Book Chapter & Symposium Paper Citations and Abstracts

Sediments and rooted aquatic plants are major components of wetland systems. Sediments in these systems act as sinks and reservoirs for organic and inorganic pollutants that are absorbed to sediment particles or contained in interstitial water that exists between the particle spaces of sediments. Sediments release toxic materials to aquatic environments through processes that include resuspension, desorption, and bioturbation. In sediment systems, aquatic plants are affected by toxic materials through direct effects on root growth or rhizosphere microorganisms, or by the assimilation and translocation of chemical substances to aerial organs, where they may injure foliage or inhibit growth and seed production. Procedures are being developed to identify the effects of contaminated sediments on aquatic plants through use of laboratory toxicity tests involving natural and artificial sediments, and a group of rooted aquatic species. In these studies, two freshwater species, Echinochloa crusgalli and Sesbania macrocarpa, and a saltmarsh species, Spartina alterniflora, are being evaluated. Artificial substrates proved useful in assessing the effects of contaminants on plant growth and development, and provided techniques for evaluating the role of sediment components on plant response.

Barkay, Tamar, Sylvie Nazaret and Wade Jeffrey. 1995. Degradative Genes in the Environment. In: Microbial Transformation and Degradation of Toxic Organic Chemicals. L.Y. Young and C. Cerniglia, Editors. Wiley-Liss, New York, NY. Pp. 545-577. (ERL,GB 862).

This book chapter briefly describes processes of gene expression and provides recent literature references for the interested reader. Authors focus on potential benefits to bioremediation strategies that might arise from understanding molecular events that lead to biodegradation in situ. Authors propose that tools to stimulate remedial treatments can be obtained by further understanding of how the environment modulates gene expression of biodegradative genes.

Devereux, Richard and Stephanie G. Willis. 1995. Amplification of Ribosomal RNA Sequences. In: Molecular Microbial Ecology Manual. A.D.L. Akkermans, J.D. van Elsas, and F.J. DeBruin, Editors. Kluwer Academic Publishers, Norwell, MA. Pp. 3.3.1:1-11. (ERL,GB 881).

This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedures for the isolation of microbial nucleic acids from pure cultures and environmental samples and preparation of DNA templates.

Trust, Beth A., James G. Mueller, Richard B. Coffin and Luis A. Cifuentes. 1995. Biodegradation of Fluoranthene as Monitored Using Stable Carbon Isotopes. In: Monitoring and Verification of Bioremediation. Robert E. Hinchee, Gregory S. Douglas and Say Kee Ong, Editors. Battelle Press, Columbus, OH. Pp. 233-239. (ERL,GB 901).

The measurement of stable isotope ratios of carbon (d13C values) was investigated as a viable technique to monitor the intrinsic bioremediation of polycyclic aromatic hydrocarbons (PAHs). Biometer-flask experiments were conducted in which the bacterium, Sphingomonas paucimobilis, designated EPA505, was grown on fluoranthene. During growth of EPA505 on fluoranthene, bacterial biomass, respired CO2 and dissolved organic carbon (DOC), as well as fluoranthene, were sampled over eight days. The concentrations and d13C values of each of these carbon pools were determined. The concentration of fluoranthene decreased from 12.1 ± 2.0(n=2) to 3.0 ± 0.9 (n=2)mg C per flask over 188 h, and CO2 increased from undetectable levels to 7.1 ± 0.3 (n=4) mg C per flask. A total of 55.5% mineralization resulted. DOC concentrations remained fairly constant with time, averaging 2.2 to 3.6 mg C per flask. The d 13C value of fluoranthene remained constant over the course of the experiment, averaging -24.5 ± 0.2 o/oo (n=8). Bacterial nucleic acids and respired CO2 took on d13C values similar to those of fluoranthene within 47 h, measuring -22.6 and -24.3 o/oo, respectively.

