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OCR for page 159
5
Bioconfinement of
Viruses, Bacteria, and Other Microbes
INTRODUCTION
The use of genetically engineered microbes can offer enormous poten-
tial benefits. Because viruses, bacteria, and fungi are natural pathogens of
insects and other pests, microbes can be harnessed and genetically enhanced
as agents of biocontrol. Bacteria and fungi also are able to degrade some
environmental pollutants, and molecular technology allows us to expand
the list to include other toxic compounds as well. There is a long human
history of using microbes in agriculture, food processing, waste treatment,
and other beneficial capacities. Modern molecular methods allow us to
broaden the range of useful applications, and all of the evidence indicates
that the methods used to generate genetically engineered organisms (GEOs)
are not intrinsically dangerous. Some caution is warranted, however, because
information about the ecology and evolution of transgenic microbes in the
wild is limited. Microbes occur in extremely large populations with short
generation times, so they adapt quickly to adverse conditions. Their envi-
ronments change constantly, resulting in unpredictable and variable selec-
tion pressures. Bacteria also can transfer DNA into unrelated microbes, and
the long-term ecological consequences of that transfer are unclear (Bushman,
2002). The consequences of releasing transgenic microbes into the environ-
ment have not been evaluated adequately. As with the plants and animals
discussed in the earlier chapters, it is impossible to generally predict the
fitness consequences of genetically engineering a microbe. The new genetic
combination could be beneficial or deleterious for a microbe's survival in
the wild, depending on the ecological context (e.g., through interactions
159
OCR for page 160
160 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
with the environment and with other microbes). Most studies show that
genetically engineered microbes are relatively less fit than are their
nonengineered counterparts, although that can be a faulty assumption.
Each case of genetic engineering must be considered on its own.
This chapter is on the bioconfinement of genetically engineered microbes,
especially bacteria, fungi, and viruses. The potential effects and need for
bioconfinement in microbes are discussed first. Then there is a section that
identifies and describes the major methods of bioconfinement for bacteria,
fungi, and viruses and discusses the strengths and weaknesses of each
method. Next are considered the effectiveness of those methods in different
spatial and temporal scales and their potential effects on biological popula-
tions and ecosystems. The needs, feasibilities, and realities of monitoring,
detecting and culling genetically engineered bacteria, fungi, and viruses are
discussed. Finally, the aforementioned topics are related to the bioconfine-
ment of microalgae.
Because the committee was not specifically asked to evaluate bio-
confinement techniques for microbes, this chapter is purposefully less sub-
stantive than are the chapters on plants and animals. However, the wide-
spread appreciation for the usefulness of transgenic microbes warrants their
treatment here. Earlier NAS reports also have dealt with genetically engi-
neered microbes (NRC, 1989a, 2002b).
The 1989 NAS report, Field Testing Genetically Modified Organisms,
extensively evaluated environmental risks associated with the release of
transgenic microorganisms. The recommendations in that publication in-
fluence policy decisions today, despite the advancement of molecular tech-
nology in the intervening years. Although portions of the present report
discuss transgenic microbes, the subject cannot be dealt with in detail be-
cause microbes were not a central focus of the committee's charge. The
committee suggests that genetically engineered microbes be reconsidered on
their own.
POTENTIAL EFFECTS OR CONCERNS, AND NEED FOR
BIOCONFINEMENT IN VIRUSES, FUNGI, AND BACTERIA
The three potential areas of concern that attend the release of geneti-
cally engineered bacteria, fungi, and viruses are similar to those for any
other class of GEO: invasion, displacement, and transfer. Together they can
be used to argue that bioconfinement measures should be considered.
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VIRUSES, BACTERIA, AND OTHER MICROBES 161
Invasion into Indigenous Populations
Viruses
Genetically engineered viruses could infect and harm nontarget hosts.
Because viruses are obligate intracellular parasites, they require metaboliz-
ing (living) cells to replicate their genomes and make progeny. The reliance
on host cells often produces strong selection for viruses to evolve more
efficient mechanisms to exploit their hosts. In turn, selection of the host
favors genotypes that excel in their ability to repel virus attack. Viruses can
gain the upper hand in these coevolutionary battles simply because they can
evolve more rapidly than do their hosts (Levin and Lenski, 1983). Thus, a
virus can be successful by evolving a greater propensity to exploit the host,
through creating more progeny per infection per unit time. Alternatively,
viruses could be evolutionarily successful by adapting to infect a greater
variety of hosts (DeFilippis and Villarreal, 2000). The latter adaptation
exemplifies the potential consequence of releasing genetically engineered
viruses into an ecological community of naïve (inexperienced) hosts. The
concern is not the introduction of engineered alleles (such as transgenes) per
se, but that the foreign strain of virus will harm a nontarget host species
that is ill-prepared to defend against the viral attack because of its lack of
resistance genes. Support for this idea comes from studies that demonstrate
elevated virulence in naïve host populations (Bull, 1994; Taylor et al.,
2001). Thus, releasing genetically engineered viruses into the environment
might result in their becoming successfully established in nontarget hosts.
Fungi
The host range of fungi also can evolve. Reports of changes in the
known host range of plant pathogenic fungi are common (Mundt, 1995).
For example, the scabrum rust, which is pathogenic on Agropyron scabrum
and Hordeum vulgare (barley) in Australia, arose from a cross between
Puccinia graminis f. sp. tritici (wheat stem rust) and P. graminis f. sp. secalis
(rye stem rust) (Burdon et al., 1981). In 1991 cultivated barley was found to
be heavily infected with a new variety of P. coronata, a fungus that was
known for more than 200 years to cause serious disease in cultivated oat.
