Risk assessment in environmental biotechnology Rita R. Coiwell University of Maryland, College Park, Maryland, USA Scientists in academia and industry concur that appropriate oversight and regulation for biotechnology are in the best interests of society. Field trials have not resulted in any uncontrolled hazard. Oversight should continue and useful methods for assessing risk associated with release of genetically engineered organisms to the environment have been proposed. Current Opinion in Biotechnology 1991, 2:470-475
Introduction In 1987, a policy statement o f the National Academy of Sciences of the US addressed the introduction o f recombinant DNA-engineered organisms into the environment. The report dealt with several key issues associated with release. In essence, potential hazards of recombinant DNA-engineered organisms were defined find an attempt was made to separate real from hypothetical risk. For example, is it inherently dangerous to use recombinant. DNA techniques to move genes between unrelated organisms? That is, are recombinant DNA technologies inherently hazardous? These technologies have been used in a large number of laboratories for more than 2 decades to produce recombinant DNA-engineered organisms on a small, experimental scale and, in recent years, in large-scale industrial fermenters. During this period of time, many genes have been transferred between many different kinds of organisms, with the creation of a large number o f individual transgenic organisms. No hazards specific to the use of recombinant DNA techniques have yet arisen. Thus, the general consensus amongst biologists is that recombinant DNA techniques are safe [1..]. The argument is made that transfer of genetic material is not necessarily novel. Genetic exchanges brought about by unconventional, non-sexual means often occur in nature, and recent advances in molecular biology suggest that the cells of most organisms can assimilate and incorporate genetic material from a wide variety of sources. Evidence that exchanges in the natural environment occur frequently is increasing. An import,ant point is that genetic signals for gene expression will function when the recipient organism is very closely related to the donor. The tools of recombinant DNA now allow alteration of these signals to enable genes to be expressed in recipient organisms that are divergent from the donor. A very useful example for discussion is Agrobacterium tumefaciens which carries genes that can be expressed only in plant cells. The bacterium can be used to transfer genes to plant cells and to direct the plant cells to express
these genes to function such that the bacterium gains a source o f nutrient and the gene is transferred to the plant. From all cases described in the literature, the consensus has developed that it is the product of the recombinant DNA experimentation that needs to be addressed, rather than the mechanism by which the product is achieved
[2].
The concern that recombinant DNA-engineered microorganisms might alter soil and/or aquatic populations has been discounted on several lines of evidence. First, the body of data on world-wide use of nitrogen-fixing soil bacteria o f the genus Rhizobium (employed since the 1890s), along with nitrogen-fixing organisms of the genus Frankia, have indicated no alteration to the soil makeup. Second, adverse effects o n the microbial community structure in a variety o f soils into which these organisms have been introduced, have not been observed. Third, improved strains of soil fungi that establish symbiotic relationships with the roots of pine and other trees (mycorrhizae) have also been introduced into forest nurseries, without adverse effects [2]. blicroorganisms have been used extensively as insectcontrol agents. Most well known is Bacillus tburingienst:s; employed widely in the US to control gypsy moths and other insects. As with the nitrogen-fixing soil microorganisms, problems have not been encountered. However, it is not to say that environmental changes cannot occur; in fact, an increase in antibiotic resistance amongst microorganisms in human populations has been well documented, and is thought to be related to the wide-spread and indiscriminant use of antibiotics. Equally familiar are algal blooms which occur when excess nutrients are introduced into an aquatic environment.
Criteria for assessing environmental risk In late 1988, the Biotechnology Science Coordinating Committee, representing the US Department of Agri-
Abbreviations EPA--Environmental Protection Agency; NAS--National Academy of Sciences;NRC--National Research Council; RCRA--Resources Conservationand RecoveryAct.
