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7 Past Experience with the Introduction of Microorganisms into the Environment Microorganisms have had many beneficial applications in agri- culture, waste treatment, and food production. In this chapter we describe these and other uses and discuss the prospects for future beneficial applications in clean-up of toxic wastes (bioremediation), mining, and mineral recovery. As discussed in Chapter 2, modern methods of producing novel genetic combinations ~ microorganisms can be very precise. This history of safe use and an understanding of genetic modification through modern methods combine to give scientists a degree of familiarity with certain rn~croorgan~sms. This chapter wiD describe the familiarity we have with certain croorgan- isms as background for use of a familiarity criterion in a framework to help in evaluating the relative safety or risk of field testing genet- icaDy modified rn~croorganisms. We also identify the scientific issues that should be considered when an application involves an unfamiliar rn~croorganism. These issues are then dealt with in greater detail in Chapters 8, 9, and 10. HISTORY OF BENEFICIAL USES OF MICROORGANISMS AND PROSPECTS FOR THE FUTURE Food Production Naturally occurring ~n~croorganisms with specialized or unique properties have been used for centuries in food production. The 77

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78 basic microbiology of bread-making has remained substantially the same for thousands of years. Egyptian balters as early as 2100 B.C. Obtained their yeasts from the settlings of beer vats, whereas the Greeks and Romans used yeasts from wine vats, and later the English used brewer's yeast ("barman) (Ayres et al., 1980~. Sourdough bread has been produced in the San Francisco area for more than 100 years, with yeasts and a sourdough bacterium that ferments maltose (Ayres et al., 1980~. Other fermentations are used ~ producing pickles, olives, and sauerkraut. Virtually every human culture that utilized domesticated m~Ik-producing animals also developed fermented milk products. Food production, including baking, brewing, and fermenting foods, is hygienic but not aseptic. Although microorganisms from food-process~ng industries enter the environment, they have been used safely for centuries. The rn~crobial flora of a food may mclude microorganisms found on the raw material, those added during pro- cessing, and those surviving preservation, treatment, or storage. The textures, flavors, and aromas of foods often depend on a defined m~x- ture of microorganisms, such as those which develop the flavors and aromas of cheeses (Banwart, 1979~. Many opportunities exist to apply genetic modification technolo- gies to microorganisms used in the food Industry. For example, the use of classical genetics to improve brewer's yeast is limited because the strains of Saccharomyces cerevisiae cannot be crossed; genetic modification by molecular methods provides a valuable alternative strategy for strain improvement (Timers et al., 1988~. Brewer's yeast is not capable of degrading starch, and the amylase needed in brewing is produced by germinating barley. A suggested target for genetic modification of brewer's yeast is construction of amylolytic yeast strains. In addition, enzymes that degrade dextrins generated by the barley amylase could be useful, and the glucoamylase gene from Saccharomyces diastaticus has been cloned and expressed in brewer's yeast. Agriculture The history of introduction of naturally occurring microorgan- isms into the environment for agricultural purposes provides exten- sive data on the release of genetically modified microorganisms. The microorganisms that have attracted the greatest attention for agri- cultural applications include: (1) Bacillus thuringiensis (the source

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79 of Bt toxin) and baculoviruses used as insecticides; and (2) bacteria that fix nitrogen ~ soil and rh~zobia that fix nitrogen in association with legurnunous plant roots. Bacutoviruses have been used since the nineteenth century to control insect pests. They specifically infect arthropods, do not pollute the environment, and are safe to handle. More than a dozen baculov~ruses have been used cornrnercially to control insect pests without any evidence of harm to the environment (Podgwaite, 1985; Entwistle and Evans, 1985, Hunter et all, 1984~. Recent studies (Bishop, 1986, 1988; Bishop et al., 1988) demonstrate the suitability of genetically marked baculoviruses for release in field tests. The improvement of baculoviruses by genetic modification has focused primarily on increasing their speed of action and altering their ability to survive over long periods. Bacteria, such as naturally occurring strains of B. thuringiensis, have also been used for years as biological control agents. Toxins in commercially produced microorganisms are effective insecticides and are used on several agricultural and forest pests. The toxins from different strams are specific to certain lepiclopteran, dipteran, and coleopteran insect pests (Hofte and Whitely, 1989~. The genes conferring production of different B. thuringiensw delta endotoxins (Bt toxin) have been cloned and partially characterized (see Lm~ow et al., 1989, and references therein). Because B. thuringiensis does not multiply on plants, effective insect control requires repeated applications. At this time, widespread use of the Bt endotox~ for biocontro! of insect pests has not had any adverse environmental or health effect. Recently, there have been attempts to overcome the spatial and economic limitations of foliar applications of B. thuringiensts by introducing the delta endotoxin gene into the chromosome of Pseu- dfomonas puorescens, an effective colonizer of corn plants (Obukowicz et al., 1987~. The organism has not been field-tested but laboratory studies indicate that this genetically modified bacterial strain does not differ from its parental strain in survival and dispersal character- istics, and it Is somewhat toxic to root cutworm but not to the corn rootworm. Transfer of the Bt endotoxin gene into internal colonists of plants, such as Ciavibacter xyli subspecies cynodontis found in the xylem elements of Bermuda grass, shows prorruse for application in corn (Lindow et al., 1989~. Field studies have been initiated with this ge- netically modified rn~croorganism for control of leaf- or stem- feeding

