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Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.

The concentration of mercury in water and sediment and in the oil extracted from water and sediment was determined for samples collected in Colgate Creek, located in Baltimore Harbor of the Chesapeake Bay. The concentration of mercury in the oil was 4,000 times higher than in sediment and 300,000 times higher than in water samples. The mercury-resistant bacterial populations of the samples studied have been shown to degrade oil, suggesting these bacteria to be a significant factor in the degradation of oil in Colgate Creek.

Crude and fuel oils were compared for ability to support growth of a mixed population of estuarine bacteria. A total of four oils, two crude and two fuel oils, were examined. It was found that each of the oils supported a unique population of bacteria and yeasts, with respect to generic composition. Low-sulfur, high-saturate, South Louisiana crude oil was found to be highly susceptible to degradation. In contrast, the dense, high-sulfur, high-aromatic, Bunker C fuel oil was strongly refractory to microbial degradation.

Prototheca zopfii is an achlorophyllous alga which degrades oil. It has been found to degrade 10 and 40% of a motor oil and crude oil, respectively, when tested under appropriate conditions. Degradation of the crude oil observed in this study compares well with the amount of degradation accomplished by bacteria. P. zopfii was found to degrade a greater percentage of the aromatic hydrocarbons in motor oil than of the saturated hydrocarbons and a greater percentage of saturated hydrocarbons in crude oil than of aromatic hydrocarbons. Resins and asphaltens were produced during degradation of motor oil, whereas these fractions in crude oil were degraded. P. zopfii did not demonstrate preferential utilization of lower homologues of cycloalkanes and aromatics as has been observed with bacteria.

Degradation of mixed hydrocarbon substrate in a system comprising water from an environment relatively free of oil and a sediment inoculum from an oil-contaminated site was significantly greater than when sediment from the non-oil-contaminated environment served as inoculum. Mixed hydrocarbon substrate, however, was observed to have a limiting effect on the growth of autochthonous bacteria from the non-oil-contaminated estuarine source. Growth and cell yield were similarly reduced when marine sediment bacteria were cultured in seawater supplemented with mixed hydrocarbon substrate. The addition of a South Louisiana crude oil or a No. 2 fuel oil to water and sediment collected from a marsh area of Chesapeake Bay showed no limiting effects on growth of the total heterotrophic microbial flora when examined over a 28-day period. However, results of these studies indicate that the effects of petroleum on microoganisms should be examined carefully under conditions closely approximating those in situ.

A variety of factors, including concentration of oil, antibiotics, dyes, and inoculum washes, were examined to determine their effect on the total counts of microorganisms on oil-containing media. The media found to be best for enumerating petroleum-degrading microorganisms contained 0.5% (vol/vol) oil and 0.003% phenol red, with Fungizone added for isolating bacteria and streptomycin and tetracycline added for isolating yeasts and fungi. Washing the inoculum did not improve recovery of petroleum degraders. Specifically, silica gel-oil medium and a yeast medium are recommended for enumeration of petroleum-degrading bacteria and yeasts and fungi, respectively. It is suggested that counts of petroleum degraders be expressed as percentage of the total population rather than total numbers of petroleum degraders per se. Incubation temperature and presence of oil was found to influence the numbers of petroleum-degrading microorganisms at a given sampling site.

Five axenic cultures and a mixed culture were examined for ability to degrade South Louisiana, Brass River Nigerian, Anaco Venezuelan, and Altamont crude oils. A wax was observed during microbial degradation of Altamont crude oil, but not during weathering of the oil. The high-boiling n-alkanes in the wax were associated with microbial degradation of the oil and appeared to be similar to components of tarballs found in the open ocean.

Rates of microbial biodegradation of components of South Louisiana crude oil were determined by computer analysis of data obtained from computerized mass spectrometry. Total residue of the oil decreased exponentially with time, with maximum decrease noted at logarithmic phase, whereas asphaltenes and resins increased at the logarithmic phase of growth. Saturates decreased continuously during growth. Microbial degradation of components of crude oil was concluded to be a dynamic process.

Chesapeake Bay sediment bacteria from oil-contaminated and oil-free environments were compared for their ability to utilize a South Louisiana crude oil. Preferential solubility, column chromatography, gas-liquid chromatography, and computerized mass spectrometry were used to provide new and useful information regarding biodegradation of fractions and components of the crude oil. Vibrio, Pseudomonas, and Acinetobacter spp. were isolated from the culture inoculated with oil-contaminated sediment, whereas coryneforms and Pseudomonas spp. were isolated from the culture inoculated with oil-free sediment. Microorganisms from the oil-free sediment produced greater quantities of polar n-pentane-insoluble components (asphaltenes) after degradation, whereas microorganisms from the oil-contaminated sediments provided greater degradation of saturated and aromatic hydrocarbons.

A total of 99 strains of petroleum-degrading bacteria isolated from Chesapeake Bay water and sediment were identified by using numerical taxonomy procedures. The isolates, together with 33 reference cultures, were examined for 48 biochemical, cultural, morphological, and physiological characters. The data were analyzed by computer, using both the simple matching and the Jaccard coefficients. Clustering was achieved by the unweighted average linkage method. From the sorted similarity matrix and dendrogram, 14 phenetic groups, comprising 85 of the petroleum-degrading bacteria, were defined at the 80 to 85% similarity level. These groups were identified as actinomycetes (mycelial forms, four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococcus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (two clusters), and Sphaerotilus natans. It is concluded that the degradation of petroleum is accomplished by a diverse range of bacterial taxa, some of which were isolated only at given sampling stations and, more specifically, from sediment collected at a given station.

Bacteria from water and sediment of an oil-polluted harbor were examined for ability to degrade petroleum. Water samples contained a greater variety of bacterial species capable of degrading petroleum than sediment. Cultures from both water and sediment contained Pseudomonas and Acinetobacter spp. Bacteria present in the water samples produced significantly greater degradation of 2-,3-,4-, and 5-ring cycloalkanes and mono-, di-, tri-, tetra-, and pentaaromatics compared with bacteria in sediment samples.