Menzer, Robert E. and James E. Harvey. 1995. Bioremediation Efficacy and Risk Assessment Research: United States Environmental Protection Agency Perspective. In: Bioremediation: the Tokyo '94 Workshop. Organisation for Economic Co-operation and Development, Paris, France. Pp. 295-307. (ERL,GB 903).

The criteria for successful implementation of bioremediation technologies are often poorly understood, provoking problems or failures in field demonstrations. The U.S. Environmental Protection Agency (EPA) pursues its legislative mandate to protect human health and the environment by advancing the understanding, development, and application of bioremediation solutions to hazardous waste problems. The Agency's approach is a balance between basic research activities and engineering. The former leads to an understanding of biological degradation processes, and the latter yields practical applications of the underlying science to accomplish environmental cleanup. Successful bioremediation trials have the following components in common: characterization of the contaminated site, examination of the feasibility of enhancing in situ microbial populations, development of treatment technologies that target specific toxic chemicals or groups of chemicals, and development of treatment systems approapriate for the wastes, the site, and the treatment sequence. A trial is not complete without a determination of the level of risk reduction resulting from the treatment. The lack of validated scale-up information, the variability of contaminated field sites, and the potential for production of metabolites more toxic then parent chemicals complicate prediction of adverse ecological and human effects. Studies examining fate and effects of degradative microorganisms, target chemicals, and metabolic by-products allow information from risk assessments to alert environmental managers and regulators to potential difficulties resulting from the use of this powerful technology.

Stackebrandt, Erko, David A. Stahl and Richard Devereux. 1995. Taxonomic Relationships. In: Sulfate-Reducing Bacteria. Biotechnology Handbooks, vol. 8. Larry L. Barton, Editor. Plenum Press, New York, NY. Pp. 49-87. (ERL,GB 927).

The last several years have seen a notable increase in basic and applied research into the dissimilatory reduction of sulfate by microorganisms. The most significant change in our knowledge of this functionally-defined assemblage has been the recognition of far greater evolutionary, genetic, and metabolic capability than previously appreciated. Sulfate-reducing bacteria (abbreviated SRB in the following text) are now known to directly or indirectly participate in the degradation of a wide variety of substrates, including saturated hydrocarbons and a variety of aromatic and xenobiotic compounds. Our intention in this chapter is to provide the reader with both a historical and contemporary perspective on the classification and phylogeny of the sulfate-reducing bacteria. The historical chronicle is a prelude to discussion of more recent molecular studies and provides an essential overview of the defining physiological attributes of these bacteria. In turn, the developing molecular phylogeny serves to frame the principal physiological features within a natural classification and provide a foundation for the development of DNA probes used for the direct exploration of environmental diversity and studies of the SRB ecology. It is the latter that remains the greatest challenge. Although we have greatly expanded our appreciation of the genetic diversity of sulfate-reducing bacteria, a complete understanding of that diversity can only come from an understanding of their ecology in relationship to their evolution.

Lewis, M.A. 1995. Algae and Vascular Plant Tests. In: Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment. Second edition.. Gary M. Rand, Editors. Taylor & Francis, Washington, DC. Pp. 135-169. (ERL,GB X804).

The objective of this chapter is to describe the current use of freshwater plants and to a lesser extent, saltwater species in ecotoxicology. The chapter includes discussions on those types of tests currently conducted which are in order of decreasing frequency: algal population growth, duckweed, algal photosynthesis, rooted whole plant macrophytes, and macrophytic seeds. Chapter also describes the use of seaweeds in saltwater toxicity tests. It should be noted that due to the broad scope of this topic and limitations on the length of this chapter that only introductory information is provided. Additional and more specific details are included in the literature cited which are important to consult before any phytotoxicity test is conducted.

Mearns, Alan, Kenneth Doe, William Fisher, Rebecca Hoff, Kenneth Lee, Robert Siron, Cornelia Mueller and Albert Venosa. 1995. Toxicity Trends During an Oil Spill Bioremediation Experiment on a Sandy Shoreline in Delaware, USA. In: Proceedings of the Eighteenth Arctic and Marine Oilspill Program (AMOP) Technical Seminar. Volume 2. Environment Canada, Ottawa, Ontario. Pp. 1133-1145. (ERL,GB X809).