From these and other examples, it appears that the pathogen can
genetically alter its host range. However, studies on the rice blast fungus
showed that many genes are required for the fungus to attain the high
fitness required for field survival on a new host (Valent et al., 1991). Those
results indicate that survival in the wild in the face of competition from
other organisms and changing environmental conditions can be far more
demanding than surviving in a laboratory under optimal conditions. Field
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162 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
studies have been conducted on a transgenic mycoinsecticide to monitor the
fate of the fungi under field conditions (Hu and St. Leger, 2002). The
fungus was released onto a plot of cabbage and survivorship was deter-
mined in nonrhizosphere and rhizosphere soils. In nonrhizosphere soils, the
fungal propagules decreased from 105 to 103 per gram after several months.
However, recombinant fungi engineered only with the gene for green fluores-
cent protein (GFP) remained at 105 propagules in the rhizosphere soil.
Fungi that contained an additional protease gene did not persist as well in
the rhizosphere as did the GFP genotypes. The observations are consistent
with what has been observed for transgenic bacteria--that adding genes to
cells often decreases the microbes' fitness, but that some ecological contexts
(such as placement in the rhizosphere) can promote their survival.
Bacteria
In theory, genetically engineered bacteria introduced into the environ-
ment can become established in a microbial community. A limited number
of studies to assess this possibility have been done on bacteria in aquatic
and terrestrial environments. In one study (Scanferlato et al., 1989) the
viability of genetically engineered and wild-type strains of Erwinia
carotovora were compared after their addition to an aquatic microcosm.
Both declined in viability at the same rate, and within 32 days neither was
detectable by viable counts. Those data suggest that the newly introduced
bacteria, whether genetically engineered or wild-type, were poorly adapted
to the new environment and therefore were unable to compete with indig-
enous species. The observations are consistent with theory and with labora-
tory experiments on resource competition. Both suggest that the competitor
that grows at the lowest concentration of a limiting resource will survive
and thereby displace all inferior competitors (Hansen and Hubbell, 1980;
Tilman, 1982).
In nature, most nutrients are present at low concentrations in terrestrial
and aquatic environments (Madigan et al., 2003); therefore, only those
microorganisms that can compete for those limited resources would be
expected to thrive. To overcome the likelihood of introduced microbes,
being poor competitors, the genetic capabilities to perform a particular
function are commonly introduced into bacteria that already are adapted to
a particular habitat (Glandorf et al., 2001). For example, bacteria are being
used for bioremediation of oil-contaminated beaches and polluted soils.
The common practice is to apply nutrients, such as nitrate and phosphate,
to the contaminated area to promote growth of indigenous bacterial popula-
tions, which likely will include microbes that can metabolize the pollutants.
As a rule, introducing genes into indigenous bacteria to perform a
specific function is preferable to introducing exotic bacteria that contain
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VIRUSES, BACTERIA, AND OTHER MICROBES 163
those genes. Although bacteria initially could be maladapted to a new
environment, they often can change and increase their growth rate signifi-
cantly. In one study, transgenic bacteria were incubated in lake water for
15 days and then reisolated. The growth rates of the reisolates were more
than 50% higher in the lake water when compared to the original strain
(Sobecky et al., 1992). The researchers concluded that bacteria can adapt to
oligotrophic environments and the fitness of GEOs for survival can increase
in aquatic ecosystems. The ability of bacteria to adapt to new environments
over time is an important concern in their release to the environment. That
adaptability also applies to non-GEOs, though introduction of non-
transgenic exotic bacteria into new environments has not raised strong
concerns.
In another study, the viability of genetically engineered Pseudomonas
putida that contained a gene for the synthesis of the fungal inhibitor
phenazine was compared with its wild-type parent in the rhizosphere of
wheat plants for two growing seasons (Bakker et al., 2002). In both seasons,
the genetically engineered and the wild-type strains decreased to below
detectability within a month after the wheat harvest, indicating that the
rhizosphere was essential to the survival of the introduced bacteria. In one
season, within days of sowing the genetically engineered strain decreased
more rapidly than did the wild-type strain. In another growing season,
however, no difference in density was observed for the two strains, indicat-
ing that the additional metabolic load on the GEO did not reduce its
ecological fitness. Those results over successive years suggest that ecological
fitness, at least in soil, depends on the variable environmental conditions
encountered by the GEOs.
Considerable data suggest that the increased genetic load that results
from introducing additional genes into a microbe usually reduces its growth
rate unless a strong selection pressure favors the added genes (Lenski and
Nguyen, 1988; Milks et al., 2001; Zund and Lebek, 1980). The observed
decrease in growth rate apparently can result from the additional products
synthesized from the DNA rather than from the replication of the DNA
(Lenski and Nguyen, 1988). However, several research groups have reported
that a genetically engineered bacterium can grow at the same rate as or even
faster than its parent does (Bouma and Lenski, 1988; Devanas and Stotzky,
1986; Edlin et al., 1984; Hartl et al., 1983; Marshall et al., 1988). Those
latter observations are not well understood, and it is unclear whether the
organisms as grown in the laboratory would be ecologically fit in a natural
environment. Also, cases have been reported in which genetically engi-
neered bacteria coexist with indigenous populations (Kargatova et al.,
2001). If the introduced bacteria were resistant to an antibiotic present in
the environment, in theory it should thrive because of reduced competition
from susceptible strains. Thus, the assumption that all genetically engi-
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164 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
neered bacteria are unfit in natural environments cannot be sustained. How-
ever, the same would be true for exotic non-GEOs.