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80 lepidopteran larvae. Leguminous crops form symbiotic associations with host-specific Rhizobium and BradyThizobium species that fix nitrogen in a form that can be used by the plant, thus increasing agricultural yield. These nitrogen-fixing bacteria have attracted considerable attention with respect to genetic improvement (Schmidt and Robert, 1985~. Soil ~ the major reservoir of free-living rh~zobia; the organisms pros literate in the root-nodules of their host plant, and organisms from the decomposed nodules persist ~ the soil (Nutman, 1975~. Due to the persistence of existing rhizobia, any new rhizobia applied to the soil as seed inoculants must function and compete successfully in an extremely dynamic and complex ecosystem (Dowling and Broughton, 1986~. Therefore, the most spectacular increases im agricultural pro- ductivity from inoculation with a new rh~zobium have occurred when rhizobia specific to a certain leguminous plant have been absent Tom soil before introduction of that legume as a new crop, as in introduc- tion of soybeans into the United States and Eastern Europe arid the introduction of pasture legumes into Australia. Commercial legume inoculants have been produced for almost a hundred years, and these rhizobia have been added to soil in enormous numbers without caus- ing undesirable effects. The success of the use of Rhizobium spp. inoculants over many years provides a good example of the safe use of genetically modified m~croorganisrns. Improvements in biological nitrogen fixation will be important for meeting future demands of agriculture, reducing requirements for fertilizer, conserving fossil fuels used in producing and applying nitrogenous fertilizer, as well as in mmirn~z~g adverse environmental effects from run-off of nitrogenous fertilizers into lakes and streams. Modern biotechnology wiD allow greater precision in tailoring m~- croorgan~sms such as the rhizobia. Waste Treatment The most extensive and intensive application of microorganisms released into the environment is ~ domestic waste treatment, where they are used to reduce the biological oxygen demand ~d often to reduce the toxicity of sewage effluents. Sludge digesters, settling ponds, trickling filters, and enhanced degraclation systems depend on microbial processes. Sewage sludge from large digesters, when pumped into an evaporation pond, represents a massive release of microorganisms into the environment. Yet effluent from a properly operated activated sludge processor or trickling filter poses neither

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81 public nor environmental health problems. The history of releasing treated sewage effluent into the environment argues convincingly that these procedures are safe. Bioremediation Biodegradation of pollutants in the environment has used both naturally occurring as well as genetically modified ~riicroorg=~suLs (Bopp, 1986; Brown et al., 1988; Focht, 1988; Frantz and Chakrabar- ty, 1986; E rick et al., 1988; Ghosal et al., 1985; Kilbane et al., 1983; Senior et al., 1976; Unterman et al., 1987~. Nonbiological methods, such as physical confinement, chemical clegradation, or incineration, are costly and may disperse toxic compounds or their derivatives into the environment. Biodegradation therefore offers a valuable al- ternative (Kobayashi and Rittmann, 1982~. In situ degradation by introducer} microorganisms represents a practical solution to certain types of environments pollution. The attractiveness of bioremedia- tion treatment is that in both surface and subsurface environments microbial processes may permanently remove organic contaminants, rather than merely contain them. Tm addition, bioremediation by introduced microorganisms may continue ~ situ even after ~ntro- auctions have ceased. Genetic tools can be used to enhance specific catabolic pathways of biodegradation of foreign compounds by ~ru- croorgan~sms under a wicle range of environmental conditions (Lin- dow et al., 1989~. ~ichIoroethylene (TCE) is the contarn~nant on the Environ- mental Protection Agency list (1985) most frequently reported at hazardous waste sites. TCE and other chlorinated alkenes present a serious groundwater contamination problem because they are sum pected carcinogens that resist biodegradation in the environment (Tnfante and Tsongas, 1982~. Unfortunately, in anaerobic subsur- face sediments and aquifers, chlorinated alkenes can be converted to even more powerful carcinogens, such as viny! chloride (Bouwer et al., 1981; Wilson et al., 1983~. However, an aerobic rn~croorganism will degrade TCE when the organism ~ sunultaneously exposed to phenol (Nelson et al., 1986~. Recently, an aerobic, methane-oxid~zing bacterium that degrades TCE has been isolated; in pure culture it degrades TCE at concentrations commonly observed in groundwa- ter (Little et al., 1988~. Destruction of TCE by microorganisms in contaminated groundwater has also been demonstrated (Fliermans et al., 1988~. Monooxygenase genes have been cloned into a strain

