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In the latter portion of the 20th century, North America experienced numerous large-scale mortality events affecting a broad diversity of aquatic animals. Short-term forensic investigations of these events have sometimes characterized a causative agent or condition, but have rarely provided sufficient insight to predict and manage aquatic animal health. Although traditional tools (e.g., microbiology, parasitology and pathology) remain essential, technological advances in remote sensing and information management can be applied to better understand local events, particularly in the context of environmental conditions extending across broad regional scales. These additional tools will allow exploration of climate, weather, ultraviolet light exposure, airborne dust, and regional land use factors on disease and disease susceptibility. Ultimately, regional environmental observing systems could be used to forecast health risks to aquatic resources. One such system is under development in the Gulf of Mexico to forecast red tide events that have extended along the Gulf coastal states of the U.S. and Mexico. Yet, such an approach requires a meaningful understanding of large-scale environmental effects on disease. Data collected from diseases and mortalities of aquatic species must be integrated and examined in broad temporal and spatial contexts for possible links with regional environmental factors. A pilot program for integrating disease and mortality data from multiple species with large-scale environmental factors has been initiated, but has not been broadly implemented. Implementation will ultimately require a comprehensive monitoring and data collection network, rather than the case-by-case, species-by-species reporting that currently exists. A pilot program for standardized disease and mortality data collection also exists, but has not been widely exploited. Regardless, these programs, combined with the new opportunities afforded by remote sensing and information technology, can be used to generate epizootiological models of large-scale environmental influences on aquatic disease, and can ultimately lead to forecasting disease and mortality events.

Bourquin, Al W. 1973. Impact of Microbial Seed Cultures on the Aquatic Environment. In: Proceedings of the First Microbiology Seminar on Standardization of Methods. EPA-R4-73-022. U.S. Environmental Protection Agency, Washington, DC. Pp. 140-142. (ERL,GB 203).

Microbial seed cultures are currently being studied for application to the environment as microbiological pesticides. Viruses have been isolated which attack selectively the cabbage boll; a bacterium has been isolated as a specific pathogen of mosquitoes; and chitinoclastic bacteria have been proposed as agents against plant predators in estuarine areas. The range of impact on the aquatic environment by seed cultures must be investigated adequately before they are used on a large scale.

Bourquin, A.W., L.A. Kiefer, N.H. Berner, S. Crow and D.G. Ahearn. 1975. Inhibition of Estuarine Microorganisms by Polychlorinated Biphenyls. In: Developments in Industrial Microbiology, Vol. 16. American Institute of Biological Sciences, Washington, DC. Pp. 256-261. (ERL,GB 230).

Over 100 isolates of representative estuarine bacteria and fungi were screened for their ability to grow in the presence of commercial preparations of polychlorinated biphenyls (PCB). Super absorbant sensitivity discs impregnated with up to 0.5 mg of PCB were placed on the surface of freshly inoculated solid media. Twenty-six bacteria, representing both gram-positive and gram-negative strains of varying morphology, showed varying degrees of sensitivity to PCB. In contrast to insensitive isolates, sensitive strains were mainly amylolytic and proteolytic. PCB had negligible effect on the growth of fungi. The sensitivity of select cultures of heterotrophic bacteria to PCB may be of considerable importance to nutrient turnover in estuarine ecosystems.

Murray, E. Donald and Al W. Bourquin, Editors. 1974. Developments in Industrial Microbiology, Vol. 15. Society for Industrial Microbiology, Washington, DC. 426 p. (ERL,GB 244).

Smith, N.G., A.W. Bourquin, S.A. Crow and D.G. Ahearn. 1976. Effect of Heptachlor on Hexadecane Utilization by Selected Fungi. In: Developments in Industrial Microbiology, Vol. 17. American Institute of Biological Sciences, Washington, DC. Pp. 331-336. (ERL,GB 255).

Various concentrations of heptachlor dissolved in hexadecane were added to cultures of fungi grown in yeast-nitrogen base prepared with synthetic seawater and with deionized water. Candida maltosa and Candida lipolytica showed greatest utilization of hexadecane (20-91%) whether heptachlor was present or absent. Isolates of Pichia spartinea, Cladosporium sp., Cephalosporium sp., and Penicillium sp. also utilized the hydrocarbon, but to a lesser extent. Species of Kluyveromyces failed to grow with hexadecane as a carbon source. Compared with low concentrations, high concentrations of heptachlor appeared to have a slight stimulating effect on utilzation of hexadecane by C. maltosa, but had no effect with C. lipolytica.

Couch, John. 1975. Discussion from Selected Papers Presented at EPA-USDA Working Symposium. In: Baculoviruses for Insect Pest Control: Safety Considerations. Max D. Summers, Rilo Engler, Louis A. Falcon, and P. Vail, Editors. American Society for Microbiology, Washington, DC. Pp. 58-62, 111-114. (ERL,GB 262).

