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The effects of large-scale poultry production operations on water quality and human health are largely unknown. Poultry litter is frequently applied as fertilizer to agricultural lands adjacent to large poultry farms. Run-off from the land introduces a variety of stressors into the surface waters including nutrients, antimicrobials, and pathogenic bacteria. The Delaware, Maryland, and Virginia (Delmarva) Peninsula has the highest concentration of broiler chickens per farm acre in the United States and provides an ideal location for studying the effects of stressors from poultry farms. We investigated potential effects by characterizing shifts in the structure of aquatic bacterial communities. DNA was isolated from microorganisms in water samples from streams and rivers at varying distances from, or having different frequencies of, litter applications. Fingerprints of 16S rDNA amplicons from bacteria in water samples collected during late summer 2001 to late spring 2002 were produced by denaturing gradient gel electrophoresis (DGGE). A statistical analysis of multiple fingerprints from each sampling location demonstrated that each site harbored a bacterial community significantly different from the communities at other sites. Similarly, the bacterial communities from each sampling time differed significantly from communities at other sampling times. Most importantly, a competitive, library-based analysis showed time of sampling (month) had a greater effect on community structure than did location. This was supported by the inability to detect several antibiotic resistance genes in the water samples using PCR.

Pritchard, P.H., L.H. Mueller, J.C. Spain and A.W. Bourquin. 1987. Degradation of Jet and Missile Fuels by Aquatic Microbial Communities. U.S. Air Force, Tyndall AFB, FL. 161 p. (ERL,GB 590).

The fate of jet fuel (JP-4) in aquatic sediments was studied concomitantly in laboratory test systems and in the field. Sediments from an estuarine pond were dosed with jet fuel and then reapplied to the pond as well as into plexiglass trays on the sediment bed and quiescent bottle tests in the laboratory. Thirty-three selected hydrocarbons in the jet fuel were followed chemically to quantitate relative hydrocarbon losses. Several hydrocarbons which biodegraded or rapidly volatilized in the bottle tests were much slower to disappear in the field and the plexiglass trays. In general, mixing of the jet fuel with sediments increased the persistence of the associated hydrocarbons. The high density missile fuels RJ-5 and JP-9 resisted biodegradation when incubated with water/sediment suspensions collected from aquatic habitats. RJ-5 and JP-9 were not toxic to the microbial communities at concentrations of 400 mg per liter, but RJ-5 was toxic to Mysidopsis bahia in 96-hour acute tests (LC50 88 µg/l).

Pritchard, P.H., C.R. Cripe, L.H. Mueller and E.J. O'Neill. 1987. Metabolism of Fenthion by Aquatic Microbial Communities. In: Pesticide Science and Biotechnology: Proceedings of the Sixth International Congress of Pesticide Chemistry, IUPAC International Union of Pure and Applied Chemistry, Ottawa, Canada, August 10-17, 1986. EPA/600/D-86/217. R. Greenhalgh and T.R. Roberts, Editors. Blackwell Scientific Publications, Boston, MA. Pp. 505-508. (ERL,GB 592). (Avail. from NTIS, Springfield, VA: PB87-102430)

The microbial metabolism of the mosquito control agent, fenthion, has been studied in shake flask systems containing water and sediment from a salt marsh. The usefulness of this information in describing the fate of fenthion in microcosms and in a field dosing experiment was determined. Our results show that microbial communities associated with the sediment, the presence of invertebrate animals in the sediment bed, and the anaerobic conditions of the sediment contribute significantly to the fate of fenthion under natural conditions.

Barkay, Tamar. 1987. Adaptation of Aquatic Microbial Communities to Hg2+ Stress. EPA/600/J-87/333. Appl. Environ. Microbiol. 53(12):2725-2732. (ERL,GB 608). (Avail. from NTIS, Springfield, VA: PB88-199401)

The mechanism of adaptation to Hg2+ in four aquatic habitats was studied by correlating microbially mediated Hg2+ volatilization with the adaptive state of the exposed communities. Community diversity, heterotrophic activity, and Hg2+ resistance measurements indicated that adaptation was associated with rapid volatilization after an initial lag period. This mechanism, however, did not promote adaptation in a freshwater sample, in which Hg2+ was volatilized slowly, regardless of the resistance level of the microbial community. Distribution of the mer operon among representative colonies of the communities was not related to adaptation to Hg2+. Thus, although volatilization enabled some microbial communities to sustain their functions in Hg2+-stressed environments, it was not mediated by the genes that serve as a model system in molecular studies of bacterial resistance to mercurials.