Displacement of Indigenous Populations
Viruses
It is theoretically possible that genetically engineered viruses could dis-
place resident species. In theory, the coevolutionary battle between viruses
and their hosts leads to a never-ending arms race; hosts evolve resistance,
viruses evolve counterresistance, and the cycle repeats with both species
constantly running to remain in place (this is the "Red Queen hypothesis";
Clarke et al., 1994). Whereas the hosts and viruses must continuously
evolve to maintain same dual-species interaction, at least this scenario pro-
duces long-term stability of biodiversity in the ecosystem. In contrast, a
newly introduced virus that can prove more virulent in nontarget host tips
the balance in its favor (Bull, 1994; Taylor et al., 2001). Subsequent devas-
tation of the nontarget host is the primary concern, but a separate concern
is that the resident virus could lose out because it is less efficient (relatively
less virulent). Extinction of the endemic virus could disrupt the ecological
community; for instance, introduction of the relatively more virulent species
could force the nontarget host to a lower equilibrium density in the commu-
nity, producing a cascade effect elsewhere in a food web. Viruses might be
underappreciated in terms of their influence on regulating large-scale eco-
system processes (Fuhrman, 1999).
Bacteria
Several studies have reported on the effect of adding genetically engi-
neered or wild-type bacteria to resident flora in aquatic and terrestrial
environments. In one study (Scanferlato et al., 1989), genetically engineered
strains of E. carotovora were added to an aquatic microcosm, and the
effects were measured in some elements of the indigenous population.
Thirty-two days after inoculation the number of total and proteolytic
bacteria was the same in the inoculated and uninoculated microcosms.
Neither did the inoculation affect the number of amylolytic and pectolytic
bacteria in the water or sediment.
In another study, genetically engineered Pseudomonas fluorescens were
released into indigenous populations near wheat plants (De Leij et al.,
1995). The results for culturable organisms can be summarized as follows:
P. fluorescens and the unmodified strains produced the same results, the
perturbations to the microbial population were small, and the release of
bacteria had no obvious effect on either plant growth or health.
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VIRUSES, BACTERIA, AND OTHER MICROBES 165
Genetically engineered P. putida that contained a gene for the synthesis
of the fungal inhibitor phenazine and its wild-type parent were added to the
rhizosphere of wheat plants (Bakker et al., 2002). Neither the transgenic
strain nor its parent affected the metabolic activity of the soil microbial
population, and only transient changes were observed in the composition of
the rhizosphere fungal microflora. Although the GEO had the greater effect,
the authors suggested that the effect of the introduced GEOs was only
minor in comparison to those that result from such common agricultural
practices as plowing or crop rotation.
Most studies that have assessed the influence of genetically engineered
microbes on microbial populations have studied effects on culturable organ-
isms alone. Yet less than 1% can be grown in culture (Madigan et al.,
2003). However, polymerase chain reaction technology and measurement
of ribosomal DNA (rDNA) patterns make it possible to analyze entire
bacterial populations. Using those techniques, Robleto and colleagues
(1998) showed that introduction of engineered strains of Rhizobium syn-
thesizing a narrow-spectrum-peptide antibiotic reduced the diversity of -
Proteobacteria; while the total bacterial population was not substantially
affected. In another study P. putida, genetically engineered for increased
activity against soilborne bacterial and fungal pathogens, were released into
the rhizosphere of wheat and their effect on indigenous microflora was
determined (Bakker et al., 2002). Effects of the genetically engineered bac-
teria on the rhizosphere fungi and bacteria were analyzed, using amplified
ribosomal DNA restriction analysis. A transient change in the composition
of the rhizosphere was noted, but several soil microbial activities, such as
soil nitrification and cellulose decomposition, were unaffected. The limited
data from all of these experiments indicate that the introduction of geneti-
cally engineered microorganisms has mostly transitory effects on indig-
enous populations that are unlikely to be significant in the field. The effects
of adding transgenic microbes are not likely to be any greater than are those
that attend the addition of nontransgenic species.
To reduce the possibility of changes in the indigenous microbial popula-
tion that result from the release of genetically engineered microbes, the
committee advises that strains be used that are likely to be poor competitors
in the local environment. The limited available data suggest that the intro-
duction of genetically engineered microbes into the environment is unlikely
to have significant long-lasting effects on microbial communities.
Horizontal Genetic Transfer into Local Populations
A third concern of introducing genetically engineered microbes is the
potential consequence of horizontal transfer of engineered genes from
introduced microbes into local populations.
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166 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
Viruses
Viruses have two distinct mechanisms for the exchange of genetic
material. When two or more DNA or RNA viruses infect the same host cell,
recombination can lead to hybrid progeny that contain genetic information
from both parents (Hershey and Rotman, 1949). In contrast, some RNA
viruses have genomes that are split into several smaller segments, and co-
infection can produce hybrids that contain a random reassortment of the
segments found in the infecting parent viruses. Such exchanges have peri-
odically led to new strains of influenza virus that have caused human
pandemics (Palese, 1984; Webby and Webster, 2003).
