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Even though oil loss was enhanced by nutrient addition, active bioremediation in the form of exogenous nutrient addition might not be appropriate in cases where background nutrient levels are already sufficiently high to support high intrinsic rates of hydrocarbon biodegradation.

A most-probable-number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish between these two groups. Separate 96-well microtiter plates are used to estimate the sizes of these two populations. The alkane-degrader MPN method uses hexadecane as the selective growth substrate and positive wells are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 degrees C. Polycyclic aromatic hydrocarbon degraders are grown on a mixture of phenanthrene, anthracene, fluorene, and dibenzothiophene in a second plate. Positive wells turn yellow to greenish-brown from accumulation of the partial oxidation products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and selective. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective substrates do not produce false positive responses even when the inoculum density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and composition of hydrocarbon-degrading microbial populations.

The rates of microbial Fe(III) reduction of three sizes of hematite nanoparticles by Geobacter sulfurreducens were measured under two H2 partial pressures (0.01 and 1 atm) and three pH (7.0, 7.5, and 8.0) conditions. Hematite particles with mean primary particle sizes of 10, 30, and 50 nm were synthesized by a novel aerosol method that allows tight control of the particle size distribution. The mass-normalized reduction rates of the 10 and 30 nm particles were comparable to each other and higher than the rate for the 50 nm particles. However, the surface area-normalized rate was highest for the 30 nm particles. Consistent with a previously published model, the reduction rates are likely to be proportional to the bacteria-hematite contact area and not to the total hematite surface area. Surface area-normalized iron reduction rates were higher than those reported in previous studies, which may be due to the sequestration of Fe(II) through formation of vivianite. Similar initial reduction rates were observed under all pH and H2 conditions studied.

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A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbon-degrading 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 method's 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.

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.