Pertinence of indicator organisms and sampling variables to Vibrio concentrations.

Vibrio-indicator relationships and effects of day, depth, and tidal levels on the density of vibrios enumerated by the most probable number technique were investigated. Counts of vibrios taken monthly from Apalachicola Bay, Fla., were either negatively correlated or showed no correlation with counts of indicator bacteria (Escherichia coli, enterococci, fecal coliforms, and total coliforms). Water samples collected on two days from the surface and bottom over a complete tidal cycle on each day were analyzed for differences in vibrio concentrations. Concentrations of vibrios in samples taken on different days, in those taken at different depths, and in those taken at different tidal levels were significantly different, indicating that these factors need to be taken into account in health-related studies.

Preservation of ATP in hypersaline environments.

High concentrations of particulate ATP were found in the anoxic brines of the Orca Basin and East Flower Garden, Gulf of Mexico. Other measurements indicative of growth and respiration suggested that the microbial community in the brines was inactive, but somehow the ATP associated with the cells persisted. Conceivably, when cells growing just above the interface sank into the brine, the increased osmotic stress could elicit an osmoregulatory response resulting in increased ATP. It was also possible that hydrolytic enzymes were inactivated, resulting in the preservation of ATP. Experiments in which a culture of marine bacteria was suspended in menstrua of different salinities comparable to those found across the Orca Basin interface revealed that as salinity increased, ATP increased three- to sixfold. Within 24 h the ATP fell to its initial level and remained at that concentration for 3 days, at which time the experiment was terminated. In contrast, the control suspensions, at a salinity of 28% (grams per liter) had 1/10th of the initial ATP concentration when the experiment was ended. Cells were also exposed to killing UV irradiation, enabling us to demonstrate with absolute certainty that cellular ATP could be preserved. At the end of the experiment, the viable component of the population was reduced by orders of magnitude by UV irradiation, but the ATP levels of the cells suspended in brine did not decrease. In certain environments it appears that the conventional analytical tools of the microbial ecologist must be interpreted with caution.

Temporal Occurrence of Vibrio Species and Aeromonas hydrophila in Estuarine Sediments.

Marine sediments were assayed for their concentration of Vibrio spp. and Aeromonas hydrophila over 1 year. A temporal variation was observed in which A. hydrophila, and to a lesser degree V. fluvialis, were found in the winter months, V. parahaemolyticus and V. vulnificus predominated in the spring and summer, with non-O-1 V. cholerae and V. alginolyticus detected in the late summer and fall. These organisms were found in greatest numbers in the top 5 cm of sediment, but were detectable down to 15 cm. Epidemiological data revealed a predominance of non-O-1 V. cholerae infections at the time the organisms were observed to flourish in the sediments.

Distribution of viruses associated with particles in waste water.

The distribution of solids-associated viruses in wastewater was studied to determine the effect of treatment processes on viruses associated with solids. Solids less than 0.3 micrometers in diameter were separated from the liquid phase of each sample by using a continuous-flow centrifuge. The percentage of virus associated with solids larger than 0.3 micrometers decreased from 28% in the influent to 3.4% in unchlorinated effluent, and this was accompanied by a 92% decrease in the total concentration of virus. These results indicate that the original solids-associated virus as well as that is secondarily adsorbed to mixed liquor-suspended solids is lost during clarification. The total concentration of virus was reduced by 82% by chlorination, and the percentage of virus associated with solids increased to 7.7% upon chlorination, indicating some protection due to association with particles larger than 0.3 micrometers. When a suspension of fecal particles and a 0.22-micrometers filtrate of a fecal homogenate were sonicated, a threefold increase in virus titer was observed in each. This demonstrated that viruses may be attached to particles smaller than 0.22 micrometers. Thus, small viral aggregates or viruses attached to submicron particles represented the major portion of solids-associated virus in treated sewage.

Water-to-Air Fractionation of Bacteria.

Differences in the ability of bacterial species to be transported from water to air by bursting bubbles were investigated. Bubbles were generated in suspensions of mixed bacterial cultures, and the concentration was measured for each species in the top jet drop ejected. This concentration divided by the concentration in the bulk menstruum is the concentration factor (CF). Bubbles were generated 2 cm below the liquid surface, and jet drops with diameters from 34 to 136 mum were studied. Serratia marinorubra and Micrococcus euryhalis had CFs which were generally 10 to 100 times greater than those of Escherichia coli, Pseudomonas bathycetes, and spores of Bacillus subtilis. P. bathycetes never had a CF significantly greater than 1 at any drop size, and spores of B. subtilis had a maximum CF of 4. E. coli had a maximum CF of 6 for 1- or 2-day old cultures, but this increased to 80 when a 5-day-old culture was used. This change in the CF with age of the cells indicates that composition of the cell may be a factor influencing its ability to concentrate in jet drops.

Survival of poliovirus within organic solids during chlorination.