Recombination in viruses, can promote linkage equilibrium (free asso-
ciation of alleles) to create the potential for engineered alleles to enter and
circulate within a local gene pool. Laboratory experiments show that gene
exchange can profoundly affect virus evolution (Rambaut et al., 2004;
Turner, 2003; Turner and Chao, 1998), and it is generally accepted that
viral recombination in natural infections is a major force in the evolution of
new viruses (e.g., Goldbach, 1986). Many viruses also can recombine with
host chromosomes, thus introducing virus-derived genes into the host genome
and the host gene pool. The fitness effects of engineered genes in the origi-
nal virus background are likely to be assessed before strains are released
into target populations of hosts. The concern is that those genes could have
unanticipated effects when they transfer horizontally from the engineered
background into new ones. Epistasis (gene interaction) between introduced
alleles and those in the gene pools of other species can hamper the fitness of
individuals in those groups.
Hammond and colleagues (1999) studied transfer of engineered genes
through virus recombination and considered the likelihood that the process
would harm natural populations. The greatest concern identified by those
authors was the creation of new virus types as a result of recombination
between wild-type viruses and unrelated transgenes in genetically engi-
neered plants. However, the rate at which this happens is unlikely to exceed
that in naturally occurring mixed infections of viruses of nonengineered
plants. More data are needed from field trials to evaluate the benefits and
risks associated with release of transgenic viruses. The most extensive survey
to date (Thomas et al., 1998) studied interactions between transgenes
derived from potato leafroll virus and viruses to which transgenic plants
were exposed. The experiments revealed no evidence of recombination,
altered transmission, or altered virus properties, suggesting that such phe-
nomena are extremely rare.
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VIRUSES, BACTERIA, AND OTHER MICROBES 167
Bacteria
Most species of bacteria have several mechanisms for horizontal gene
transfer: DNA-mediated transformation, in which "naked" DNA is trans-
ferred to recipient cells; generalized or specialized transduction, in which
donor DNA is enclosed in the coat of a bacteriophage; and conjugation, in
which DNA--primarily through plasmids--is transferred from donor to
recipient cells after contact between the two. Bacterial gene exchange can
affect the persistence of a strain or its engineered alleles, and a genetically
engineered bacterium can be the donor or the recipient of genetic informa-
tion by horizontal gene transfer. Only a new genetic combination with
higher fitness than the indigenous genotype under the specific environ-
mental conditions has a high likelihood of persisting (Koonin et al., 2001).
In the laboratory, the transfer of genetic material is most efficient
between members of the same species. However, transfer in nature has been
observed between widely different genera, and even domains, of bacteria.
Comparative analysis of bacterial, archeal, and eukaryotic genomes sug-
gests that a significant fraction of the genes in prokaryotic genomes have
been involved in horizontal transfer over evolutionary time (Koonin et al.,
2001). At least one bacterium, Agrobacterium, can transfer DNA into
plants, and it is the workhorse of plant genetic engineering (Chilton et al.,
1977). The broad-host-range plasmid RSF1010, when in Agrobacterium,
can mediate its own transfer into plants as well as into other Gram-negative
bacteria (Buchanan-Wollaston et al., 1987). Thus, plants have ready access
to the gene pool of Gram-negative bacteria, thereby expanding the possi-
bilities of horizontal gene transfer from prokaryotes to eukaryotes. Many
varieties of tobacco (Nicotiana tabacum) contain genes transferred from
Agrobacterium over evolutionary time (Furner et al., 1986). In the labora-
tory, Agrobacterium can transfer DNA into a variety of fungi (Bundock et
al., 1995; de Groot et al., 1998; Piers et al., 1996) and because Agrobacterium
and many fungi occupy the same habitat in soil, transfer between the bac-
terium and fungi might also occur in nature. Escherichia coli (E. coli) can
transfer plasmid DNA into yeast in the laboratory (Heinemann and Sprague,
1989). It also has been reported that Agrobacterium can transfer DNA into
mammalian cells (Kunik et al., 2001) and that E. coli can transfer plasmids
into mammalian cells (Waters, 2001).
Several studies have examined the possibility of gene transfer from
transgenic plants to bacteria (Gebhard and Smalla, 1999; Schlüter et al.,
1995). The conclusion has been that such an occurrence would be extremely
rare, although plant DNA can persist in the soil under field conditions for
up to 2 years. Nielsen and colleagues (1998) emphasized that, although
gene transfer can be a rare event, it is critical to understand the selective
forces that act on the outcome of any transfer.
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168 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
Plasmids are a common vector for cloning and moving transgenes from
one organism to another, and conjugation in particular would allow these
genes to move easily into other bacteria in the environment. Some conjuga-
tive plasmids are highly promiscuous in their ability to transfer horizontally
between unrelated bacteria, and that has contributed to widespread dis-
semination of antibiotic-resistance genes, which often reside on plasmids.
In contrast, other plasmids are nonconjugative and thus unable to be trans-
ferred. However, they can be transferred if another plasmid in the same cell
provides the missing functions. The probability of horizontal transfer can
be reduced by cloning genes into nonconjugative plasmids and by using
bacteria that contain no other plasmids. The risk of transfer can be further
minimized if the engineered genes are integrated into the bacterial chromo-
some rather than remaining on a plasmid. Further, the enzyme transposase--
required for gene movement inside a cell, and frequently used in construct-
ing transgenic bacteria--should be disabled. All introduced plasmids should
be defective in conjugation functions. If any antibiotic resistance loci are
used to mark strains, the resistance loci should not involve antibiotics that
currently are in clinical use.