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Risk assessment in environmental biotechnology Colwell culture, Environmental Protection Agency, National Institutes of Health, National Science Foundation, and Food and Drug Administration [3,4], asked the National Academy of Sciences (N_AS)and National Research Council (NRC) of the US to evaluate the scientific information that is important for making decisions about the introduction of genetically engineered microorganisms and plants into the environment. One of the purposes of the analysis was to identify criteria for defining risk categories and to recommend ways of measuring risks that may be associated with introduction of genetically modified organisms into the environment. The report of the Committee on Scientific Evaluation of the Introduction of Genetically Modified Microorganisms and Plants into the Environment was r~leased in 1989 [5"] and addressed field testing of genetically modified organisms, providing specifically a framework for decision-making concerning release. The fundamental principle enunciated in the report of the NAS [2], is that assessing safety of recombinant DNAmodified organisms, "should be based on the nature of the organism in the environment into which it will be introduced, not on the method by which it was modified". This .principle that the product, and not the process by which the product is obtained, should be the basis of evaluation, was adopted in the subsequent report of the NRC [5.°]. The latter report focused on: the properties of the genetic modification; the phenotypic properties of the donor organism and its genetically modified recipient; and the properties of the environment into which the organisms are to be released. The low probability that moving one or a few genes from a known pathogen to an unrelated non-pathogen might confer pathogenicity on the recipient was also reaffirmed. Obviously, a pathogen acting as recipient might increase in virulence for a particular host as a result of the transfer, but this was not considered to be conversion to pathogenicity per se.
as electroporation), which render the cells permeable to DNA molecules. The most valuable contribution of the NRC report [5"'] is the framework that was developed to e ~ u a t e risk based on criteria such as familiarity with previous introductions and the ability to confine the introduced organism or genetic trait to avoid persistence longer than intended, or inadvertent spread to non-target environments. When the familiarity standard for a genetically modified microorganism has been met, that is, sufficient assurance exists that both the organism and the environment into which it is to be introduced are very similar to and extrapolatable from previous introductions, the introduction is assumed to be suitable for field testing. Thus, the familiarity criterion is a fundamental aspect of the framework of evaluation of risk [1"',5"',6"']. The value of this approach is that it permits decision-makers to use the databank of experience from previous introductions of non-engineered plants and microorganisms into the environment, and it offers flexibility in considering whether or not to carry out field tests for which permission is requested. Obviously, familiarity alone does not automatically confer safety, but the point is that familiarity with all of the parameters surrounding the introduction (or lack thereoO provides sufficient information for judging whether the introduction is safe or risky.
Applications of environmental biotechnology Agriculture
It is clear that the molecular methods now available permat genetic modifications to be fully characterized. In some cases, even the specific base alteration in the DNA nucleotide sequence and structure can be determined. In general, molecular methods are enormously powerful because they allow genes to be isolated and transferred across biological barriers. However, a significant gap in our knowledge has been identified [5"°]. Information concerning the ecology of microorganisms and the introduction of genetically modified microorganisms into the environment is extremely limited, particularly when compared with that available for plants.
The history of introduction of naturally occurring microorganisms into the environment for agriculture provides an enormous body of data suitable for estimating risk that may be associated with the release of genetically modified microorganisms. The organisms used most commonly in agricultural applications include Bacillus tbur#tgienstk and baculoviruses, which are generally used as insecticides and nitrogen-ftxing bacteria in soil, and Rbizobia, which fix nitrogen in association with leguminous plant roots. For years, these organisms have been used as biological control agents or enhancers of agricultural production. The success of the use of Rbizobium spp. inoculants for almost a century provides the best model for risk assessment of genetically modified Rbizobia. Recent studies have been carried out on genetically engineered pseudomonads in soil and the rhizospheres [7,8] and on assessing survival of genetically engineered organisms in air [9] and in water [10,11].
A wide range of highly sophisticated methods for mutating bacteria are now available. It is now quite routine to mutate a specific gene via insertion mutagenesis, as well as to alter specific nucleotides within a gene (site-directed mutagenesis). The range of microorganisms amongst which DNA can be transferred has expanded enormously, and the potential for transforming cells has been facilitated significantly by the development of methods for physically altering cell envelopes (such
The most extensive and valuable application of microorganisms released into the environment is for domestic waste treatment, for example, the reduction of biological oxTgen demand and toxicity of sewage effluents. It is well known that microbial processes are the fundamental components of sludge digestors, settling ponds, trickling filters, and enhanced degradation systems. Bioremedia-
Waste treatment
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Environmental biolechnology tion o f pollutants in the environment, during waste treatment, is a rapidly developing industry. Bioremediation is particularly attractive in view of the fact that non-biological treatment or incineration is very expensive and may generate toxic compounds or derivatives, hz sittt degradation by introduced microorganisms is a very practical solution to environmental pollution. Genetic modification of organisms can be carried out to speed up or extend specific catabolic pathways for biodegradation o f toxic compounds under a wide range of environmental conditions [12]. Good examples of the application are degradation of tricholoroethylene and other chlorinated alkenes. These are serious groundwater contaminants, not only because they are suspected carcinogens, but also because they are highly resistant to biodegradation. In anaerobic sediments and aquifers, the chlorinated alkenes can be converted to vinyl chloride, an even more powerful carcinogen. Polychlorinated biphenyls, once considered to be refractory to biodegradation, can now be degraded, especially those containing fewer than four chlorines per molecule [13]. Thus, the issues involved in field testing o f microorganisms in agriculture or waste treatment that are of ecological importance are: the functional role or niche of microorganisms in the microbial community and ecosystem; the potential for gene exchange between microbial taxa; the ability to monitor persistence and spread of microorganisms; the potential ecological consequences of the persistence and spread of such microorganisms; and the potential measures to control, if necessary, the effects of introduced microorganisms [5-.,6.-,14].