In conclusion, I would like to point out the difficulty of working with some of these noninsect invertebrates. It is very hard to work with some of the marine invertebrates, for which there are no cell lines; they are not even amenable to culturing of the whole organism. One therefore can anticipate running into problems, extreme problems, in testing or applying tests of the NPVs and GVs to these organisms. This is a pioneering field with regard to aquatic organisms, but I think it has great promise. I would emphasize my original point that we should start close to the source from a conceptual point of view in testing some of the NPVs and GVs and look critically at the effect on other arthropods, particularly crustacea. . . The major new evidence that emerges here is that many groups of invertebrates are capable of harboring viruses that formerly were studied only in more obvious insect hosts. Thus, we may need to broaden our views on virus-host concepts and seek more widely for host-virus interactions.

Couch, John. 1975. Evaluation of In Vivo Specificity of Insect Viruses: Discussion. In: Baculoviruses for Insect Pest Control: Safety Considerations. Max Summers, Reto Engler, Louis A. Falcon, and Patrick V. Vail, Editors. American Society for Microbiology, Washington, DC. Pp. 60-62. (ERL,GB 262a).

In conclusion, I would like to point out the difficulty of working with some of these noninsect invertebrates. It is very hard to work with some of the marine invertebrates, for which there are no cell lines; they are not even amenable to culturing of the whole organism. One therefore can anticipate running into problems, extreme problems, in testing or applying tests of the NPVs and GVs to these organisms. This is a pioneering field with regard to aquatic organisms, but I think it has great promise. I would emphasize my original point that we should start close to the source from a conceptual point of view in testing some of the NPVs and GVs and look critically at the effect on other arthropods, particularly crustacea.

Couch, John. 1975. Evaluation of the Exposure of Fish and Wildlife to Nuclear Polyhedrosis and Granulosis Viruses: Discussion. In: Baculoviruses for Insect Pest Control: Safety Considerations. Max Summers, Reto Engler, Louis A. Falcon, Patrick V. Vail, Editor. American Society for Microbiology, Washington, DC. Pp. 111-114. (ERL,GB 262b).

The major new evidence that emerges here is that many groups of invertebrates are capable of harboring viruses that formerly were studied only in more obvious insect hosts. Thus, we may need to broaden our views on virus-host concepts and seek more widely for host-virus interactions.

Bourquin, A.W., M.A. Hood and R.L. Garnas. 1977. Artificial Microbial Ecosystem for Determining Effects and Fate of Toxicants in a Salt-Marsh Environment. In: Developments in Industrial Microbiology, Vol. 18. EPA-600/J-77-075. Society for Industrial Microbiology, Washington, DC. Pp. 185-191. (ERL,GB 309). (Avail. from NTIS, Springfield, VA: PB-277 181)

An artificial laboratory environment designed to determine microbial interactions with pollutant chemicals is proposed. The system is designed to obtain maximum reproducibility between replicates by dividing a single tank into separate closed chambers. Radiolabeled toxicants are added directly to the core-chambers and monitored for metabolic breakdown. Further information is obtained easily on changes in microbial, physiological indexes induced by the toxicants. Techniques for monitoring effects of the methyl parathion on the microbial population and the fate of this chemical are given.

Bourquin, A.W. and D.G. Ahearn. 1976. Microbiology and Chemistry of Estuarine Surface Microlayers. In: Proceedings of the International Symposium on Marine Pollution Research. EPA-600/9-76-032. Samuel P. Meyers, Editor. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 89-96. (ERL,GB 313). (Avail. from NTIS, Springfield, VA: PB-267 601)

Organic microlayers occur at the air-water interface of most bodies of water. The microlayer or "sea slick" formation appears to be related mainly to decay of naturally occurring aquatic organisms or to their production of lipodial by-products. Sea slicks are known to influence wave action and height, air-water temperature exchange, stability of bubbles and foams, and the concentration of salt-containing micro-droplets in the marine atmosphere. In coastal regions particularly, the direct activities of man are of increasing importance in the generation of surface slicks. Industrial and municipal sewage effluents constitute the major source of films and foams, but crude oil spillage appears to be a major contributor in localized areas. In studies from our laboratories on a yeast isolated from a freshwater oil layer, the chlorinated pesticide heptachlor either stimulated or inhibited hexadecane utilization dependent upon cultural conditions. The heptachlor was bound to the yeast cells but no direct evidence for its metabolism was obtained. Stimulation of oxidation of hexadecane in the presence of a non-utilizable substrate by a fungus (Cladosporium resinae) has been shown also by Walker and Cooney (1975). Growth inhibition of estuarine microorganisms by polychlorinated biphenyl formulations has been demonstrated in laboratory studies and field and laboratory studies have shown that crude oil may diversely affect the various species of the estuarine microecosystem. The potential for alteration of the estuarine surface film microflora by hydrocarbons can be expected to increase with the advent of superports and the extension of offshore oil fields. For a better understanding of the composite phenomena affected by surface films, and their role in pollutant concentration or metabolism, a substantial increase in our knowledge of the chemical identity of surface films and of the changes in the film with microbial activity needs to be developed.