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82 of Escherichia coli, and this genetically modified organism has been shown to degrade TCE completely (Winter et al., 19893. PolychIorinated biphenyls (PCBs) are ubiquitous pollutants that are physically inert and poorly soluble in water. Although PCBs were believed to be refractory to biodegradation, many PCBs containing fewer than four chlorines per molecule can be degraded biologically (Focht and Brunner, 1985; Rochkind-Dubinsky et al., 1987; Shields et al., 1985~. Field studies on degradation of PCB In soil treated with nutrients and inoculated with PC~degrad~g bacteria have been conducted near Oak Ridge, Tennessee. Biodegradation increased markedly ~ the inoculated soils. Gene-probe analyses and growth of the microorganism In culture demonstrated that the added microbic strain survived (Hill et al., 1989~. Anaerobic degradation of PCBs has been demonstrated by Quensen et al. (1988~. Genes involved in PCB degradation have been cloned by Taira et al. (1988) and Kimbara et al. (1989), an accomplishment that should allow future development of new genetically modified PCB-degrad~ng microorganisms. Investigators have sought, by genetic modification, to combine several degradative steps in a single microorganism to be used for de- stroyingpollutants (Ghosalet al., 1985; Tomasek et al., 1989; Focht, 1988; Sangodkar et al., 1988; Golovievaet at., 1988; Timmis et al., 1988~. Timmis et al. (1988) tested biodegradation of substituted aro- matic compounds (chiorm and methyla~omatics) in activated sludge; mollified microorganisms survived and functioned with no adverse effects on the rest of the microbial community and with no trance fer of the new genetic information to indigenous microorganisms. Genetic modifications also may provide significant unproYements in the rates of degradation and the range of toxic pollutants subject to degradation. Mining and Mineral Recovery The principles of microbial biogeochem~stry (Ehrlich, 1981) and the potential for recovering minerals through improved microbial processes may be exploited withm the next decade. ThiobaciHi are autotrophic and c" derive their energy from the oxidation of sulfur compounds or ferrous iron. Techniques for manipulating the genes of tints group of microorganisms to enhance leaching of copper and uranium are being developed (Yates et al., 1988~. Although genetic technology as applied to this group of microbes is less advanced than that for other microorganisms, plasmids have been constructed that