A 13-week, refereed, inter-agency toxicity testing program involving five bioassay methods was used to document the effectiveness of shoreline bioremediation to accelerate toxicity reduction of an oiled sandy shoreline at Fowler Beach, Delaware, USA. The study was part of an international oiling experiment using a randomized complete block design with repeated measures. Bioremediation - treatment with nutrients or nutrients and oil-degrading bacteria - did not accelerate toxicity reduction. Nor did treatment increase toxicity at weeks 0. 6 or 12-13. However, results of one high-frequency test suggested there may have been a substantial delay in toxicity reduction due to treatment during the first few weeks of treatment. All tests provided information but the most sensitive tests were the 10-day sediment amphipod and grass shrimp embryo bioassays. Standardized sediment and water toxicity tests can play a valuable role in evaluating the effectiveness and effects of oil spill shoreline countermeasures.

Klaine, Stephen J. and Michael A. Lewis. 1995. Algal and Plant Toxicity Testing. In: Handbook of Ecotoxicology. David J. Hoffman, Barnett A. Rattner, G. Allen Burton, Jr. and John Cairns, Jr., Editors. Lewis Publishers, Boca Raton, FL. Pp. 163-184. (ERL,GB X818).

The evaluation of the phytotoxicity of a chemical is an essential component of the ecological risk assessment of that compound. Primary producers form an essential trophic level of any ecosystem. Further, since all chemicals introduced into the environmental ultimately find their way into aquatic ecosystems, aquatic algal and plant toxicity evaluations are particularly critical. This chapter presents the current state of phytotoxicity testing, with particular attention paid to algal and vascular plant bioassays. The algal bioassay section focuses on test methods due to the relatively long history of algal toxicity testing. The vascular plant section focuses on the different plants used for bioassays and the various endpoints used in these bioassays.

Morris, Pamela J., Michael E. Shelton and Peter J. Chapman. 1995. Co-Contaminated Sites: Biodegradation of Fossil Fuels in the Presence of PCBs. In: Bioremediation of Recalcitrant Organics. Robert E. Hinchee, Daniel B. Anderson and Ronald E. Hoeppel, Editors. Battelle Press, Columbus, OH. Pp. 123-130. (ERL,GB X823).

Polychlorinated biphenyl (PCB)-contaminated sites are often co-contaminated with fossil fuels making biodegration more difficult. Our current studies examine biodegradation of the fossil fuel components of two PCB-contaminated sites: (1) a former racing Drag Strip soil contaminated with Aroclor 1242 and (2) a sediment from Silver Lake contaminated with Aroclor 1260. The sandy surface soil at the Drag Strip site contains 1.9% organic carbon and 1.5% fossil fuel component. Analysis of the solvent-extractable organic fraction, by alumina column chromatography, shows the distribution of organics to be 91.2% hydrocarbons, 7.8% polars, and 1.1% asphaltenes. This oil is extremely weathered and contains few readily biodegradable components. Enrichments have yielded undefined mixed cultures of bacteria capable of extensive degradation of components of both the Drag Strip and Silver Lake site materials. One culture, enriched from a creosote-contaminated soil adjacent to a utility pole, transformed approximately 28% and 37% (by weight) of the Drag Strip and Silver Lake oils, respectively. While the presence of fossil fuels has been shown to inhibit aerobic PCB degradation, our studies show that the presence of PCBs negatively impacts fossil fuel biodegradation. continuing studies will examine the nature of PCB inhibition of fossil fuel biodegradation.

Lantz, Suzanne, Jian-Er Lin, James G. Mueller and Parmely H. Pritchard. 1995. Effects of Surfactants on Fluoranthene Mineralization by Sphingomonas paucimobilis Strain EPA 505. In: Microbial Processes for Bioremediation. Robert E. Hinchee, Fred J. Brockman, Catherine M. Vogel, Editor. Battelle Press, Columbus, OH. Pp. 7-14. (ERL,GB X824).