Bourquin, A.W., P.H. Pritchard and W.R. Mahaffey. 1978. Effects of Kepone on Estuarine Microorganisms. In: Developments in Industrial Microbiology, Vol. 19. EPA-600/J-78-075. Society for Industrial Microbiology, Washington, DC. Pp. 489-497. (ERL,GB 345). (Avail. from NTIS, Springfield, VA: PB-290 049)

Low concn of the insecticide Kepone, approaching those found in contaminated James River sediment, were shown to be inhibitory to the growth and oxygen uptake of microorganisms randomly selected from estuarine environments. No significant correlations were noted between growth inhibition by Kepone and cell morphology, aliphatic hydrocarbon utilization, pesticide tolerance, selected enzyme activities, nitrate reduction, and urea hydrolysis. Oxygen uptake by pure cultures grown on glucose or hydrocarbons at cell densities equivalent to 103 - 104 cells/ml was decreased by 60-100% at Kepone concn of 0.02-2.0 mg/liter. Total viable counts from estuarine water or sediments grown aerobically on agar media containing 0.02 mg/liter Kepone were reduced by 8-78%. The inhibitory effect was eliminated partially when sediment populations were grown anaerobically.

Lucyszyn, E. and P.H. Pritchard. 1979. Characteristics of Bacteria Adapted to Low Nutrient Conditions in Lake Ontario. In: Developments in Industrial Microbiology, Vol. 20. EPA-600/J-79-054. Society for Industrial Microbiology, Washington, DC. Pp. 579-589. (ERL,GB 375). (Avail. from NTIS, Springfield, VA: PB80-184302)

The fate of organic pollutants in aquatic ecosystems depends, in part, on metabolic activities of the indigenous microflora. Knowledge is therefore needed of the growth characteristics of aquatic bacteria in low nutrient conditions typical of many aquatic environments. We have studied changes in bacterial populations from Lake Ontario using continuous culture enrichments, with lactose as the sole carbon and energy source. Enrichment studies have shown that the concn of lactose, the type of water utilized for inflowing media, and the inoculum source affected the outcome of continuous culture enrichments from fresh-water samples. Two isolates from enrichment experiments, an Aeromonas sp. and a Pseudomonas sp., were obtained using concn of 50 and 5 mg/liter lactose, respectively. These isolates were shown to be competitive with each other at different lactose concn. The Pseudomonas sp. was more sensitive to starvation and heat treatment than the Aeromonas sp. and did not take up oxygen in the presence of glucose when cell suspensions were prepared from cultures grown in peptone broth.

Mahaffey, W.R., P.H. Pritchard and A.W. Bourquin. 1979. Phenylacetic Acid Metabolism by Three Aquatic Bacteria Isolated from Continuous Culture Enrichments. In: Developments in Industrial Microbiology, Vol. 20. EPA-600/J-79-052. Society for Industrial Microbiology, Washington, DC. Pp. 489-495. (ERL,GB 377). (Avail. from NTIS, Springfield, VA: PB80-163736)

The fate of toxic organic compounds in aquatic environments depends, in part, on the capacity of microorganisms to metabolize these compounds at very low concn. Little information exists on the fate of aromatic ring compounds at low concn (< 100 mg/l) in aquatic environments. Therefore, we attempt to characterize the degradation of the aromatic compound phenylacetic acid (PAA) at low concn by aquatic bacteria. Three bacterial isolates, PAL-1, PAL-10, and PAL-100 obtained from continuous culture enrichment experiments at PAA concn of 1, 10, and 100 mg/liter, respectively. Washed cell suspensions of each isolate metabolized PAA, as measured by oxygen uptake, without lag regardless of whether cells were grown in acetate or PAA minimal salts medium, suggesting PAA metabolism was constitutive. Acetate-grown PAL-1 and PAL-100 cultures showed greater oxygen uptake activity in the presence of PAA than did PAA-grown cultures. All three isolates had high activity in the presence of meta-hydroxy-PAA. Incubation of washed cell suspensions at 25 C for 12 h showed that only the PAL-1 isolate lost activity (75%) relative to cell suspensions incubated at 4 C. Our studies provide physiological criteria that may characterize the types of bacteria that transform aromatic compounds at low concn in aquatic environments.

Couch, John A. 1986. Diseases and Parasites of Marine Fishes (Book Review). Estuaries. 9(3):229. (ERL,GB 601).

