Microbial inhibitors for U.S. EPA drinking water methods for the determination of organic compounds.

Preservation of chemical analytes in drinking water samples is necessary to obtain accurate information concerning contaminant occurrence. Sample preservation to prevent biodegradation is important for most samples and analytes. With the unique demands of environmental methods, it is not always possible to kill all microorganisms without having undesirable effects. To find a suitable preservative, the sample, analysis, and preservation needs should be considered. During method development of U.S. Environmental Protection Agency (EPA) Methods 526 (for unstable semivolatile compounds) and 532 (for phenylurea pesticides), a number of studies were conducted to identify compatible microbial inhibitors. Copper sulfate was successfully used in Method 532 and is an excellent first-choice antimicrobial agent for many applications. Copper sulfate can catalyze hydrolysis reactions for some pesticides such as those analyzed in Method 526. Under these conditions, a nonmetal compound of antimicrobial activity must be considered. During the development of Method 526, a survey of alternate organic based antimicrobial compounds found that diazolidinyl urea worked well in the method. Several other candidate microbial inhibitors were identified that could have application to other environmental methods. A general approach to selecting antimicrobial compounds in future environmental methods in water matrixes is discussed.

Enumeration of Cryptosporidium spp. in water with US EPA method 1622, USA.

The occurrence of Cryptosporidium parvum or other pathogenic Cryptosporidium species in water must be known in order to assess risk and determine the treatment needed to reduce Cryptosporidium oocysts to acceptable levels in finished drinking water. Because Cryptosporidium oocyst occurrence may be sparse, methods must concentrate a large volume of water and correctly identify oocysts in the concentrate. The U.S. Environmental Protection Agency Information Collection Rule (ICR) protozoan method gives low and variable recoveries of Cryptosporidium oocysts, making risk assessment difficult. Therefore, a method giving better oocyst recovery and more consistent results was needed. Method 1622 was developed with existing materials and procedures, and improvements were made in filtration, cleanup, and detection. Absolute porosity filters were used, with cleanup by immunomagnetic separation and detection by direct fluorescent antibody stain with 4',6-diamidino-2-phenylindole (DAPI) staining for additional cell structures. Both the level and consistency of oocyst recovery were improved compared to recovery with the ICR method.

Total system costs and the case of the executive's hernia.

This article describes the advantages of taking a total system cost approach in the development of a benefits plan that improves employee health, productivity and profitability. It discusses the benefits and points out the possible pitfalls to the total system cost approach in providing employee health care. A research model is used to assess the value of certain medical procedures and technologies. To exemplify the point more clearly, the author uses a most interesting case study alluded to in the title of the article.

Protozoa in subsurface sediments from sites contaminated with aviation gasoline or jet fuel.

Numbers of protozoa in the subsurface of aviation gasoline and jet fuel spill areas at a Coast Guard base at Traverse City, Mich., were determined. Boreholes were drilled in an uncontaminated location, in contaminated but untreated parts of the fuel plumes, and in the aviation gasoline source area undergoing H(2)O(2) biotreatment. Samples were taken from the unsaturated zone to depths slightly below the floating free product in the saturated zone. Protozoa were found to occur in elevated numbers in the unsaturated zone, where fuel vapors mixed with atmospheric oxygen, and below the layer of floating fuel, where uncontaminated groundwater came into contact with fuel. The same trends were noted in the biotreatment area, except that numbers of protozoa were higher. Numbers of protozoa in some contaminated areas equalled or exceeded those found in surface soil. The abundance of protozoa in the biotreatment area was high enough that it would be expected to significantly reduce the bacterial community that was degrading the fuel. Little reduction in hydraulic conductivity was observed, and no bacterial fouling of the aquifer was observed during biotreatment.

In situ biodegradation: microbiological patterns in a contaminated aquifer.

Conventional approaches for proving in situ biodegradation of organic pollutants in aquifers have severe limitations. In the approach described here, patterns in a comprehensive set of microbiological activity and distribution data were analyzed. Measurements were performed on sediment samples gathered at consistent depths in aquifer boreholes spanning a gradient of contaminant concentrations at a buried coal tar site. Microbial adaptation to polyaromatic hydrocarbons (PAHs) was demonstrated by mineralization of naphthalene and phenanthrene in samples from PAH-contaminated, but not adjacent pristine, zones. Furthermore, contaminant-stimulated in situ bacterial growth was indicated because enhanced numbers of protozoa and their bacterial prey were found exclusively in contaminated subsurface samples. The data suggest that many convergent lines of logically linked indirect evidence can effectively document in situ biodegradation of aquifer contaminants.

Distribution and activity of microorganisms in subsurface sediments of a pristine study site in Oklahoma.

Distribution and activity of microorganisms in surface soil and subsurface sediments were studied in depth profiles of six different microbial biomass and activity indicators (total direct counts, number of cells capable of electron transport system activity, viable cell plate counts, most Probable numbers of protozoa, and 4-hydroxybenzoate-degrading microorganisms, and ATP content). The profiles showed the same general trends on two different dates (January and June 1985). Seasonal variations were noted, but they were not extreme. Biomass and activity values declined sharply with depth in the unsaturated zone, reaching minima in a clay confining layer in the interface zone between 3 and 4 m. Contiguous 10-cm samples from the interface zone showed significant textural and microbiological variability. Higher and more stable biomass and activity values were detected in the saturated zone, the highest being a very permeable gravelly loamy sand layer at approximately 7.5 m. In this layer, viable counts were nearly equal to total counts and they approached the viable counts in surface soil. Surface-type protozoa and cyanobacteria also were detected in this layer, suggesting that it was connected hydrologically to a nearby river. Lowest values were detected in an underlying bedrock clay layer at 8 m, which, despite its impermeability and low viable counts, did contain measurable total counts, 4-hydroxybenzoate-degrading microorganisms, and ATP. Correlations were noted between sediment texture and microbial activity (i.e., sandy texture=high activity, clayey texture=low activity), but other hydrogeological and geochemical factors probably also influenced microbial distribution and activity in the profile.