Past results from surfactant-enhanced biodegradation studies have been equivocal because of inhibitory effects of the surfactants and a poor understanding of the characteristics of PAH-degrading microorganisms that make them responsive to surfactants. We have studied the mineralization of 14C-radiolabeled fluoranthene by high cell masses of Sphingomonas paucimobilis, strain EPA 505, and have shown that initial rates of mineralization can be enhanced by concentrations of the surfactant Triton X-100 as high as 2%. Mass balances are reported that show complete degradation of fluoranthene. The presence of soil stimulated biodegradation of fluoranthene in the same manner as surfactants, presumably because of increased dissolution rates from soil particulates. The usefulness of this bacterium in the bioremediation of PAH-contaminated soil is discussed.

Lin, Jian-Er, Suzanne Lantz, Warren W. Schultz, James G. Mueller and Parmely H. Pritchard. 1995. Use of Microbial Encapsulation/Immobilization for Biodegradation of PAHs. In: Bioaugmentation for Site Remediation. Robert E. Hinchee, Jim Fredrickson, and Bruce C. Alleman, Editors. Battelle Press, Columbus, OH. Pp. 211-220. (ERL,GB X825).

Bioaugmentation as a strategy in bioremediation has great potential but has had little success to support its use. Problems have arisen because of a general inability to support the growth and/or activity of the introduced organism in the environment because of competition factors, poor survival of the inoculum, and grazing by protozoa. A specialized technique that has been used to overcome these problems is cell immobilization or encapsulation, in which the inoculant can be placed in environmental media in a way that reduces competition from the indigenous microflora and allows expression of the specific introduced metabolic function. Packaging of specific bacterial or fungal cells in a porous polymeric material potentially improves storage of inocula, and enhances the capability of directly introducing viable and active cells into environmental material at some future time without the need to regrow the cells. We have been experimenting with encapsulation/immobilization procedures for use in the bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil. In this paper, we demonstrate the potential usefulness of polyurethane foam and vermiculite for this purpose and show that optimal PAH degradation can be maintained with immobilized cells

Mueller, James G., Michael D. Tischuk, Mitchell D. Brourman and Garet E. Van De Steeg. 1995. In Situ Bioremediation Strategies for Organic Wood Preservatives. In: In Situ Aeration: Air Sparging, Bioventing, and Related Remediation Processes. Robert E. Hinchee, Ross N. Miller and Paul C. Johnson, Editors. Bettelle Press, Columbus, OH. Pp. 571-579. (ERL,GB X826).

Laboratory biotreatability studies evaluated the use of bioventing and biosparging plus groundwater circulation (UVB technology) for their potential abililty to treat soil and groundwater containing creosote and pentachlorophenol. Soils from two former wood-treatment facilities were used in these studies. These studies provided useful, site-specific data demonstrating enhanced biodegradation of all monitored organic constituents. The results suggest that the introduction and delivery of co-reagents (i.e., oxygen and nitrogen) essential to in situ biodegradation of organic wood preservatives represents an important component of effective in situ bioremediation. Full-scale implementation strategies are being considered based on the findings of these studies.

Pritchard, P.H., J.G. Mueller, S.E. Lantz and D.L. Santavy. 1995. Potential Importance of Biodiversity in Environmental Biotechnology Applications: Bioremediation of PAH-contaminated Soils and Sediments. In: Microbial Diversity and Ecosystem Function. D. Allsopp, R.R. Colwell, and D.L. Hawksworth, Editors. CAB International, Wallingford, UK. Pp. 161-182. (ERL,GB X827).