Recent and up-to-date review of the literature, outstanding illustrations and photomicrographs, and clarity in writing make this book a useful reference to students and researchers of marine fish diseases. The volume covers infectious, parasitic, and noninfectious causes of marine fish diseases in a systematic approach. Considerable emphasis is placed on the microbial and metazoan parasites of fishes, with a fairly general review of diseases associated with environmental and/or pollution problems. Over 200 photographs, many in color, are presented with good communicative value, and the line drawings are adequate to excellent in the sections on parasites. This book should be very useful to fish pathologists, parasitologists, microbiologists, aquatic ecologists, and environmental specialists. It could serve as a partial text for both undergraduate and graduate courses in the disciplines just mentioned. Because of the diversity of disciplines and subject matter covered, some of the discussion of cause and effect relationships in disease, and roles of parasites in the ecology of marine fishes are quite brief. Perhaps in future efforts it will be necessary to separate the pure parasitology and microbiology of marine fishes from their environmental- and pollution-related disorders in order to provide adequate discussion of each subject. Generally, this first attempt by two young authors is quite successful and provides another needed reference for investigators and students concerned with marine fishes' infectious and noninfectious diseases.

Walsh, Gerald E. 1988. Principles of Toxicity Testing with Marine Unicellular Algae. EPA/600/J-88/279. Environ. Toxicol. Chem. 7(12):979-987. (ERL,GB 606). (Avail. from NTIS, Springfield, VA: PB89-208599)

Toxicity testing with unicellular algae requires application of the principles of phycology and microbiology to culturing, handling, and exposing the organisms. This brief review describes major aspects of algal toxicity testing, including growth curves, factors that influence population growth in culture (light, temperature, medium composition, pH, and salinity), choice of test species, measurement of population density, enumeration of living and dead cells, numerical expression of toxic effects, and bioaccumulation.

Barkay, Tamar. 1992. Mercury Cycle. In: Encyclopedia of Microbiology, Volume 3. Academic Press, San Diego, CA. Pp. 65-74. (ERL,GB 761).

The mercury cycle describes fluxes of mercury among different compartments in the biosphere. Microbial (and abiological) transformations play a key role in this cycle by altering the chemical form of mercury compounds. Mercury as a toxicant is of most concern in its methylated form in aquatic environments where it is available for bioaccumulation by aquatic organisms. Thus, transformations of mercury that favor methylmercury formation and its accumulation have far reaching effects on human and animal health.

Devereux, Richard, Matthew D. Kane, Janet Winfrey and David A. Stahl. 1992. Genus- and Group-Specific Hybridization Probes for Determinative and Environmental Studies of Sulfate-Reducing Bacteria. EPA/600/J-93/064. Syst. Appl. Microbiol. 15(4):601-609. (ERL,GB 787). (Avail. from NTIS, Springfield, VA: PB93-168987)

A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. Four probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus multivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by hybridizations against closely related nontargeted species. In addition, each probe was screened by using a "phylogrid" membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The value of these probes to studies in environmental microbiology was evaluated by hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.

Barkay, Tamar. 1993. Old Concepts, New Approaches: Environmental Microbiology (Book Review). BioScience. 43(5):342-343. (ERL,GB 843).

The author reviews the book "Environmental Microbiology," edited by Ralph Mitchell and published by Wiley-Liss, New York, in 1992. The reviewer suggests that readers of the book will benefit from the insights found in chapters that tie basic processes in microbial physiology with enviromental problems and their solutions.

Devereux, R., J. Kurtz and G. Mundfrom. 1993. Molecular Phylogenetic Explorations of Natural Microbial Community Composition and Diversity. In: Trends in Microbial Ecology. EPA/600/A-94/115. R. Guerrero and C. Pedros-Alio, Editors. Spanish Society for Microbiology, Barcelona, Spain. Pp. 387-390. (ERL,GB 886). (Avail. from NTIS, Springfield, VA: PB94-190832)

Comparative sequence analysis of ribosomal RNA molecules has led to a phylogenetic-based approach to characterize natural microbial communities. The approach has been applied to study natural communities of sulfate-reducing bacteria. Hybridization probes were used to measure relative amounts of specific sulfate reducer rRNAs in an estuarine sediment. Selective amplification, cloning, and comparative sequence analysis of 16S ribosomal RNA gene sequences have revealed new diversity among sulfate-reducing bacteria.

Hines, Mark E., Pieter T. Visscher and Richard Devereux. 1996. Sulfur Cycling. In: Manual of Environmental Microbiology. Christon J. Hurst, Editor. ASM Press, Washington, DC. Pp. 324-333. (ERL,GB 926).