Problems associaled with persistence One of the most important phenotypic properties of genetically engineered microorganisms is persistence. This can be described as the surviwal of introduced modified microorganisms, or the maintenance of specific genetic traits resulting from gene transfer. Both persistence and spread are very. important, as organisms released to the environment cannot be 'recalled' and simply ceasing addition of the organism to the environment does not allow for mitigation of an environmental effect, if the microorganism persists. Thus, fitness (any factor or component reflecting the rate at which a particular kind of organism increases or decreases in number) may be affected if the organism develops an increased resistance to a toxic chemical or some other compound in the environment, or the ability to metabolize a nutrient in the environment. Laboratory tests usually permit determination of such fitness. Most commonly, however, the bacterium that is not indigenous to the environment (including strains genetically modified by both classic and recombinant techniques), is not detectable within a short time after being introduced into soil or water. In fact, it has been accepted as a principle that long-established microbial communities will successfully resist invasions by foreign organisms [15].
It is possible that gene transfer can affect persistence and, in fact, the genetic material may itself persist even though the donor and recipient d o not. For instance, ff the recipient remains viable but becomes non-culturable, the genetic material may still persist [16o]. Other properties that might be associated with persistence include competitiveness between microorganisms, such as growth rates, and the ability to use very rapidly the available nutrients. It is incorrect to say that there is a penalty associated with carrying and expressing additional functions, that is, additional genetic information, even though there is evidence to support that premise. Some microorganisms are exceptions, however, in that they show increased fitness as a result of carrying new genetic information. It is also possible that an introduced microorganism might exclude another organism that is more valuable in its activities. For example, Bradyrhizobium japonicum serogroup 123 is believed to be more competitive in certain soils, thus excluding other more effective nitrogen fixers [5°,]. It can be hypothesized that the major biogeochemical cycles o f carbon (lignin and cellulose decomposition), nitrogen (nitrogen fixation, nitrification, dentritication), sulphur (sulphur oxidation and sulphate reduction), and the phosphorous, silicon, manganese, iron and trace metal cycles, may be affected by introduction of genetically engineered microorganisms into the environment. Similarly, the distribution of gases of the atmosphere, such as CH4, NO2, H 2 and CO2, may be disturbed. Thus, it is important to consider the environment into which the genetically engineered organism is to be introduced. For example, nutrient status, presence of toxic chemicals or metabolites, physical-chemical parameters and biological factors should all be taken into account. It has been shown that seawater surrounding oil spills is limiting in nitrogen and phosphorous nutrients and that introduced microorganisms simply cannot exceed resource limits. Additional nutrients have to be added either to stimulate or to enhance degradation [17,18,19]. Very high concentrations o f heavy metals, acids and inorganic compounds, which are pollutants in a given environment, can be toxic to microbial cells. Such toxins might cause cells to go into stasis or, in some cases, actually to be cidal. Certainly, one effect is an alteration of species composition and community diversity, with selection for those organisms that are able to degrade the compounds that have been introduced. Physical-chemical parameters that must be considered before release include salinity, temperature, pH, oxidation-reduction potential, light, and a wide range of other factors. It must be understood, however, that microbial predators, parasites, symbionts and competitors will influence microbial species composition and community structure, as well as persistence and spread of the introduced microorganism. The ability to persist also involves the ability of microorganisms to disperse by attachment to soil particles or
Risk assessment in environmental biotechnology Colwell 473 particulates in water, or their ability to resist ultraviolet irradiation during aerial transport [9]. Monitoring is necessary and gene probes have been or are being developed, which permit detection at the level of a single cell or genome. With the polymerase chain reaction, sensitivity has been increased enormously. In any case, nucleic acid methods can improve specificity and sensitivity, if culturing is not a limiting factor [20.,21.].
