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Experiments were carried out to develop methods to generate well-characterized, polycyclic aromatic hydrocarbon (PAH)-spiked, aged but minimally altered sediments for fate, biodegradation, and bioavailability experiments. Changes in indigenous bacterial populations were monitored in mesocosms constructed of relatively clean San Diego Bay sediments, with and without exposure to gamma radiation, and then spiked with five different PAHs and hexadecane. While phenanthrene and chrysene degraders were present in the unspiked sediments and increased during handling, PAH spiking of nonirradiated sediments led to dramatic increases in their numbers. Phenotypic characterization of isolates able to grow on phenanthrene or chrysene placed them in several genera of marine bacteria: Vibrio, Marinobacter or Cycloclasticus, Pseudoalteromonas, Marinomonas, and HALOMONAS: This is the first time that marine PAH degraders have been identified as the latter two genera, expanding the diversity of marine bacteria with this ability. Even at the highest irradiation dose (10 megarads), heterotrophs and endospore formers reappeared within weeks. However, while bacteria from the unirradiated sediments had the capacity to both grow on and mineralize 14C-labeled phenanthrene and chrysene, irradiation prevented the reappearance of PAH degraders for up to 4 months, allowing spikes to age onto the sediments, which can be used to model biodegradation in marine sediments.

In-situ bioremediation of benzene, toluene, ethylbenzene and the xylenes (BTEX) was carried out in an O2-poor (approx. 1 mg O2/l) fuel-contaminated aquifer. Extracted groundwater, enriched with ammonium polyphosphate (nutrients) and KNO3 (electron acceptor), was piped to an infiltration gallery over the contaminated site. Before, during and after infiltration, BTEX, nitrate and different populations of culturable bacteria were measured. BTEX declined by 78% in water from the monitoring well which was most contaminated initially and by nearly 99% in water from one of the extraction wells. These declines persisted after cessation of nutrient and nitrate addition. During the second half of the nutrient and nitrate addition period (weeks 107 to 160.5), nitrate appeared in the monitoring well, denitrifying bacteria increased about 50-fold and bacteria degrading benzene, toluene and xylenes (BTX) and phenanthrene (enumerated aerobically) increased 16- and 121-fold, respectively. At one of the extraction wells, down-gradient of the monitoring well, nitrate appeared in significant concentrations after week 124; this appearance coincided with a marked decline (> 90%) in BTEX concentration and 21- and 10-fold increases, respectively, in BTX- and phenanthrene-degrading bacteria. Low concentrations of BTEX and nitrate in down-gradient, off-site wells showed that water washing did not mobilize BTEX from the aquifer. The data indicate that the BTEX in this nitrate-enriched aquifer was biodegraded in-situ under denitrifying conditions.

Bacteria that are capable of degrading polycyclic aromatic hydrocarbons were enumerated by incorporating soil and water dilutions together with fine particles of phenanthrene, a polycyclic aromatic hydrocarbon, into an agarose overlayer and pouring the mixture over a mineral salts underlayer. The phenanthrene-degrading bacteria embedded in the overlayer were recognized by a halo of clearing in the opaque phenanthrene layer. Diesel fuel- or creosote-contaminated soil and water that were undergoing bioremediation contained 6 x 10(6) to 100 x 10(6) phenanthrene-degrading bacteria per g and ca. 5 x 10(5) phenanthrene-degrading bacteria per ml, respectively, whereas samples from untreated polluted sites contained substantially lower numbers. Unpolluted soil and water contained no detectable phenanthrene degraders (desert soil) or only very modest numbers of these organisms (garden soil, municipal reservoir water).

Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macrophages and macrophage-like tumor cells, and for cells of the slime mold Dictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogen-supersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 x 10(7) Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed.

It is generally assumed that hydrophobic surfaces play a role in bubble formation in vivo, but no tests of this assumption seem to exist. Model systems for both in vitro and in vivo study of the bubble nucleation properties of hydrophobic surfaces were developed. First, aqueous suspensions of particles were exposed to gas supersaturations, and the numbers of bubbles that formed were determined. Although the supersaturation thresholds for spontaneous bubble nucleation in pure water exceeds 175 atmospheres gas tension, gas tensions of only a few atmospheres caused the profuse formation of bubbles with the most effective particles. Some or most of this latter effect seemed to be caused by gas trapped in irregularities on the particles. Second, particles that were especially effective bubble promoters were added to suspensions of ciliates. Upon their ingestion, all of the particles lost their ability to induce bubble formation in the cells with supersaturations equal to or exceeding the threshold for spontaneous nucleation in water. These results indicate that intracellular bubble formation may not occur readily in vivo.