Sulfur(S) is an important element biochemically and geochemically. It constitutes about 1% of the dry mass of organisms where it serves many structural and enzymatic functions. S also acts as a significant electron donor and acceptor during many bacterial metabolisms (28). S can be found in a range of valence states from the highly reduced sulfide (-2) to the most oxidized form in sulfate (SO42-) (+6). There are several intermediate valence forms of S that can act as both electron donors and acceptors depending on environmental conditions, the most notable being elemental sulfur (S0) and thiosulfate (S2O 32-) (44). Microbial S transformations are closely linked with the carbon cycle in which S reduction coupled with organic matter utilization is a major mineralization pathway in anaerobic habitats, while S oxidations, some of which are autotrophic and/or phototrophic (28, 65), can occur aerobically and anaerobically. Transformations in the S cycle can uncouple carbon and energy flow since reduced S can carry a significant portion of the detrital energy. Many S compounds are highly reactive, and microorganisms often must compete with abiotic reactions. This competition makes study of certain physiological types of S bacteria difficult. In addition, the S cycle is complicated further by the reactivity of sulfide with metals and the oxidation of metal sulfides by bacteria. In general, the high chemical and biological reactivity of S compounds results in a tight coupling of the oxidative and reductive portions of the S cycle in aquatic habitats, particularly at the redoxcline where S cycling can be extremely rapid.

Borawski, Jason, Stephanie D. Friedman, Diane F. Yates, Alfred P. Dufour and Fred J. Genthner. 2001. Enterococci in the Water Column and Shoreline Interstitial Waters at Beaches on the Gulf of Mexico and Santa Rosa Sound, Pensacola, Florida. Presented at the 2001 Annual Meeting of the American Society for Microbiology, 20-24 May 2001, Orlando, FL. 1 p. (ERL,GB R801).

Interstitial water in the swash zone, that area of a beach where waves continuously wash up on the sand, is suspected of accumulating microbes. If pathogens are concentrated in the interstitial water or if they grow, they may pose a health risk, especially for children. This study compared densities of enterococci in the swash zone intersitial water to densities of enterococci in the adjacent bathing water at selected locations along Pensacola Beach, Florida. At each site, water samples were collected from the water column at a depth of 0.5 m, where the depth of the water column was 1.0 m. Swash zone water samples were obtained from holes 50 to 70 cm deep, that were dug in the sand 1.0 to 1.5 m from the shoreline. Enterococci, salinity, temperature, pH, dissolved oxygen and dissolved organic carbon were measured. At all sites, dissolved organic carbon and enterococci densities were higher in the swash zone interstitial water than in the water column. Analysis of paired swash zone/water column enterococci densities indicated that one of six beaches sampled possessed enterococci densities in the interstitial water of the swash zone that were significantly (p<0.05) higher than those in the water column. These data suggest that microbes in the swash zone interstitial water may pose an increased risk to bathers.

Bourquin, A.W. 1980. Discussion - Aquatic Microbial Ecology. In: Microbiology--1980. David Schlessinger, Editor. American Society for Microbiology, Washington, DC. Pp. 390-391. (ERL,GB X157).

We have been discussing the problem of how to assess environmental stress on ecosystems and the microbiological processes therein. Basically, what we have been discussing are methods--methods for measuring biomass, heterotrophic activity, biodegradation, and other indicators of environmental quality. What I would like to see discussed now is the question: 'What can we get out of these methods that can be used by regulatory agencies?' I am not sure that we are any closer to agreement on any particular method or methods that are more acceptable than others to the scientific community for assessing any environmental stress. However, I think that we have learned a lot about the various methods, including their limitations and their possible benefits. We have addressed various research needs and have just heard a very excellent presentation on this subject. What I would like to add to this is a request that we begin to apply some of this information, particularly in terms of the limitations and benefits of the various methods, to assess the so-called health of a given environment. There are environmental decisions which must be based on the available technology, because we really do not have any time and must make some decisions now. We can modify those decisions later, but a decision is needed now based on the current technology. If people such as those at this conference do not aid in determining which methods should be used with what qualifications, then less qualified people will make those decisions.

Portier, R.J. and S.P. Meyers. 1981. Chitin Transformation and Pesticide Interactions in a Simulated Aquatic Microenvironmental System. In: Developments in Industrial Microbiology, Vol. 22. EPA-600/D-81-081. Society for Industrial Microbiology, Arlington, VA. Pp. 543-555. (ERL,GB X224). (Avail. from NTIS, Springfield, VA: PB82-205840)

Interactions between the structural amino-polysaccharide, chitin, and the organophosphate pesticide, azinphosmethyl (Guthion), have been studied in a controlled continuous flow-through microcosm. Pesticide-induced microbial population changes and increases in substrate utilization of chitin are noted. Significant increases in total heterotrophs and chitinoclasts occurred relative to continuous application of azinphosmethyl. Rapid uptake and utilization of chitin and its derivatives, enhanced by controlled azinphosmethyl additions, were examined using a tagged chitin breakdown product, N-(glucosamine-1-14C)-acetyl-D Rates of utilization coincided with rapid 14CO2 release, assimilation of 14C by microbial and enzymatic criteria, inoculate the application of the benchtop microcosm in analyses of compound impact and substrate turnover.