A sensible choice should be made for the bacterial strain introduced
into the environment to carry out a specific function. For example, it would
be unwise to introduce a close relative of a disease-causing bacterium
because nonpathogenic relatives conceivably could differ only by the presence
of a genetic element (plasmid, transposon, prophage) on which virulence
genes reside. Thus, in theory, inadvertent acquisition of one or more func-
tions might convert the introduced strain into a dangerous pathogen of
humans, animals, or plants. This is because bacteria, unlike viruses, are not
obligate parasites. That is, bacterial pathogenicity depends on an array of
characteristics that relatively few bacteria have acquired through extended
coevolution with a particular host (Salyers and Whitt, 2002). Therefore, it
is unlikely that minor genetic modifications would convert a nonpathogenic
strain into a pathogen, and laboratory experiments to achieve it--at least
with E. coli--have thus far been unsuccessful (S. Moseley, University of
Washington, personal communication, 2003).
Fungi
Evidence is weaker for horizontal gene transfer between fungi than it is
between bacteria (Rosewich and Kistler, 2000). In one case, however,
sequence analysis of a gene for chymotrypsin synthesis in a fungus in which
chymotrypsins had never been observed revealed that the sequence is related
to that of a soil bacterium (Screen and St. Leger, 2000). As more sequences
become available, it should become clearer whether horizontal gene trans-
fer can occur.
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VIRUSES, BACTERIA, AND OTHER MICROBES 169
To reduce the possibility of horizontal transfer of genetically engi-
neered alleles, it is advisable to use microbial strains in which transgenes are
integrated into chromosomes. The enzyme transposase, required for gene
movement inside a cell and frequently used in constructing transgenic bac-
teria, should be disabled. All introduced plasmids should be defective in
conjugation functions. If any antibiotic resistance loci are used to mark
strains, the resistance loci should not involve antibiotics that are currently
in clinical use.
BIOCONFINEMENT OF BACTERIA, VIRUSES, AND FUNGI
Because they are small, easily dispersed, and numerous, genetically
engineered microbes will require bioconfinement approaches that are dif-
ferent from those for other GEOs. Control centers on fitness reduction.
Fitness Reduction
Phenotypic Handicapping
One potential consequence of releasing transgenic microbes to the envi-
ronment is that they could perpetuate by invading or displacing natural
populations in competition for resources. The limited experimental data
suggest that, in general, genetically engineered bacteria and viruses will be
competitively less fit than their wild-type counterparts because of burdens
associated with carrying and expressing additional functions coded by
transgenes. As noted already, microbes generally fare poorly when intro-
duced into a new environment, although numerous cases could be cited in
which the genetically engineered microbe did not appear to be significantly
handicapped compared with the parental strain (Bouma and Lenski, 1988;
Devanas and Stotzky, 1986; Hartl et al., 1983; Marshall et al., 1988). One
solution to the uncertainty of whether transgenic microbes or their genes
will persist is to use strains with phenotypic handicaps, such as reduced
survival capability, reduced reproductive capacity, low resistance to a pre-
dictable change in the environment (such as seasonal heat or cold), or a
tendency to lose the specific function of concern (NRC, 1989a).
Viruses
Phenotypic handicapping is widely applied in the design of live viruses
for use as vaccines (Murphy and Chanock, 2001). They do not cause dis-
ease but they stimulate the host's immune system to produce antibodies
against wild-type viruses to fight subsequent infection. An ideal live vaccine
is an attenuated (weakened) form of the virus that is phenotypically handi-
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170 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
capped, thus ensuring that its competitive inferiority will prevent it from
persisting in nature. That is, phenotypic handicapping hampers the vaccine
or its engineered alleles from influencing the evolution of wild populations
through entry of those elements into the natural gene pool. Similarly,
attenuated viruses can be engineered to carry proteins of unrelated virus
pathogens, and those chimeric vectors elicit immune responses without
posing the threat of long-term persistence (Rose et al., 2001).
Recent studies have attempted to engineer herpes simplex virus-1 and
other well-described viruses for therapeutic interventions, such as combat-
ing cancer through gene therapy (e.g., Advani et al., 2002). Overall, few
data exist regarding the long-term persistence of genetically engineered
viruses. The effectiveness of phenotypic handicapping of viruses as a con-
finement measure is not clear.
Bacteria
Phenotypically handicapped live bacteria also have been used in induc-
ing cellular immune response (Stocker, 1990). When a gene that codes for a
particular epitope (a short, linear peptide sequence that is a portion of a
larger protein antigen) was inserted into a gene that codes for flagellin in
Salmonella auxotrophic for aromatic acids, a cellular immune response to
the epitope was generated (Verma et al., 1995). The bacteria did not multi-
ply because of a lack of required compounds in the environment. Pheno-
typic handicapping of bacteria as a confinement measure already has been
alluded to: One form involves the rapid decline of nonindigenous microbial
strains (including genetically altered ones) after they are introduced into
soil or aquatic environments (e.g., Glandorf et al., 2001; Scanferlato et al.,
1989). The data support the widely accepted view that long-established
microbial communities are able to resist invasion by foreign organisms
(Liang et al., 1982).
However, one challenge to phenotypic handicapping is the evidence
that genetically engineered strains can persist for long periods by quickly
adapting to a local environment. For instance, Kargatova and colleagues
(2001) observed that recombinant E. coli strains can persist for one year or
more in aquatic microcosms, and that they can coexist with indigenous
microflora. The strains adapted by decreasing their expression of cloned
genes, suggesting that the genetically engineered bacteria tended to lose the
genes of concern. Similarly, addition of plasmids does not necessarily lead
to a long-lived handicap to the bacterial host. Bacteria can adapt through
the mutation of genes that are not associated with the plasmid and thereby
restore their growth rate to that of the original parental strain (e.g., Bouma
and Lenski, 1988; Hartl et al., 1983).