Barkay, T. and P.H. Pritchard. 1988. Adaptation of Aquatic Microbial Communities to Pollutant Stress. EPA/600/J-88/169. Microbiol. Sci. 5(6):165-169. (ERL,GB 625). (Avail. from NTIS, Springfield, VA: PB89-120901)

The importance of microbial adaptation in the removal of environmental pollutants and in maintaining active microbial communities in impacted ecosystems is discussed using the biodegradation of p-nitrophenol and the volatilization of mercuric mercury as examples. A molecular mechanism of adaptation is suggested by enrichment of mercury resistance (mer) genes in some communities upon exposure to mercury.

Liebert, Cynthia and Tamar Barkay. 1988. Direct Viable Counting Method for Measuring Tolerance of Aquatic Microbial Communities to HG(II). EPA/600/J-88/351. Can. J. Microbiol. 34(9):1090-1095. (ERL,GB 628). (Avail. from NTIS, Springfield, VA: PB89-237176)

Direct counts of Hg2+ resistant cells in natural waters were obtained by fluorescence microscopy after incubation for 20 h in the presence of a growth substrate, nalidixic acid, to halt cell division, and inhibiting concentrations of Hg2+. This method discriminated Hg2+ resistant from sensitive Escherichia coli strains. Estuarine samples were used to compare this procedure with two other toxicity measurements that determine the effect of Hg2+ on colony growth and on rates of radioactive thymidine incorporation into cellular material. Toxicity measurements based on direct viable counts and thymidine incorporation rates had comparable sensitivities, and both were 3-4 orders of magnitude more sensitive than the method that utilized colony counts. Thus, the direct enumeration of Hg2+ resistant cells is useful for predicting the potential of aquatic communities to sustain heterotrophic activity, an essential microbial process, in the presence of Hg2+.

Barkay, Tamar, Cynthia Liebert and Mark Gillman. 1989. Environmental Significance of the Potential for mer(Tn21)-Mediated Reduction of Hg2+ and Hg0 in Natural Waters. EPA/600/J-89/166. Appl. Environ. Microbiol. 55(5):1196-1202. (ERL,GB 660). (Avail. from NTIS, Springfield, VA: PB90-129511)

The role of mer(Tn21) in adaptation of aquatic microbial communities to Hg2+ was investigated. Elemental mercury was the sole product of Hg2+ volatilization by freshwater and saline microbial communities. Bacterial activity was responsible for biotransformation because most microeukaryotes did not survive the exposure conditions, and removal of larger microbes (>1 µm) from adapted communities did not significantly (P >0.01) reduce Hg2+ volatilization rates. DNA sequences homologous to mer(Tn21) were found in 50% of Hg2+ resistant bacterial strains representing two fresh water communities, but in only 12% of strains representing two saline communities (the difference was highly significant; P < 0.001). Thus, mer(Tn21) played a significant role in Hg2+ resistance among strains isolated from freshwaters where microbial activity had a limited role in Hg2+ volatilization. In saline environments where microbially mediated volatilization was the major mechanism of Hg2+ loss, other bacterial genes coded for this biotransformation.

Barkay, T. and R. Turner. 1989. Gene Probes to Predict Responses of Aquatic Microbial Communities to Toxic Metals. In: Heavy Metals in the Environment: International Conference. J.P. Vernet, Editor. CEP Consultants, Edinburgh, Scotland. Pp. 57-64. (ERL,GB 678).

Relationships between rates of Hg2+ reduction and CH3Hg+ demethylation in natural waters, and the frequency of bacterial genes coding for these reactions (mer genes) in gene pools of active microbial communities, are demonstrated. This finding could be developed as a tool in management of polluted environments.

Liebert, C.A., T. Barkay and R.R. Turner. 1991. Acclimation of Aquatic Microbial Communities to Hg(II) and CH3Hg+ in Polluted Freshwater Ponds. EPA/600/J-91/329. Microb. Ecol. 21(2):139-149. (ERL,GB 709). (Avail. from NTIS, Springfield, VA: PB92-129667)

The relationship of mercury resistance to the concentration and chemical speciation of mercurial compounds was evaluated for microbial communities of mercury-polluted and control waters. Methodologies based on the direct viable counting (DVC) method were developed to enumerate mercury-resistant communities. Elevated tolerance to Hg(II) was observed for the microbial community of one mercury-polluted pond as compared to the community of control waters. These results suggest an in situ acclimation to Hg(II). The results of the methylmercury resistance-DVC assay suggested that minimal acclimation of CH3Hg+ occurred since similar concentrations of CH3HgCl inhibited growth of 50% of organisms in both the control and polluted communities. Analyses of different mercury species in pond waters suggested that total mercury, but not CH3Hg+ concentrations, approached toxic levels in the polluted ponds. Thus, microbial acclimation was specific to the chemical species of mercury present in the water at concentrations high enough to cause toxic effects to non-acclimated bacterial communities.