Pritchard, P.H., R.J. Larson and L.S. Clesceri. 1980. Synopsis of Discussion Session: Extrapolation. In: Biotransformation and Fate of Chemicals in the Aquatic Environment. Alan W. Maki, Kenneth L. Dickson and John Cairns, Jr, Editors. American Society of Microbiology, Washington, DC. Pp. 99-104. (ERL,GB X266).

The discussion resulting from the session on the extrapolation of laboratory data to natural environmental situations dealt with three major problem areas: (I) the steps and decision points involved in providing definite exposure concentration estimates, (II) the experimental limitations to biodegradation rate constants, and (III) the assessment of the effects of environmental parameters on degradation rates and their integration into predictive mathematical models. All of these problems indicated a need for more research before definitive extrapolations can be made. It appears, however, that our approaches to the problem of extrapolation are headed in the right direction and that the results coming from various research laboratories lead to a certain degree of optimism. As discussed in the papers presented in this session and others, the potential of reducing biodegradation rates to simple descriptive rate constants which may be applicable over a large range of environmental variables is indeed exciting. Certainly, the ensuing discussions indicated that the rate constant approach was a provocative concept which should greatly stimulate and orient a data-gathering effort. This effort could eventually culminate in an appreciation of biodegradation rates which would substantially increase our confidence in making exposure concentration predictions through the extrapolation of laboratory data to the environment.

Portier, R.J. and S.P. Meyers. 1982. Monitoring Biotransformation and Biodegradation of Xenobiotics in Simulated Aquatic Microenvironmental Systems. In: Developments in Industrial Microbiology, Vol. 23. EPA-600/J-82-261. Society for Industrial Microbiology, Washington, DC. Pp. 459-475. (ERL,GB X432). (Avail. from NTIS, Springfield, VA: PB83-153718)

Microbiological studies coupled with physiochemical analysis of environmentally significant xenobiotics were conducted in continous flow-through and carbon metabolism microcosms to determine the behavior of these toxic substances in soil and sediment-water systems typical of coastal wetlands. The organophosphate, 14C-methyl parathion, and the chlorinated insecticide, 14C-Kepone, were examined for stress indexes based on microbial response and compound turnover. Significant increases in microbial populations, ATP, and specific enzyme systems (i.e., phosphatase and dehydrogenase), coinciding with rapid 14CO2 expiration and 14C-assimilation by the cellular component, pointed to both rapid biotransformation and biodegradation of methyl parathion. Minimal rates of 14CO2 release were noted for Kepone, with no significant variation seen in total microbial response or ATP levels. However, 14C-assimilation of Kepone was detected. Degradation rates for methyl parathion were comparable to in situ rates at experimental plots. Fungal biotransformation is suggested as important in methyl parathion degradation, with a negative response for Kepone. Similar activity was discerned for both compounds in axenic flask studies. Statistically valid correlations are evident between microcosm and field data.

Sayler, Gary S., Rakesh K. Jain, Andrew Ogram, Charles A. Pettigrew, Laura Houston, James Blackburn and William S. Riggsby. 1986. Applications for DNA Probes in Biodegradation Research. In: Perspectives in Microbial Ecology. EPA/600/D-86/249. F. Megusar and M. Gantar, Editors. Slovene Society for Microbiology, Ljubljana, Yugoslavia. Pp. 499-508. (ERL,GB X531). (Avail. from NTIS, Springfield, VA: PB87-145322)

The use of DNA:DNA hybridization technology in biodegradation studies is investigated. The rate constants for sediments exposed to synthetic oils could be calculated from the NAH+ genotypes and this approach would be useful in predicting the kinetics of aromatic hydrocarbon degradation. Gene probes prepared from NAH7 plasmid were also used to monitor and enumerate the naphthalene-degrading populations in a continuous mixed culture bioreactor and this analysis demonstrated at least one order of magnitude difference in the naphthalene-degrading population over the conventional plate analysis. It was also shown that using pSS50 (a chlorobiphenyl mineralizing plasmid) as probe DNA, other polychlorinated biphenyls degrading organisms can be identified from the environment. Further, the maintenance and stability of a genetically modified Pseudomonas putida (carrying plasmids TOL and RK2) over an 8-week period in chemically contaminated groundwater aquifer material was established. Results demonstrate the wide applications, signficances, sensitivity, and accuracy of DNA probes in environmental biodegradation research.

Miller, Robert V., Tyler A. Kokjohn and Gary S. Sayler. 1990. Environmental and Molecular Characterization of Systems Which Affect Genome Alteration in Pseudomonas aeruginosa: Chapter 25. In: Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology. EPA/600/D-91/118. S. Silver, A. Chakrabarty, B. Iglewski, and S. Kaplan, Editors. American Society for Microbiology, Washington, DC. Pp. 252-268. (ERL,GB X634). (Avail. from NTIS, Springfield, VA: PB91-199877)

Pseudomonas aeruginosa was used as a model organism to study mechanisms that lead to genome alteration in freshwater microbial populations. Our studies demonstrated horizontal transmission by both transduction and conjugation in freshwater ecosystems and provided data that suggest that intracellular genome instability may be increased due to environmental stresses encountered by the cell in this habitat.