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VIRUSES, BACTERIA, AND OTHER MICROBES 171
Most experiments on phenotypic handicapping have been performed in
the laboratory. But in natural environments such as lake water, the fitness
consequences of added genetic material are more difficult to evaluate and
apparently depend on many factors associated with the genetics of the
bacteria and the environment, with its usually obscure and variable selec-
tion pressures. For instance, in a study involving prototrophic strains of
P. putida, one with and the other without a plasmid, the plasmid-bearing
strain was maintained in a lake system over a period of 2 months (Sobecky
et al., 1992). The plasmid was lost within 24 hours if the strains were
amino acid auxotrophs. Variations in weather, such as rainfall and tem-
perature, can affect the population density of transgenic microbes intro-
duced into the soil (Glandorff et al., 2001). In addition, it is possible that an
altered microbe can become immediately more fit than the wild-type if the
phenotypic change increases resistance to a noxious substance in the envi-
ronment or increases the ability of the microorganism to metabolize a
substrate in the environment. Expression of additional functions and their
effects on fitness reduction have not been clearly defined, and a greater
effort to examine this phenomenon in field tests is warranted (e.g., Palmer
et al., 1997).
A second identifiable complication of phenotypic handicapping is that
indigenous bacteria generally do not exist as free-living, individual cells.
Rather, in their natural environment, bacteria often form highly structured
clumps, called biofilms, with properties that are quite different from those
of bacteria growing in the laboratory. For this reason, phenotypic handi-
capping of transgenic bacteria growing in liquid medium in the laboratory
might not be relevant to performance in a natural setting. In particular,
bacteria in biofilms are far more resistant to noxious chemicals, including
antibiotics and heavy metals, than are individual cells (Madigan et al.,
2003). Biofilms attach to inanimate objects in the environment and their
formation requires the action of several genes. It could be undesirable for
genetically engineered bacteria to persist long term, but they must live long
enough to perform the intended function. Unless those bacteria form
biofilms by attaching to such objects as rocks, soil particles, and teeth, they
could be washed away by rain or other fluids. Several genes have been
identified in E. coli and other microorganisms that are necessary for biofilm
formation, so mutations in those genes could debilitate the organisms to the
extent that they would not persist even for short periods with the indig-
enous flora (O'Toole et al., 2000; Pratt and Kolter, 1998).
A third difficulty of phenotypic handicapping is that the GEO could be
so handicapped that it is not practical to use. Perhaps the best example is
strain 1776 (see Box 5-1).
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172 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
BOX 5-1
1776
The most extreme case of phenotypically handicapping a microbe was carried
out in the laboratory of Roy Curtiss after the 1975 International Conference on
Recombinant DNA Molecules. The idea was to disable the K-12 strain of E. coli,
considered safest for use in cloning experiments, and make it even safer, against
the possibility of its escape from the laboratory. The thinking that went into the
disabling process serves as a guideline for genetically handicapping other
organisms.
Curtiss and his colleagues (1977) introduced mutations that precluded coloni-
zation of and survival in the intestinal tract, prevented biosynthesis of the rigid
layer of the bacterial cell wall in nonlaboratory conditions, led to the degradation of
DNA in any organisms that escaped the laboratory environment, permitted moni-
toring of strains, and inactivated DNA repair mechanisms. The key mutations were
deletions or independent mutations. Thus, the traits were stable and unlikely to
revert. The strain had a generation time twice to four times longer than the wild-
type E. coli K-12 strain and likely would not compete well with healthy microbes in
the environment. The strain also was resistant to most known E. coli-transducing
phages and defective in inheriting many conjugative plasmids. Thus, the strain
could not transmit genetic information by transduction or conjugation at detectable
frequencies.
To celebrate the nation's bicentennial, the strain was named 1776. It was
used for the industrial production of insulin after the cloning of the eukaryotic insulin
gene into the nonconjugative plasmid pSC101. However, the strain, with its multi-
ple auxotrophic markers, sensitivity to detergents, and increased generation time,
proved so difficult to grow that widespread use was clearly impractical. Accordingly,
as studies with recombinant DNA became more routine and the guidelines for
biocontainment were relaxed, wild-type strains of E. coli K-12 or HB101 were used
as the hosts for DNA cloning. 1776 is now just a memory of a bygone era.
Fungi
Two methods have been proposed to phenotypically handicap fungi.
One is to isolate auxotrophic mutants that can exist on the pest host, in the
case of a biocontrol agent, but that would not survive outside the host. Such
mutants should be isolated using a physical mutagen, such as gamma or
neutron radiation, that fragments genes and thereby prevents reversion.
Another proposed technique is to render the fungi asporogenic (unable to
produce spores), thereby helping not only to prevent their spread but also
inhibiting the formation of dormant resting structures that resist heat, cold,
desiccation, and other harsh environmental conditions (Gressel, 2001).
Asporogenic mutants would be handicapped both in persistence and in the
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VIRUSES, BACTERIA, AND OTHER MICROBES 173
major structures dispersed by wind, water, or animals. Spores of some
fungi, however, are required for pathogenesis. Thus, if the fungus is to be
used in biocontrol, an asporogenic mutant would not be suitable, and this
form of handicapping would be inappropriate.
Suicide Genes
Suicide genes can be used to confine bacteria and fungi under two
circumstances. The first ensures that bacteria growing in a closed container
(such as a vat) are unable to survive if they escape. The strain should die as
quickly as possible after escape. In many situations this is best achieved by
chemical sterilants. The second circumstance is to combat a perceived threat
to the environment should released microorganisms persist beyond the
intended period of usefulness, for example, for bioremediation of Superfund
sites or for biocontrol of plant pests in agriculture. In that case, the GEO
must be able to carry out its function before it expires. In either scenario,
the microorganism would carry a suicide gene that is repressed when the
microbe is at work and becomes active immediately thereafter (Curtiss,
1988; Molin et al., 1987).