Nazaret, Sylvie, Wade H. Jeffrey, Erwan Saouter, Robin Von Haven and Tamar Barkay. 1994. merA Gene Expression in Aquatic Environments Measured by mRNA Production and Hg(II) Volatilization. Appl. Environ. Microbiol. 60(11):4059-4065. (ERL,GB 891).

The relationship of merA gene expression (specifying the enzyme mercuric reductase) to mercury volatilization in aquatic microbial communities was investigated with samples collected at a mercury-contaminated freshwater pond, Reality Lake, in Oak Ridge, Tenn. Levels of merA mRNA transcripts and the rate of inorganic mercury (Hg[II]) volatilization were related to the concentration of mercury in the water and to heterotrophic activity, in field samples and laboratory incubations of pond water in which microbial heterotrophic activity and Hg(II) concentration were manipulated. Levels of merA-specific mRNA and Hg(II) volatilization were influenced more by microbial metabolic activity than by the concentration of mercury. merA-specific transcripts were detected in some samples which did not reduce Hg(II), suggesting the levels of merA may not always be proportional to rates of mercury volatilization in environmental samples.

Spain, J.C. and C.C. Somerville. 1985. Biodegradation of Jet Fuel by Aquatic Microbial Communities. In: Microbes and Oil Recovery. EPA/600/D-85/084. J.E. Zajic and E.C. Donaldson, Editors. Bioresearch Publications, El Paso, TX. Pp. 345-356. (ERL,GB X485). (Avail. from NTIS, Springfield, VA: PB85-191971)

This paper describes laboratory experiments that studied the fate of jet fuel in several types of situations that could be encountered in the field. Benzene, toluene, and p-xylene were the only components of the fuel that dissolved in the water to significant concentrations. All three compounds volatilized within 24 h and, thus, did not remain in the water long enough for microbial degradation to affect their fate. Inclusion of sediment (500 mg/l dry weight) did not retard the disappearance of the fuel components, and rates of disappearance were identical in controls sterilized with HgCl2.

Shimp, Robert J. and Frederic K. Pfaender. 1985. Influence of Easily Degradable Naturally Occurring Carbon Substrates on Biodegradation of Monosubstituted Phenols by Aquatic Bacteria. EPA/600/J-85/023. Appl. Environ. Microbiol. 49(2):394-401. (ERL,GB X494).

The influence of readily degradable, naturally occurring carbon substrates on the biodegradation of several monosubstituted phenols (m-cresol, m-aminophenol, p-chlorophenol) was examined. The natural substrate classes used were amino acids, carbohydrates, and fatty acids. Samples of the microbial community from Lake Michie, a mesotrophic reservoir, were adapted to different levels of representatives from each natural substrate class in chemostats. After an extended adaptation period, the ability of the microbial community to degrade the monosubstituted phenols was determined by using a radiolabeled substrate uptake and mineralization method. Several microbiological characteristics of the communities were also measured. Adaptation to increasing concentrations of amino acids, carbohydrates, or fatty acids enhanced the ability of the microbial community to degrade all three phenols. The stimulation was largest for m-cresol and m-aminophenol. The mechanism responsible for the enhancement of monosubstituted phenol metabolism was not clearly identified, but the observation that adaptation to amino acids also increased the biodegradation of glucose and, to a lesser extent, naphthalene suggests a general stimulation of microbial metabolism. This study demonstrates that prior exposure to labile, natural substrates can significantly enhance the ability of aquatic microbial communities to respond to xenobiotics.

Barkay, Tamar. 1990. Studies on Conjugal Transfer of Plasmids from GEMs to Indigenous Aquatic Bacteria. In: Review of Progress in the Biotechnology-Microbial Pest Control Agent Risk Assessment Program. EPA/600/9-90/029. U.S. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, OR and Environmental Research Laboratory, Gulf Breeze, FL. Pp. 64-66. (ERL,GB X677).

The horizontal transfer of recombinant DNA from introduced species to indigenous microbes may result in the establishmnet of new genes in organisms that are better fitted to survive and reproduce in the environment. Such events may have harmful consequences if the foreign genes are expressed and alter the ecology of the affected microbial community. The well characterized bacterial mer operon has been used to study how the spread of mer genes affects the response of aquatic microbial communities to Hg2+ stress.