Suspensions of human erythrocytes or of unicellular microorganisms (Tetrahymena pyriformis, Euglena gracilis, Escherichia coli, and Microcyclus aquaticus) were equilibrated with nitrogen gas pressures up to 200 atm and rapidly decompressed to hypobaric pressures below the vapor point of water. The intracellular environments proved to be very tolerant to the gas supersaturations induced. None or only a few cells were damaged in each case, and bubbles were never observed intracellularly after decompression. In view of such extreme tolerances, it is doubtful that bubbles originate intracellularly during decompression of multicellular organisms, in which bubbles occur with far lower gas supersaturations, unless the tolerances are greatly affected by extensive mechanical deformations of the cells or by the presence of internalized particles with bubble-promoting properties.

Intact mammalian, avian, and amphibian erythrocytes were saturated with up to 300 atm nitrogen or argon gas and rapidly decompressed. Despite the profuse nucleation of gas bubbles in the suspending fluid, no evidence of intracellular gas bubble nucleation was found; all or most of the cells remained intact and little or no hemoglobin escaped. Internal bubbles were similarly absent from resealed ghosts of human erythrocytes as shown by lack of disintegration and by retention of an entrapped fluorescent compound. The absence of bubbles may indicate that much of the internal water does not have the same nucleation properties as external water.

Using a new approach, we estimated the physical strength of the cell envelopes of three species of gram-negative, gas vacuolate bacteria (Microcyclus aquaticus, Prosthecomicrobium pneumaticum, and Meniscus glaucopis). Populations of cells were slowly (0.5 to 2.9 h) saturated with argon, nitrogen, or helium to final pressures up to 100 atm (10, 132 kPa). The gas phases of the vesicles remained intact and, upon rapid (1 to 2 s) decompression to atmospheric pressure, expanded and ruptured the cells; loss of colony-forming units was used as an index of rupture. Because the cell envelope is the cellular component most likely to resist the expanding intracellular gas phase, its strength can be estimated from the minimum gas pressures that produce rupture. The viable counts indicated that these minimum pressures were between 25 and 50 atm; the majority of the cell envelopes were ruptured at pressures between 50 and 100 atm. Cells in which the gas vesicles were collapsed and the gas phases were effectively dissolved by rapid compression tolerated decompression from much higher gas saturations. Cells that do not normally possess gas vesicles (Escherichia coli) or that had been prevented from forming them by addition of L-lysine to the medium (M. aquaticus) were not harmed by decompression from gas saturation pressures up to 300 atm.

Cells of the ciliate Tetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1-0.5 X 10(7) Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.

Eucaryotic unicellular (a yeast, a cellular slime mold, and various protozoans) and two multicellular (aschelminths) microorganisms were saturated with gas at high pressures and rapidly decompressed. No effect was observed with pressures of argon up to 125 atm, nitrogen up to 175 atm, and helium up to 350 atm, showing that the induced gas supersaturations did not cause intracellular bubbles to form. With 25--50 atm higher gas pressures, the decompression usually produced killing and cell rupture, although differences in tolerances existed among the various organisms. Substantial fractions of the populations survived gas supersaturations well above the threshold values for massive spontaneous nucleation of bubbles in the water. When killing occurred, external rather than internal bubbles appeared to be the cause. Even with the 300 atm argon or nitrogen pressures, yeast cells were unaffected, apparently because of the external protection provided by their cell wall. It is concluded that the gas supersaturations required for intracellular formation of bubbles generally are at least equal to and probably higher than the bubble nucleation thresholds for water or aqueous solutions.

Electron microscopic investigations of thin sections of Nitzschiaalba revealed that dividing cells yield both Nitzschia and Hantzschia daughter cells, suggesting that these genera are not taxonoinically distinct.