Bacteria
The key to designing an effective suicide containment system rests on
regulating gene expression from one of a variety of controllable promoters.
They can be divided into two categories: those that function when a trigger
is present and those that function until repressed (see Molin et al., 1993 for
review). Systems that have been devised in the first category include the PL
promoter of phage lambda and a thermosensitive lambda repressor (Ahrenholz
et al., 1994) and the lac promoter from E. coli. The PL promoter is induced
by raising the temperature to inactivate the lambda repressor; the lac pro-
moter is activated by the chemical isopropyl--D-thiogalactopyranoside
(IPTG) (Bej et al., 1988; Knudsen and Karlstrom, 1991; Knudsen et al.,
1995). Although such systems work in the laboratory under controlled
conditions, it is unrealistic to apply heat to fields or to irrigate fields with a
chemical inducer such as IPTG. As a solution, Molin and colleagues (1993)
suggest manipulating the regulated system such that growth of the cells in
the laboratory leads to the synthesis of a compound that is toxic to the
microbe. When cells are introduced into the environment, several genera-
tions of growth would be needed before the repressor would be diluted and
the toxin synthesized. Because generation times in the wild can be just days
or weeks long, the engineered cells should survive only long enough to
achieve the goal. The approach is speculative, but it seems promising. For
bioremediation, a possible approach involves repressing transcription from
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174 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
a promoter that functions only in the presence of the target substrate. Once
the substrate is exhausted, transcription from the promoter ensures that
suicide is induced (Contreras et al., 1991).
The systems that function when the activator is absent include the trp
promoter from E. coli, which represses the synthesis of a toxic compound
when tryptophan is present. If the microorganisms escape, their new envi-
ronment would likely contain insufficient supplies of this amino acid, result-
ing in transcription of the toxin gene from the active trp promoter and
synthesis of the toxin.
Two goals are paramount in the design of suicide systems. First, the
gene product should extend beyond mere growth inhibition; the best candi-
dates are killing functions whose targets are likely to be found in essentially
all bacteria. Second, the toxicity of the gene product should be high, so that
a high efficiency of killing is achieved at a low concentration. Therefore,
putative killing functions should be assayed in various bacteria at several
ranges of induction (Molin et al., 1993).
The strengths of suicide genes in bioconfinement are their high specificity
and the variety of potential targets and activators. Molin and colleagues
(1993) reviewed the major systems of suicide genes developed in bacteria.
The hok/sok (host killing/suppression of killing) system originally was
observed in bacterial plasmids (Gerdes et al., 1986). That system and others
in the gef gene family consist of genes that encode for a toxic polypeptide
that both attacks the cytoplasmic membrane and is the antidote to that
polypeptide. If a cell spontaneously loses the plasmid (or if the gene inserted
in the chromosome mutates) it dies because the leftover mRNA is translated
into a toxic protein that degrades the bacterial membrane. One advantage
of using this class of toxic factors is that their target, the cytoplasmic
membrane, is similar in structure in many bacteria.
Other killing systems include nucleases, which target destruction of
genetic material (e.g., Ahrenholz et al., 1994). Those systems are highly
promising. Not only would they kill the engineered bacterium but they also
destroy its DNA, which might otherwise be transferred from the dead
organism to living cells via transformation. Genes that code for nucleases
from Serratia marcescens and Staphylococcus aureus have been fused to an
inducible lac promoter to create such killing systems. It is unclear to what
extent the DNA repair systems in cells would make it difficult for the
nucleases to degrade the DNA to the extent necessary to kill the cells. Lysis
genes from bacterial viruses also have been considered as a source of killing
genes. Those genes have been cloned and fused with regulated gene expres-
sion systems, with promising results (Molin et al., 1993). It is noteworthy
that research in the development of suicide genes as a means of bioconfine-
ment appears to have stopped about ten years ago for reasons that are not
clear to the committee.
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VIRUSES, BACTERIA, AND OTHER MICROBES 175
Fungi
The same methods that are being used to control the spread of trans-
genic bacteria are being applied to fungi. The object is to prevent fungal
persistence and spread through the formation of various kinds of spores. If
the spore is necessary for the fungus to execute its intended function, such
as the infection of an insect, it is necessary to suppress sporulation after that
goal is accomplished. Genes that inhibit sporulation could be put under the
control of an inducible promoter and then engineered into the fungus. The
spores would be treated with a chemical or environmental inducer before
they are applied to the target pest.
Some fungi are being genetically engineered to contain genes that
increase their virulence in specific insect pests (Hu and St. Leger, 2002;
St. Leger et al., 1996). To prevent the creation of hypervirulent organisms,
it has been proposed that the genes be flanked by antisense forms so as not
to affect the virulence of the strain but to target genes in the recipient cells
that might inadvertently receive the virulence genes. Such targets could
involve reproduction, spore formation, and spore germination. To prevent
vegetative spread of the mycelium, suicide genes could be engineered into
cells under the control of an inducible promoter.