Lessie, T.G., M.S. Wood, A. Byrne and A. Ferrante. 1990. Transposable Gene-Activating Elements in Pseudomonas cepacia: Chapter 27. In: Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology. S. Silver, A. Chakrabarty, B. Iglewski, and S. Kaplan, Editors. American Society for Microbiology, Washington, DC. Pp. 279-291. (ERL,GB X646).

The authors exploited the failure of pGC91-14 and pGC210 to confer ability to utilize lactose to select for insertions into Tn951 of P. cepacia elements able to increase lac gene expression. Bacteria in which such insertions had occurred grew rapidly on lactose. The increase in gene expression was due to increased formation of lac m-RNA. Isolation of broad-host-range plasmids carrying activated lac genes afforded an opportunity to examine the influence of P. cepacia IS elements on gene expression in other bacteria. Certain insertion sequences were more effective in turning on lac gene expression in other bacteria than in P. cepacia. The elements exhibited some differences in specificity. For example IS406 promoted constitutive formation of beta-galactosidase in Acinetobacter calcoacetisus, but not in the cellulose-forming bacterium Acetobacter xylinum. In contrast IS407 activated lac gene expression in A. xylinum, but not in A. calcoacetisus.

Miller, Robert V. and Tyler A. Kokjohn. 1990. General Microbiology of recA: Environmental and Evolutionary Significance. EPA/600/J-90/383. Annu. Rev. Microbiol. 44:365-394. (ERL,GB X657). (Avail. from NTIS, Springfield, VA: PB91-163964)

The RecA protein, a molecule of 38,000 Mr (125,126), is a multifunctional polypeptide directing a number of activities, none of which is completely understood in detail. It's central role in homologous recombination and DNA-damage repair has created considerable interest. Numerous studies in the last eight years which identify and characterize this gene have revealed the widespread distribution and evolutionary conservation of recA. In this review, authors explore the current knowledge of the general microbiology of recA and its protein product.

Walter, M.V., R.H. Olsen, V. Prince, R.J. Seidler and F. Lyon. 1989. Use of Catechol Dioxygenase for the Direct and Rapid Identification of Recombinant Microbes Taken from Environmental Samples. In: Rapid Methods and Automation in Microbiology and Immunology. A. Balows, R.C. Tilton, and A. Turano, Editors. Brixia Academic Press, Brescia, Italy. Pp. 69-77. (ERL,GB X697).

A study to (1) determine if the xylE gene could be utilized to detect and enumerate target microorganisms from the environment and (2) compare the sensitivity of detecting recombinant organisms with the xylE gene on nonselective media and on media containing antibiotics. Use of a selective medium in combination with the xylE gene on nonselective media and on media containing antibiotics. Use of a selective medium in combination with the xylE technique proved to be highly effective in detection and enumeration of recombinant organisms.

Eaton, Richard W., Peter J. Chapman and P. Hap Pritchard. 1990. Degradation of Heterocyclics. In: Bioremediation of Hazardous Wastes. EPA/600/9-90/041. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 46-48. (ERL,GB X722).

Many contaminants of ground water are heterocyclic compounds that are potentially hazardous to human health and to the environment. These classes of compounds along with many others are formed as combustion products in the generation of synthetic fuels from fossil fuels. Groundwater contamination by these chemicals has been shown in the vicinity of coal gasification plants, oil shale-retorting facilities, and at wood treatment facilities where coal-tar derived creosote has been used. To develop biological approaches to treat sites contaminated with these and other chemicals, the microbiology and biochemistry of their biodegradation must be understood. This research is designed to provide such insights.

Shields, Malcolm S., Michael Reagin, Robert Gerger, Rhonda Schaubhut, Robert Campbell, Charles Somerville and P. Hap Pritchard. 1993. Field Demonstration of a Constitutive TCE Degrading Bacterium for the Bioremediation of TCE. In: Symposium on Bioremediation of Hazardous Wastes: Research, Development, and Field Evaluations. EPA/600/R-93/054. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 73-79. (ERL,GB X764).