Mitigation In general, persistence is difficult to control. For soils, procedures such as fumigation, sealing the surface, or providing barriers to penetration from the introduced site externally, are usually carried out. Methylbromide has proved to be useful for controlling plant pathogenic bacteria and in some cases fungi. However, antibiotics and non-volatile organic biosides are not very effective because they bind to the soil material (A Vidaver and G Stotzky, personal communication). . r
For microorganisms, it is clear that, by considering familiarity with a specific introduction of a specific organism to a given environment, an estimate of control of the persistence and spread of the introduced organism (i.e. the risk) can be achieved. Knowledge of potential environmental effects, including a reasonable assessment of potential adverse effects associated with introduction, allows a workable framework for risk assessment and regulation. For example, in a small-scale field test, a reasonable assessment of safety can be made if the microorganism, its intended function, and the environment into which it is to be introduced are reasonably similar to a prior introduction with a very long history of safety, such as those involving Rhizobium spp. If all of the familiarity criteria are not satisfied, then an estimate o f risk requires careful and critical review of the available methods for controlling the persistence and dissemination of the microorganism, as well as its potential for causing adverse environmental effects. In conclusion, there is a strong contention that transgenic organisms to be released into the environment should be assessed for risk and accordingly regulated on the basis of their biological properties (phenotype), rather than on the genetic methods employed to produce the organism [6°°]. Yet, factors such as fitness, scale and frequency of introductions to the environment, and fate and potential for delayed effects, have been assembled in matrices for consideration in risk evaluation. Workable guidelines have been devised by several investigators [1.°,5°°,6°o].
Future applications of environmental biotechnology The major developments in environmental biotechnology will be in microbially mediated bioremediation. In gen-
eral, industrial and domestic sewage wastes are usually either immobilized in stationary landfills or waste lagoons, diluted by addition to water above or below ground, or subjected to chemical neutralization a n d physical processes, such as incineration. Combustion products are disposed of in a similar manner to wastes, that is, either to the air or into water. In general, the strategy for disposing o f a pollutant is to dilute it. Microorganisms are employed increasingly to detoxify landfills in situ, as well as water sites and effluents from industrial processes. Genetically engineered microorganisms are highly attractive, mainly because of the precision of genetic manipulation via plasmid mediation, transformation, and transduction, yielding more accurate and more precisely controlled results than mutation with non-recombinant DNA. Prior to 1984, microbial waste reduction processes were not Federally regulated, mainly because these treatments (usually sewage treatment processes) were thought of as being 'natural' and therefore safe. Risk assessment evolved with the consideration of the source of the original chemical and the environment into which it was being placed, usually in terms of transport, fate and effects. This changed o n December 31st, 1984, when the US Environmental Protection Agency (EPA) stated that commercial genetically engineered microorganisms to be used in the environment should b e reviewed under the Toxic Substances Control Act. Thus, risk assessment of microorganisms in microbial processes is required. The review procedure now includes the microbial products and the processes by which they are generated at the early research and developmental stages. The pre-manufacmre notification processes affect products reviewed and regulated under the Resources Conservation and Recovery Act (RCRA) and thus requires clearance from both offices within EPA [22,23]. The RCRA reauthorization also requires waste minimization and hazardous waste site operators to agree to clean up contamination at the site. The question is whether biotechnology will provide the answer to satisfy the best demonstrated available technology requirements under RCRA. In 1986, the Superfund Law (Public Law 9 9 4 9 9 ) was amended and reauthorized. This law requires treatments for permanently and significantly reducing waste toxicity, mobility and volume to be carried out, as well as a research, evaluation and testing program (Superfund Innovative Technology evaluation; the SITE program) to be followed. The development of RCRA review criteria and procedures are going to be required if biotechnology products are to be employed. Clearly, much effort is needed to streamline and develop a consistent regulatory process [22,23]. An interesting experiment has been carried out in Alaska in an oil spill bioremediation project [17]. The US EPA's Alaska Bioremediation Project was initiated in the aftermath of the EXXON VALDEZ oil spill on March 24th 1989. The objective of the project was to demonstrate a method of enhancing the clean-up of oil-contaminated shore lines by adding nutrients to stimulate the growth of naturally occurring oil-degrading microorganisms. After
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Environmentalbiotechnology planning, mobilizing staff and facilities, and selecting test sites in Prince William Sound, Alaska, nutrient application began on June 8th, 1989. Nitrogen- and phosphorousrich nutrients were added to the oR-contaminated shore line sites in the form of oleophilic, slow-release and water-soluble fertilizers.
highly promising and very exciting. It is critical that sufiqcient knowledge of molecular microbial ecology be gathered in the near furore to allow useful and beneficial applications of environmental biotechnology to be made.