The biodiversity of hydrocarbon degraders is indeed extensive. This is due, in part, to the ubiquitous nature of hydrocarbons, from both natural and anthropogenic sources. It is proposed that this biodiversity be investigated further because (a) it will improve our understanding of the role that diversity plays in maintaining hydrocarbon mineralization within carbon cycling processes in the environment, and (b) it can provide a strong scientific basis upon which the effectiveness and environmental safety of environmental biotechnologies can be implemented. This chapter has focused on the aromatic hydrocarbon degraders, because of their current attention as widespread environmental contaminants. Relatively little is known about the ecology and diversity of microorganisms that degrade the polycyclic aromatic hydrocarbons (PAHs), especially those of four or more condensed rings. The importance of the PAHs as worldwide contaminants dictates that further biodiversity characterization of microbial population in soil be carried out as a means to predict more precisely the fate and ecological effects of the PAHs. If the degradation of the high-molecular-weight PAHs involves a unique group of microorganisms because of the special characteristics associated with the chemistry and environmental distribution of PAHs, then further research may reveal new elements of biodiversity that can be useful as a measure of ecosystem health.

Santavy, D.L. 1995. Diversity of Microorganisms Associated with Marine Invertebrates and Their Roles in the Maintenance of Ecosystems. In: Microbial Diversity and Ecosystem Function. D. Allsopp, R.R. Colwell and D.L. Hawksworth, Editors. CAB International, Wallingford, UK. Pp. 211-229. (ERL,GB X828).

The marine environment, especially coral reef environments with its vast biodiversity, offers many unique niches for opportunities to explore and discover novel microorganisms and the significant roles they assume. A number of microbial floras have already been described and shown to yield genetically novel microorganisms, although most are non-culturable at this time. The search for increasing our knowledge of microbial biodiversity should not exclude microorganisms associated with higher organisms, including descriptions and examination of functional roles of mutualistic symbioses, pathogenic associations, and other categories of assemblages. Microorganisms are fundamentally important to the cycling of nutrients among different trophic levels, remineralization of recalcitrant substrates, and survival of higher organisms by imparting unique survival qualities. All of these organisms are necessary to maintain the ecological balance of marine environments; the disruption or loss of certain components of the community may permanently alter entire ecosystems. One of the most germane issues to address is the immense scale, intensity and irreversibility of habitat modification and subsequent loss of species diversity. Synergistic effects of global climate change and burgeoning anthropogenic stresses induced by increased development in coastal regions are difficult to discern and often compounded. The need to assess entire ecosystem responses to perturbations is critical since long-term consequences may affect overall reproduction, recruitment, community structure and composition of the entire species assemblages and perhaps, ultimately the survival and fate of entire complex and diverse living communities. Potential causes and effect of species decline and the concomitant loss of biodiversity and habitat should be identified

Genthner, Fred J. and Douglas P. Middaugh. 1995. Nontarget Testing of an Insect Control Fungus: Effects of Metarhizium anisopliae on Developing Embryos of the Inland Silverside Fish Menidia beryllina. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 250-268. (ERL,GB X853).

Developing embryos of the inland silverside fish Menidia beryllina, were exposed to conidiospores of the entomopathogenic fungus, Metarhizium anisopliae. Several adverse effects were observed in both embryos and newly hatched larvae. These included transitory effects on the heart resulting in decreased cardiac output or circulation velocity, rupture of the chorion, fungal growth on the mandibles of larvae, focal vertebral abnormalities in larvae and teratogenic expressions in embryos and larvae. An ordinal ranking system was used to enumerate responses to conidiospores. This ranking system allowed significance to be determined by nonparametric analysis of variance. Responses were highly variable with significant (p less than or equal to 0.05) adverse effects observed in five of the six experiments conducted. Heat-killed spores failed to cause significant adverse effects, indicating that viable spores were required for adverse effects to occur.

Burand, John P., Greg Dwyer, Vincent D'Amico and Joseph S. Elkinton. 1995. Methods for Monitoring the Environmental Fate of Genetically Engineered Baculoviruses. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 293-306. (ERL,GB X854).