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83 may enhance the recovery of gold from ores by Thiobacill?~s ferroox- idans and increase the org~n~sm's resistance to arsenic compounds (see Lindow et al., 1989, and references therein). Commercial bioleaching operations in the mining industry rem resent another application of microorganisms-treatment of ores in heaps or pits. Adverse environmental effects have not been reported from the introduction of improved bacteria] strains for mining appli- cations of biotechnology (Nicolaides, 1987~. SIMILARITIES AND DIFFERENCES BETWEEN CLASSICAL AND MOLECULAR METHODS The preceding section illustrates a long history of beneficial ap- plications of microorganisms in food processing, agriculture, waste treatment, and bioremediation. In many instances, such applica- tions can be performed even more effectively with rn~croorganisms that have been genetically modified, by either classical methods or molecular techniques (NAS, 1977; OTA, 1984, 1988; GiDett et al., 1985; Korwek and de la Cruz, 1985; Olson, 1986; Timmis et al., 19883. Among the products of classical methods of genetic mod- ification are spontaneous mutants and many recombinants formed by natural processes such as conjugation, transduction, and trans- formation In bacteria. As mentioned in Chapter 2, site-directed mutagenesis, DNA cloning, and cell fusion are included among the molecular techniques. Why then employ the recently developed molecular techniques to produce rn~croorga~sms to be introduced into the environment? First, the newer methods permit greater precision in the construction and characterization of the desired genotypes. Enhanced precision means scientists know more exactly the changes that have been ef- fected and can make better judgments about the safety of genetically modified organisms (Brill, 1985; Davis, 1987; Tie~je et al., 1989~. Sec- ond, molecular methods (either alone or In conjunction with classical approaches) may permit the formation of novel combinations from distantly related genomes, combinations that would be extremely dif- ficult or even impossible to obtain by classical methods. In essence, molecular techniques allow scientists to bypass natural barriers to genetic exchange (Tie~je et al., 1989; Wang et al., 1988, Jaynes et al., 1987~. With the precision and power of new techniques, researchers have begun to develop new genetically modified microorganisms in an attempt to enhance and extend beneficial applications (Orser et

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84 al., 1984; McCorTn~ck, 1985; Obukowicz et al., 1987; OTA, 1988; Gillett et al., 1985, and references therein; Olson, 1986; Korwek and de la Cruz, 1985; NAS, 1977; Timmis et al., 1988; Lindow et al., 1989~. These genetically modified microorganism can be used in the production of food, pharmaceuticals, and industrial chemicals as weD as in agriculture, pollution control, and mining. CONSIDERATIONS ASSOCL\TED WITH UNEAMI1;IAlL APPLICATIONS OF MICROORGANISMS It is apparent from the examples ~ the preceding sections that microorganisms have already provided many benefits when intro duced into the environment and that molecular techniques to mod- ify microorganisms hold great promise for enhancing and extending these benefits. The experience gained from the use of m~croorgan- ism~ over the years provides scientists with considerable familiarity as they design certain types of field tests. For example, we know from experience that many microorganisms can be used safely in the environment even if we are uncertain of their precise roles in the microbial community or ecosystem. Were our knowledge of the genetically modified microorganism and the environment perfect, we could assess all risks precisely and make a thoroughly informed judgment based on the magnitudes of the anticipated benefits and potential costs. Because we lack perfect knowledge, we must base our predictions of responses to the release of genetically modified microorganisms on sound scientific inference and on a long record of safety. Microbial ecology is rapidly developing field that has the potential to add greatly to our knowledge of how to ensure the safe and effective environmental application of microorganisms. Unfamiliar microorganisms or instances of substantial uncer- tainty about the interaction of the microorganism and the environ- ment into which it is to be introduced require careful evaluation prior to field testing. Issues pertaining to the biology of a field test that have ecological significance include the following: the functional role or niche of the rn~croorganism 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 m~croorganisrns, and the potential measures to control, if necessary, the effects of introduced microorganisms. In Chapters 8

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85 through 10, we exarn~ne these and other issues that may be sources of ecological uncertainty for some microbial field tests. SUMMARY POINTS 1. Microorganisms have a Tong history of use in food produc- tion, agriculture, and waste treatment. Many opportunities exist to use genetically modified m~croorganisrns to enhance these and other applications, including the clean-up of environmental pollutants and the recovery of minerals. Familiarity with particular m~croorgan- isrrm, their functions, and their target environments ~ unportant to consider in assessing potential environmental effects. Familiarity has been incorporated as the first criterion ~ the framework presented in Chapter 11 for the evaluation of the safety or risk of field-testing - mlcroorgamsms. 2. Both similarities and differences exist between classical and molecular methods for genetically modifying microorganisms. Com- parable modifications can often be accomplished with either method, and each type follows procedures to selectively enrich those genotypes that have the desired phenotypic properties. Molecular methods of- ten provide greater precision in generating the desired genotype and greater power in producing novel genetic combinations. 3. Much has been learned from past experience with ~rucroor- ganisms, such as those used as biocontro} agents or for nitrogen fixation, and the information provides a basis for assessment of rel- ative safety or risk. It may be difficult to assess all potential risks precisely, especially for unfamiliar microorganisms. Although some uncertainties may persist, they can be resolved scientifically as our knowledge of microbial ecology increases.