Poliovirus in fecal homogenates was used to determine the protection against inactivation by chlorination afforded virus that was occluded within particulates. Virus that was closely associated with or occluded within small fecal particulates was protected. A fourfold increase in combined residual chlorine was required to achieve the same degree of inactivation for occluded virus as for free or secondarily adsorbed virus. A combined chlorine residual of 6.6 mg/liter was necessary to achieve 50% inactivation in 15 min at pH 8.0 and 22 degrees C in a particulate suspension containing occluded virus compared to 1.4 mg/liter for free virus. These differences were found to be relatively small compared to differences due to the presence of dissolved organics or between free and combined chlorine residuals. The results suggest different mechanisms of protection due to adsorption and occlusion.

Microbial biomass and activity distribution in an anoxic, hypersaline basin.

The Orca Basin is a hypersaline depression in the northern Gulf of Mexico with anoxic conditions observed in the lower 200 m of the water column. Measurements of adenosine 5'-triphosphate, heterotrophic potential, and uridine uptake made above and across the interface into the anoxic zone revealed the presence of an active microbial population approximately 100 m above the interface. Biomass and activity decreased at and just below the interface but increased near the bottom, consistent with similar observations made in the Cariaco Trench. The maximum adenosine 5'-triphosphate concentration above the interface of 5.9 ng/liter (2,173 m) is about eight times greater than the value found in oxygenated waters of corresponding depth in the absence of an anoxic zone. The maximum adenosine 5'-triphosphate concentration in the anoxic zone is approximately 15 times greater than that found in oxygenated water of similar depth, suggesting anoxia will support the development of a larger bacterial population. Our findings suggest that autotrophic bacteria may be the dominant physiological group in the region just above the interface.

Radioisotope assay for the quantification of sulfate-reducing bacteria in sediment and water.

A radioisotope enrichment culture method was developed to estimate the physiologically active component of a population of sulfate-reducing bacteria in environmental water and sediment samples. Aliquots of water or sediment were added to 50-ml serum bottles filled with 35-S-sulfate broth incubated for approximately 30 h. After incubation, the disintegration rate per milliliter of spent medium was measured, and the percentage of loss of activity resulting from bacterial sulfate reduction was determined. This loss of sulfate from the medium was then translated to a specific number of Desulfovibrio desulfuricans cells that would reduce an equivalent amount of sulfate in the same incubation time. This comparison was done using a series of growth curves of D. desulfuricans covering a range of inoculum densities between 10-2 and 10-7 cells. The radioassay was used to follow the effects of a pulp mill on a small anoxic river in Florida. The activity of the sulfate-reducing bacteria in the river was greatly suppressed when the mill was closed for annual maintenance. The initiation of waste treatment resulted in improved water quality in 1 week, but the river sediments required a month to show a 10-fold reduction in the population of sulfate-reducing bacteria.

Effect of water on the thermal death of a hydrocarbon bacterium in a nonaqueous fluid.

A bacterium that grows in oil was tested for survival at elevated temperatures in menstruums of varying water content. For each doubling of the water concentration, the surviving fraction decreased by a factor of approximately 3.0. A minimum value of 0.02% water is required before enhanced killing occurs.

A radioisotope assay for the quantification of hydrocarbon biodegradation potential in environmental samples.

An enrichment culture method is described for quantifying the activity of hydrocarbon oxidizing bacteria in water and sediments. Application of the procedure indicated that the hydrocarbon oxidizing potential of environmental samples reflects the hydrocarbon burden of the area, the ability of the microflora to utilize hydrocarbons, and that lakes with large aquatic plant communities contain populations of hydrocarbon bacteria comparable to those found in oil-polluted harbors.

Observations of bacterial microcolonies on the surface of ferromanganese nodules from blake plateau by scanning electron microscopy.

Examination of the surface of freshly collected ferromanganese nodules by scanning electron microscopy revealed the presence of microcolonies of rod- and coccus-shaped bacteria which appeared to be anchored to the nodule surface by slime. The attachment of microcolonies by slime to the surface of freshly collected nodules argues against their being contaminants introduced during nodule collection or processing. These results corroborate cultural and biochemical detection of bacteria on ferromanganese nodules.

Thermal death of a hydrocarbon bacterium in a nonaqueous fluid.

A hydrocarbon-utilizing Brevibacterium which grew into the oil phase of an oil-water system was tested for survival at elevated temperature. Cells suspended in oil and cells that had been resuspended in aqueous solution were tested by placing 1-ml samples of the cell suspension in small test tubes immersed in a controlled-temperature water bath. The resultant survival curves in oil consisted of two parts, a flat shoulder obtained in the first half of the heating period, followed by a break indicating rapid die-off. The break in the curves occurred after 50% of the cells were killed. This occurred at exposures of 25, 15, and 8 min for 78, 88.6, and 96.2 C, respectively. The survival curve for 63.5 C in the aqueous solution was a rapid, exponential die-off. The actual increase in survival of the organism in oil is reflected by the length of the shoulder portion. The shoulder occurs only in an oil medium and is increased by decreasing temperature and increasing age of the culture.