A genetically modified gypsy moth baculovirus carrying a marker gene for B-galactosidase activity was released on an 8 hectare plot to monitor virus movement in the insect population and to test the strategy of co-occlusion for limiting virus fate. Analysis of virus killed insects for presence of the marker gene allowed us to follow the movement of virus in the population. Deletion of the EGT gene results in a gypsy moth virus with enhanced virulence. An EGT-gypsy moth virus is being used in greenhouse experiments to measure parameters that effect fitness and the ability of the virus to compete with wild type virus in the field. These measurements will be used in our computer model of the interaction of the gypsy moth virus and its host to predict the fate of EGT-virus in the environment.

Kokjohn, T.A., J.O. Schrader, J.J. Walker and H.S. Schrader. 1995. Effects of Stress on Bacteriophage Replication. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 79-89. (ERL,GB X855).

While bacteriophages have been the subjects of intense scientific study for decades, knowledge of their replication potential in situ is limited. If optimal environmental conditions for host cell growth are prevalent only on an infrequent basis, the production of bacteriophages may be severely limited and sporadic. Models of the dynamics of bacteriophages created by the simple extrapolation of laboratory observations of systems maintained under artificially optimal conditions may not faithfully represent situations in natural ecosystems. At the present time, because of the highly limited nature of the knowledge base, it is impossible to make accurate predictions of the magnitude of the effect of bacteriophages on bacterial populations in marine ecosystems. In order to more accurately model bacteriophage dynamics in marine ecosystems, we have undertaken a study of the effects of environmental stresses on marine and freshwater bacteria. The effects of solar ultraviolet (UV) irradiation and starvation on the replication of bacteriophages and host cell survival have been quantified. These experiments have examined both the kinetics of progeny phage production as well as burst sizes subsequent to application of stress. Prophage induction due to DNA damage has been quantified in lysogens exposed to solar UV or starvation stress. In addition, the effects of broad-band solar UV irradiation on the survival of free virions have been quantified.

Jeffrey, Wade H., David L. Mitchell and Richard B. Coffin. 1995. Molecular Studies of Ultraviolet Radiation Effects on Marine Bacteria. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 90-104. (ERL,GB X856).

To determine the environmental effect of stratospheric ozone depletion, research has focused on the responses of marine microorganisms to ultraviolet radiation (UVR). The majority of this research has focused on the base of marine food chains, phytoplankton, while substantially fewer studies have examined bacteria. Bacteria mediate geochemical cycles that support the activity of the entire marine food chain. An evaluation of the effect of UVR on bacteria will complement work that has been conducted on phytoplankton and significantly contribute to our understanding of the control UVR exerts on plankton community dynamics. Our studies have focused on two processes; the formation of DNA photoproducts (cyclobutane pyrimidine dimers and pyrimidine (6-4)pyrimidone dimers) and induction of damage repair mechanisms determined by measurements of recA transcription and translation. UVB (280-320 nm) survival curves and photoproduct dose response curves have been determined for two marine bacteria, Vibrio natriegens and Pseudomonas stutzeri. V. natriegens is significantly more sensitive to UVB than P. stutzeri and higher photoproduct formation per UV dose was observed in V. natriegens. Gulf of Mexico waters have been used for studies of indigenous bacterioplankton populations. Diel patterns have been observed in RecA expression and levels of RecA have been found to be higher in surface waters than in deeper waters. Preliminary data suggests that solar radiation does cause photoproduct formation in surface water bacterial populations. We are currently examining diel formation and repair of photoproducts in surface waters and photoproduct formation in waters collected at various depths. Future experiments will investigate the relationships between photodamage and RecA expression in indigenous bacterioplankton populations.

Chowdhury, M.A.R., J. Ravel, R.T. Hill, A. Huq and R.R. Colwell. 1995. Physiology and Molecular Genetics of Viable but Non-Culturable Microorganisms. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 105-122. (ERL,GB X857).