From field and laboratory studies, it was possible to confirm that both the oleophilic fertilizer and inorganic nutrients increased extent and rate of oil degradation, compared with untreated shore line sand, cobble and gravel [17]. Laboratory analyses of the oil indicated that the mechanism of action was indeed biodegradation and not merely chemical removal or dispersal of the oil. It was also observed that a high rate of natural oil biodegradation occurs, arising from nutrients in the seawater and fresh water at the sites tested. Results of this carefully designed study, which has been subjected to extensive and critical review, indicate the potential of bioremediation. Obviously, with improved procedures employing genetically engineered microorganisms, one can foresee an enormous potential for bioremediation.
References and recommended reading
Tailor-made cultures will surely play a major role in the future to abate pollution. Waste water from industrial effluents or domestic sewage are particularly suitable for directed fermentations because the starting material is reasonably well-defined being of high concentration, with specific composition, pH, temperature and mineral content. In addition, fermentation conditions are sufficiently specialized to be selective for a narrow range of microorganisms. These factors offer great opportunity for success in bioremediation. Obviously, pure cultures provide guidelines for large-scale treatment, although in situ treatment processes have also proven suflqciently successful such that very large-scale fermentation treatments are being considered by some industrial firms in the US [12,13]. The genetics of xenobiotic degradation indicate that molecular microbial ecology will provide very useful tools for studying structure and function of biodegradative microbial communities for in situ removal of environmental pollutants [24,25].
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest oo of outstanding interest 1. .•
MILLER HI, BURRISRH, VIDAVERAK, WP,'ELNA: Risk-Based Oversight of Experiments in the Em'ironment. Science 1990, 250:490--491. An algorithm, based on the nature of the organism and of the site into ~ c h the organism is to be introduced, is proposed for risk assessment. An expanded version is available from the senior author. 2.
NAS (NAxqO.X-XLACADEM'YOF SCIENCES): Introduction of Recombinant DNA-Engineered Organisms into the Environment. Key Issues. Washington DC: National Academy Press 1987, pp 1-24.
3.
OFFICEOF SCIENCE AND TECI-LNOLCKS, Y POLICY: Coordination Framework for Regulation of Biotcchnology: Establishment of the Biotechnology Science Coordinating Committee. Fed eral Register 1985, 50:47-174.
4.
OFFICE OF SCIENCE AND TECI~NOLOGY POLICY: Coordinated Framework for Regulation of Biotectmology. Federal Regi~ ter 1986, 51:23302.
5. •.
NA'nOX-LL RESEARCttCOUNCIL: Field Testing Genetically Modified Organisms: Framework for Decisions. Washington DC: National Academy Press 1989, pp 1-170. An anal~is of the issues associated with the release of genetically engineered organisms to the emironment. A relatively thorough study of the issues is proxSded. 6. +*
TmDJEJM, COLWELLRK, GROSSXt~NYL, HODSON RE, LENSKI RN, ]kIACKRN, REGALPJ: The Planned Introduction of Genetically Engineered Organisms: Ecological Considerations and Recommendations. Ecology 1989, 70:298-315. An excellent overview of the ecological aspects of introduction of geneticalb, engineered organisms into the environment. 7.
YEUNGK-HA, SCtIELLMA, HARTELPG: Growth of Genetically Engineered Pseudomonas aemginosa and Pseudomonas putida in Soil and Rlfizosphere. Appl Environ Microbiol 1989, 55:3243-3246.
8.
TREVOP6JT, V&N ELSASJD, V&N OVERBEEKIS, STARODUBM-E: Transport of a Genetically Engineered Pseudomonas fluorescens Strain Through a Soil Microcosm. Appl Environ Microbiol 1990, 56:401--408.
9.
KNRJDSENGR: Model to Predict Aerial Dispersal of Bacteria During EmSronmental Release..4ppl Environ Microbiol 1989, 55:2641-2647.
10.
AWONGJG, BRITON G, CttA.'~'DHRYGR: Microcosm for Assessing Survival of Genetically Engineered Microorganisms in Aquatic EmSronments. Appl Environ Microbiol 1990, 56:977-983.
11.
