extent to which the treated area had previously been submerged by water at high tide: submergence resulted in nearly complete removal of dissolved compounds from the bioremediation zone. This high rate of nutrient washout was confirmed by daily monitoring of nutrient concentrations in the bioremediation zone during an oil-spill bioremdiation field study that was conducted on a nearby beach.