Gram-negative bacteria are known to enter a viable but non-culturable (VBNC) state, in which they no longer grow on conventional media, but remain intact and retain viability. This phenomenon has been regarded, in many ways, to be analogous to sporulation in Gram-positive bacteria. We have investigated the presence of spoO-like genes in Vibrio cholerae and Escherichia coli by polymerase chain reaction using primers based on conserved regions of Bacillus subtilis spoOA and spoOF genes. We did not identify regions of V. cholerae or E. coli that exhibited complete homology to spoO genes from B. subtilis and, therefore, concentrated on an alternate approach of transposon mutagenesis. Over 2,500 transposon mutants of V. cholerae were screened under low nutrient conditions in artificial sea water for an altered VBNC response, compared to the wild type. Mutant JR09H1 entered the VBNC state more rapidly than the wild type at both 25 degrees C and 4 degrees C. Physiological investigations, done in parallel with the genetic analysis of control of the VBNC state, focused on two aspects of the VBNC response. Metabolic activity of V. cholerae and E. coli cells at the point of entry into the VBNC state was investigated in substrate uptake of 3H-labeled thymidine and 14C-labeled glucose and acetate were measured. Uptake of substrate decreased as cells entered the VBNC state and dramatically increased with temperature upshift from 4 degrees C to 30 degrees C, with recovery of culturable cells of V. cholerae. The increased uptake was not observed with E. coli, which did not recover culturability. Transition from the VBNC state, after temperature upshift was due to regrowth of a few culturable cells rather than true transition of VBNC cells to the culturable state. However, we do not exclude the possibility that VBNC cells converted to culturable cells as a first step in the process. Elucidation of the mechanism(s) of genetic control of VBNC state will be required before this phenomenon can be fully understood.

Swannell, Richard P.J., Joe E. Lepo, Kenneth Lee, P.Hap Pritchard and D. Martin Jones. 1995. Bioremediation of Oil Contaminated Fine-Grained Sediments in Laboratory Microcosms. In: Proceedings of the Second International Oil Spill Research and Development Forum, 23-26 May 1995. Volume 1. International Maritime Organisation, London, England. Pp. 45-55. (ERL,GB X899).

The potential of bioremediation to treat oiled soft sediments taken from a mud flat was investigated in laboratory microcosms subjected to a vertical tidal cycle. The velocity and duration of the tidal cycle was determined from a site survey. The effect of 3 different bioremediation strategies on the biodegradation of a weathered and emulsified Arabian Light crude oil were investigated. The weekly addition of sodium nitrate and potassium dihydrogen orthophosphate was found to be the most effective strategy. This method stimulated microbial respiration rates substantially, increased the total numbers of chemoheterotrophs and the total number of hydrocarbon-degraders significantly, and resulted in a substantial degradation of the aliphatic and aromatic components of oil. These preliminary results suggest that bioremediation may have a role to treat oil contamination on soft sediments.

Ho, Yifong, Yujing Yang and Parmely H. Pritchard. 1995. Classification of PAH-Degrading Bacteria by PAH Utilization Patterns and the Comparison of Metabolic Products. In: Eighth International IGT Symposium on Gas, Oil and Environmental Biotechnology, December 11-13, 1995, Colorado Springs, Colorado. Institute of Gas Technology, Des Plaines, IL. 18 p. (ERL,GB X912).

Bacterial strains capable of using either phenanthrene, fluoranthene, or pyrene as sole carbon and energy sources were isolated from 16 different soil samples collected from the United States, Germany, and Norway. Thirty one strains were isolated on fluoranthene and the other twenty one strains were isolated on pyrene. Two other strains (CO6 and UMB), isolated on phenanthrene, are also included in this study. Co-metabolic degradation of PAHs, including fluorene, phenanthrene, anthracene, fluoranthene (FLA), pyrene (PYR), B[b]fluorene, B[a]anthracene, chrysene and B[a]pyrene were tested for each isolated using a plate screening technique. From our studies, the bacterial strains isolated on pyrene can use either pyrene or fluoranthene as sole carbon and energy source while most of the thirty one strains isolated on fluoranthene could not grow on pyrene as sole carbon source. However, these isolates are able to transform pyrene into other products co-metabolically. GC-FAME results suggest that many PYR degraders are Mycobacterium sp. and FLA degraders are Sphingomonas sp. Preliminary isolation of the pyrene metabolites produced by the fluoranthene degraders showed one major product by HPLC. This peak matches the pyrene metabolite produced by the pyrene isolates.