The degree to which trichloroethylene (TCE) has been recognized as a significant environmental pollutant is reflected by the amount of research into methods for its remediation. Despite the demonstrated environmental hazards, its industrial use continues apace because few alternatives exist. TCE owes its environmental behavior partly to its physical properties (i.e. high density and water solubility and low chemical reactivity), and partly to its biological recalcitrance. Both contribute to its notoriety as a persistent point source pollutant, despite numerous reports of both anaerobic and aerobic bacterial transformation capabilities. Aerobic bacteria are more rapid TCE metabolizers, but do so only in a cooxidative fashion. TCE serves as a cooxidative substrate for various bacterial oxygenases, but not as an inducer of them. These bacteria require co-inducers that include, toluene, phenol, methane, ammonia, isoprene, 2,4-dichlorophenoxyacetic acid (2,4-D), and propane. Our research has centered on the microbiology of P. cepacia G4, which expresses a unique toluene ortho- monooxygenase (Tom) in response to various aromatic inducers. Tom carries out the cooxidative metabolism of TCE by this strain. We have developed a non-recombinant derivative of G4, called G4 PR1, that constitutively expresses Tom, and consequently degrades TCE without the need for co-inducer. This communication deals with out characterization, alteration and application of this constitutive derivative.

Kidambi, Saranga P., Melissa G. Booth, Tyler A. Kokjohn and Robert V. Miller. 1996. RecA-Dependence of the Response of Pseudomonas aeruginosa to UVA and UVB Irradiation. Microbiology. 142(4):1033-1040. (ERL,GB X838).

The responses of the autochthonous soil and aquatic organism, Pseudomonas aeruginosa to UV radiation wavelengths (UVA, 320-400 nm, and UVB, 280-320 nm) has been investigated in this study. P. aeruginosa recA mutants were found to be more sensitive to both UVA and UVB radiation than were their isogenic RecA+ parents. Introduction of a low-copy-number plasmid containing the cloned wild-type P. aeruginosa recA gene restored UVA and UVB resistance to recA mutants.The concentration of RecA protein increased twofold 120 min after exposure to either UVA or UVB radiation, suggesting induction of expression of the recA gene by these wavelengths. In this study, we found that a functional RecA protein is required for activation of D3 prophage in lysogenic cells following exposure to UVB radiation. Prophage were not induced by exposure of their hosts to UVA radiation. Induction of damage-inducible (din) genes in response to UVA or UVB irradiation was also shown to be RecA dependent. These data indicate that the recA gene plays a role in the response of P. aeruginosa to exposure to wavelengths of UV radiation found in the solar spectrum.

Wackett, Lawrence P. 1996. Biodegradation of Halogenated Solvents. In: Manual of Environmental Microbiology. Christon J. Hurst, Editor. ASM Press, Washington, DC. Pp. 784-789. (ERL,GB X852).

Chlorinated solvents have been used industrially in vast quantities as degreasing agents, heat transfer agents, and chemical intermediates in synthesis. Their usage has decreased because of public health concerns, but they are still widely used. Most heavily in use are C1 compounds plus C2 alkanes and alkenes containing chlorine and, in some cases, fluorine substituents in place of hydrogen atoms. Most notable with respect to negative environmental impact are chlorofluorocarbons, implicated in ozone layer destruction, and CCl4 and vinyl chloride, which have been demonstrated to be carcinogenic in mammals. The biodegradability of the compounds shown in Fig. 1 varies widely, but a general rule is that biodegradability decreases with increasing halogen content. This reflects the relatively low chemical reactivity of polyhalogenated compounds and the necessity for multiple dehalogenation mechanisms for complete metabolism of the compound. In general, chlorinated alkenes are less biodegradable than the corresponding chloroalkanes. It is sometimes erroneously stated that low-molecular-weight chlorinated compounds are only of human origin when, in fact, chlorinated natural products are found ubiquitously. For example, chloromethane is made in significant quantities by soil fungi. However, CCl4 and chlorofluorocarbons are principally, if not exclusively, of anthropogenic origin. In this context, these latter compounds have been in the environment for only a short time on an evolutionary time scale, and this may reflect the rarity of microorganisms that can metabolize them.

Mueller, James G., Carl E. Cerniglia and P. Hap Pritchard. 1996. Bioremediation of Environments Contaminated by Polycyclic Aromatic Hydrocarbons. In: Bioremediation: Principles and Applications. Ronald L. Crawford and Don L. Crawford, Editors. Cambridge University Press, New York, NY. Pp. 125-194. (ERL,GB X881).

Bioremediation is described herein as the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state, or to levels below concentration limits established by regulatory authorities. In past reviews, we have focused on the basic biochemical and microbiological principles of polycyclic aromatic hydrocarbon (PAH) biodegradation (Cerniglia, 1984, 1992, 1993; Cerniglia et al., 1992; Cerniglia & Heitkamp, 1989), and the applicability of these processes toward the bioremediation of PAH-contaminated environments (Mueller et al., 1989a, 1993b; Pritchard, 1992). In this review, we have attempted to combine our perspectives on PAH biodegradation to yield a comprehensive overview of myriad factors impacting the efficacy of bioremediation of environments contaminated by PAHs. As such, this review helps unite technical insight and practical experience from several scientific disciplines including biochemistry, environmental chemistry, environmental microbiology, microbial ecology, and toxicology. Together, these factors interact to influence the scientifically valid, safe and effective use of bioremediation technologies for restoration of PAH-contaminated environments.