Distribution of protozoa in subsurface sediments of a pristine groundwater study site in oklahoma.

Sediment core samples were obtained at a groundwater study site in Oklahoma in January and June 1985. Most-probable-number estimates showed that protozoan numbers declined steeply with depth in subsoil. Flagellates and amoebae dominated the protozoan population, which declined to a most probable number of 28 . g (dry weight) in a clay loam layer at the bottom of the unsaturated zone. Samples from a texturally variable interface zone between 3 and 4 m down also were variable in their content of protozoa. Four contiguous clay loam samples in a single core from this zone contained variable numbers of amoebae ranging from 0.2 to 44 . g (dry weight). However, a sandy clay loam layer at the bottom of the core contained a mixture of flagellates and amoebae with a combined population density of 67 . g (dry weight). A slow-growing filose amoeba was isolated from interface zone samples and was tentatively classified in a new family in the order Aconchulinida. Protozoa were not detected in the saturated zone except in a very permeable gravelly, loamy sand layer at a depth of approximately 7.5 m. Low numbers (4 to 6 . g [dry weight]) of surface-type flagellates and amoebae, as well as the filose amoeba seen in the interface zone, were observed in this layer. Acid-treated and untreated samples contained equivalent numbers of protozoa, showing that the majority of protozoa in the layer at 7.5 m and the interface zone samples were encysted. Increased numbers of bacteria also were found in the layer at 7.5 m, indicating that it was biologically more active than other saturated-zone layers. Cyanobacteria grew in illuminated samples from this layer, suggesting that it may be connected hydrologically to a nearby river.

Role of resistance to starvation in bacterial survival in sewage and lake water.

A study was conducted to determine the significance of starvation resistance to the ability of a species to survive in sewage and lake water. Tests were conducted for periods of up to 14 days. Rhizobium meliloti and one fluorescent and one nonfluorescent strain of Pseudomonas were resistant to starvation because their population sizes did not fall appreciably in buffer and sterile lake water, and the first two maintained high numbers after being added to sterile sewage. Cell densities of these bacterial species dropped slowly in nonsterile sewage, and more cells of these three organisms than of the other test organisms remained in nonsterile lake water. Rhizobium leguminosarum was moderately resistant to starvation because its numbers fell slowly in buffer and sterile lake water and did not change appreciably in sterile sewage. The abundance of Micrococcus flavus added to buffer and sterile lake water did not change, but the density of M. flavus declined in nonsterile lake water. The abundance of R. leguminosarum fell in nonsterile lake water and nonsterile sewage. Streptococcus faecalis, Staphylococcus aureus, an asporogenous strain of Bacillus subtilis, and Streptococcus sp. were susceptible to starvation because their populations were markedly reduced in buffer. Populations of the last three species declined rapidly in nonsterile and sterile samples of lake water and sewage. S. faecalis declined rapidly when added to nonsterile lake water and sewage and sterile lake water but not when added to sterile sewage, the persistence in the last instance probably being associated with the availability of organic nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Fate in model ecosystems of microbial species of potential use in genetic engineering.

The changes in populations of Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Klebsiella pneumoniae, Agrobacterium tumefaciens, Rhizobium meliloti, and Saccharomyces cerevisiae were measured after their introduction into samples of sewage, lake water, and soil. Enumeration of small populations was possible because the strains used were resistant to antibiotics in concentrations and combinations such that few species native to these ecosystems were able to grow on agar containing the inhibitors. Fewer than 2 cells per ml of sewage or lake water and 25 cells per g of soil could be detected. A. tumefaciens and R. meliloti persisted in significant numbers with little decline, but S. aureus, K. pneumoniae, S. typhimurium, S. cerevisiae, and vegetative cells of B. subtilis failed to survive in samples of sewage and lake water. In sterile sewage, however, K. pneumoniae, B. subtilis, S. typhimurium, A. tumefaciens, and R. meliloti grew; S. cerevisiae populations were maintained at the levels used for inoculation; and S. aureus died rapidly. In sterile lake water, the population of S. aureus and K. pneumoniae and the number of vegetative cells of B. subtilis declined rapidly, R. meliloti grew, and the other species maintained significant numbers with little or a slow decline. The populations of S. aureus, K. pneumoniae, A. tumefaciens, B. subtilis, and S. typhimurium declined in soil, but the first four species grew in sterile soil. It is suggested that some species persist in environments in which they are not indigenous because they tolerate abiotic stresses, do not lose viability readily when starved, and coexist with antagonists. The species that fails to survive need only be affected by one of these factors.

Nitrogen Mineralization by Acanthamoeba polyphaga in Grazed Pseudomonas paucimobilis Populations.

Nitrogen mineralization was studied in a simple grazing system in which the protozoan Acanthamoeba polyphaga was grown with the bacterium Pseudomonas paucimobilis (two soil organisms isolated from the shortgrass prairie in northern Colorado). In different experiments, either carbon or nitrogen was adjusted to be in limiting amounts. When carbon was limiting, grazers were almost entirely responsible for nitrogen mineralization, with bacteria themselves contributing little. When nitrogen was limiting, nitrogen mineralization by grazers permitted continued growth by the grazed bacteria and a greater bacterial biomass production. The increased growth of the grazed bacteria did not result in an increased total amount of carbon used, but the grazed bacteria used carbon more efficiently than the ungrazed bacteria.