ORVOSDR, LACYGH, CAIRNSJR. J: Genetically Engineered Erwtnia c a r o t o v o r a - Survival, Intraspecific Competition and Effects Upon Selected Bacterial Genera. Appl Environ Microbiol 1990, 56:1689-1694.
12.
JA~ RK, SAYLERGS: Problems and Potential for In Situ Treatment of EmSronmental Pollutants by Engineered Microorganisms. Microbiol Sci 1987, 4:59-63.
13.
EATONRW, Tt~t~tt5 KN: Genetics of Xenobiotic Degradation. In Current Perspectives in Microbial Ecology edited by Klug
Conclusions In summary, pragmatically, it must be recognized that zero risk and uncertainty in environmental biotechnology cannot be achieved. Finite risk will remain. However, there are mechanisms for assessing risk and determining the balance in favor of benefits to be achieved. A critical aspect of risk assessment is the need to gather more extensive data on microbial ecology under natural, as well as polluted, systems. It must be remembered that genes move between members of loosely or tightly structured microbial communities, as well as witlfin mixed populations and natural ecosystems. Significant gene rearrangements can occur under natural conditions. Nevertheless, the genetic engineering of microorganisms for environmental applications, notably bioremediation, is
Risk assessment in environmental biotechnology Colwell MJ, Reedy CA [book]. Proc Third Intemat Symp Microbial Ecology, Amer Soc Microbiolog), Washington DC 1984, pp 694-703. 14.
RATHERM: Barriers to Field-Testing Genetically Modified Organisms. Biotechnology 1990, 8:196--199.
15.
LIgNG LN, SINCLAIR JL, i~.tAU.ORY I.~|, ALEXANDER ~,~: Fate in Model Ecosystems of Microbial Species of Potential Use in Genetic Engineering. Appl Environ Microbiol 1982, 44:708-714.
BYRDJJ, COL~XLLRPC Maintenance of Hasmids pBR322 and pUC8 in Nonculturable Escherichia colt in the Marine Environment. Appl Environ Microbiol 1990, 56:2104-2107. Demonstrates maintenance of plasmids when ceils are non-culturabte in seawater s)~tems.
Population of Soil Bacteria. Appl Environ Microbiol 1990, 56:776-781. A strong correlation between phenotypic and genotypic diversity of cultumble bacteria from soil ~as demonstrated. 21. TORS~ V, GORSOYRJ, DAAE FL: ttigh Diversity in DNA of , Soil Bacteria. Appl Environ Microbiol 1990, 56:782-787. Demoustmtion of the diversity of the total bacterial community in a deciduous forest soil, mostly in non-culturable bacteria. 22.
ROGULM, I.L~x'qM: Regulation of Biotechnology by the Environmental Protection Agency. In Assessing Risks of Bioted~ nology edited by Ginzberg L [book]. Boston: Butterworths 1990, pp 233-265.
23.
MtLEWSKI E: EPA Regulations Governing Release of Genetically Engineered Organisms. In Biotechnology of Plant--Microbe hzteractions edited by Nakas JP, Hagedom C [book]. New York. McGraw-Hill Publishing Co. 1990, pp319-340.
24.
MORGANP, \VATKL'qSONRJ: Microbiological Methods for the Cleanup of Soil and Ground Water Contaminated with Halogenated Organic Compounds. Ft~IS Microbiol Rev 1989, 63:277-300.
25.
SOSG H-G, WANG XP, BARTHA Pc Bioremediation Potential of Terrestrial Fuel Spills. Appl Environ Microbiol 1990, 56:652-656.
16. •
17.
18.
PP,ITCHARD PIt, GLASERJ, KREMER F, ROGERS J, VENOSA A, CIIIANELLI R, I-ILNTONS, PRLNCER, MCMILLENS, REQUEJOA: Oil Spill Bioremediation Project. US Environmental Protection Agency Interim Final Report December 8, 1989, pp 1-194. FOX JI2 Native Microbes' Role in Alaskan Clean-up. Biotecb nology 1989, 7:852-855.
19.
HARVEYS, ELASHVlLI 1, VALDESJJ, KK~mLY D, CHAKRABARTY AM: Enhanced Removal of Exon Valdez Spilled Oil From Alaskan Gravel by a Microbial Suffactant. Biotechnology 1990, 8:228--232.
20.
TORSVlK V, SALTE K, SORHELM R, GOKSOYR J: Comparison of Phenotypic Diversity and DNA Heterogeneity in a
RR Colwell, Mar~tand Biotechnology" Institute, University of Marylandl College Park, Mar~tand 20742, USA_
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