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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.
Representative terms from entire chapter:
genetic modification