Mueller, James G., Susanne Borchert, Cherie Heard, Fayaz Lakhwala, Suzanne Lantz, Eric Klingel, William Langley, Mitchell Brourman, Richard Coffin, Cheryl Kelley, Beth Trust, Luis Cifuentes, Michael T. Montgomery, Thomas Boyd, Barry J. Spargo and Warren Schultz. 1995. Management of PAH-Impacted Sites via In Situ Chemical Containment and Monitoring. In: Eighth International IGT Symposium on Gas, Oil and Environmental Biotechnology, December 11-13, 1995, Colorado Springs, Colorado. Institute of Gas Technology, Des Plaines, IL. 23 p. (ERL,GB X913).

On a world-wide basis the magnitude of environmental contamination problems involving polycyclic aromatic hydrocarbon (PAHs) is unmatched by any other group of organic chemicals. Despite the recognized limitations to PAH biodegradation (e.g., intrinsic chemical stability of the higher-molecular-weight PAHs), in situ bioremediation is frequently recommended out of practical and economic necessity. This is especially true when depth of contamination, magnitude of the problem, the presence of free product, or nature of contaminated material preclude other remedial actions. From our perspective, in situ chemical containment often represents an effective, safe and scientifically-valid management strategy, especially when these practices are combined with unequivocal approaches for monitoring in situ bioremediation of target contaminants. To field validate one approach to this strategy, we have completed the full-scale installation of a microbiologically-enhanced ground water circulation (UVB) system coupled with an in situ bioreactor at an operating wood preserving site in the southeast United States where constituents of coal tar creosote are present in soil and ground water. Data collected from a continuing sampling and analysis plan demonstrate that this sytem enhances in situ biodegradation of creosote and provides cost-efficient, effective in situ chemical containment and source management.

Borchert, Susanne M., James G. Mueller, Eduard J. Alesi and Christoph A. Leins. 1995. Application Strategies and Design Criteria for In Situ Bioremediation of Soil and Groundwater Impacted by PAHs. In: Eighth International IGT Symposium on Gas, Oil and Environmental Biotechnology, December 11-13, 1995, Colorado Springs, Colorado. Institute of Gas Technology, Des Plaines, IL. 8 p. (ERL,GB X914).

Biotreatability studies conducted in our laboratory used soils from two former wood-treatment facilities to evaluate the use of in situ bioventing and biosparging applications for their potential ability to remediate soil and groundwater containing creosote. The combination of physical, chemical and microbiological analytical data from these studies suggested that biodegradation or creosote polycyclic aromatic hydrocarbons (PAHs) could be stimulated by adding oxygen and nitrogen to the indigenous microflora. Over the relatively short time frame of these studies, however, biodegradation of potentially carcinogenic PAHs (pc PAHs) was limited, which is in accordance with much of the existing scientific literature. Nevertheless, on a site-specific basis, appropriately designed in situ bioremediation systems may represent effective strategies for degrading PAHs and affecting in situ chemical containment. Critical to the future success of bioventing/biosparging applications for PAh-impacted environments is the continued development and refinement of effective implementation tools. These must offer better means of delivering essential co-reagents, such as oxygen and nitrogen, and must possess the potential to integrate biotechnological advances. One such system attempting to increase the efficiency of site remediation is the "Multifunctional Well" Technology. To maximize remediation progress, different technologies are combined to operate in one multi-process remediation well. Thus, various remediation processes are integrated to remove certain types and/or suites of contaminants. For example, vapor extraction/in situ air stripping removes volatile (e.g. naphthalene) organics, while bioremediation of more persistent organics (e.g. fluoranthene) is enhanced by circular air and water flow. Remediation success can be maximized by applying these complementary processes. Depending on the extent and degree of contamination, individual treatment steps can be omitted or operated simultaneously. All four treatments can be carried out in one remediation well in situ which offers significant advantages over other systems.