A major weakness of suicide genes in fungi and bacteria is the occur-
rence of mutations that prevent the system from operating. In large part,
their usefulness has been demonstrated only in laboratory studies and it is
not clear how they will function in the field. Some suicide systems are
intriguing ideas that have yet to work even in the laboratory. Laboratory
experiments show that killing by suicide gene systems is never absolute; a
surviving subpopulation can continue to grow even in the presence of
inducer (Molin et al., 1993). The survivors result from mutations in the
killing gene, mutations in the expression system that inactivate the suicide
function, or mutations in other parts of the cell that confer resistance to the
action of the killing agent (Knudsen et al., 1995). Suicide systems also can
be lost from the cell if they are located on a plasmidthe plasmid can be
lost after transfer to only one daughter cell during cell division. One way to
reduce the problem is through redundancy, provided by the use of two
identical systems or the combining of different suicide systems (Jensen et
al., 1993; Knudsen et al., 1995). Those efforts will lower, but not eliminate,
the probability of mutations, resulting in resistance. Thus, suicide systems
can reduce, but not eliminate, a genetically engineered population.
Viruses
To the committee's knowledge, suicide gene systems per se have not
been applied in the production of genetically engineered viruses. However,
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176 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
viruses can mutate spontaneously into temperature-sensitive or ts mutants
such that the mutant genotype cannot replicate at some temperatures. If a
genetically engineered virus featured a ts mutation, that system could be
harnessed in a fashion similar to the physical control of bacterial suicide
genes. However, ts mutants typically are much less fit than are wild-type
viruses, and using them might be more accurately described by the fitness
reduction method known as phenotypic handicapping.
Failed or Inappropriate Methods of Microbial Bioconfinement
Chapters 3 and 4 describe the major bioconfinement methods used in
higher organisms, and most of them are inappropriate for use in microbes.
Because bacterial reproduction is strictly asexual, for instance, sterilization
cannot be used to confine transgenic bacteria. Although some viruses can
reproduce through reassorting chromosomal segments when multiple virus
particles infect the same cell, the same viruses also can reproduce clonally.
Thus, unlike most eukaryotes, they are not bound to obligate sexual repro-
duction. As a consequence, confinement of genetically engineered microbes
must be limited to fitness reduction methods such as the induction of suicide
genes or phenotypic handicapping.
Effectiveness of Methods at Different Temporal and Spatial Scales
Temporal scales could influence the effectiveness of bioconfinement in
bacteria, fungi, and viruses. Although those microbes can grow rapidly
under ideal laboratory conditions (up to one generation per hour), typically
they grow much more slowly in nature (Madigan et al., 2003). Nutrients in
the wild usually are limiting, and their scarcity can prevent microbes from
achieving rapid exponential growth. In nature, bacteria often experience
"feast or famine;" periods of rapid growth are interspersed with longer
periods of retarded growth. In the transition, bacteria undergo dramatic
changes in physiology and morphology, which adapt them to poor growth
conditions. In periods of slow growth bacteria are much more resistant to
environmental assault than are rapidly growing cells (Siegele and Kolter,
1992). The process of bacterial sporulation, in which bacteria enter a dor-
mant, nonmetabolizing, highly resistant state, is an extreme example.
Fungi develop spores in the course of sexual or asexual reproduction.
The spores, which are readily dispersed, are hardier than mycelia but not
nearly as resistant to harsh environmental conditions as bacterial spores
can be. Viruses are nonmetabolizing entities, so they do not have the luxury
of regulating metabolism as do bacteria and fungi. Rather, as obligate
parasites, viruses are at the mercy of their hosts (the biotic environment),
and their ability to grow in adverse conditions (the abiotic environment)
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VIRUSES, BACTERIA, AND OTHER MICROBES 177
depends on host metabolism. Because the ideal growth conditions of host
cells are likely to be separated in time, many viruses can infect their hosts
latently until the hosts resume growth or active metabolism, which then
would allow productive infection to occur again.
Fitness reduction methods are designed to hamper the reproductive
potential of genetically engineered strains of bacteria, fungi, and viruses,
placing them at a growth disadvantage relative to wild-type strains. These
debilitated strains should fare no better and likely far worse than their wild-
type counterparts in terms of ability to survive in the natural environment.
However, insufficient field testing has been done in a variety of environ-
ments with different organisms and genotypes to confirm that expectation.
A more important consideration for the effects of temporal scale on fitness
reduction methods in any microbe is the possibility that the microbe will
become latent (for viruses) or sporulate (for bacteria and fungi).
Effects of spatial scale on the confinement of bacteria and viruses are
difficult to gauge; relatively little is known about dispersal of microbes in
the wild. Most current data concern dispersal of pathogenic bacteria and
viruses through physical processes (such as flow of water) or geographic
movement of their host organisms (such as air travel by infected humans).
Phenotypic handicapping and other fitness reduction methods are designed
to reduce local survival of introduced bacteria, fungi, and viruses. Should
those microbes become dispersed to distant locales, one might assume that
the methods would be effective there as well. But this is not necessarily true,
especially if the microbes are dispersed to different kinds of environments.
Some viruses can inflict very different degrees of damage (becoming more
virulent) when the host population is naïve to virus attack because of an
absence of resistance alleles or antibodies in the host population (Bull,
1994; Taylor et al., 2001). Although highly speculative, a potential concern
is that migration of genetically engineered bacteria, fungi, and viruses to
new places could release them from phenotypic handicapping and from
other mechanisms designed to hinder fitness.
Because the effects of sporulation and germination traditionally have
not been evaluated in field tests, it would be wise to avoid, if possible,
genetic modification and release of sporulating microbes as a way to mini-
mize the risk of long-term survival and dispersal and allay the fear that
those strains would transfer their genetic material to local populations.
Ecosystem and Population Effects
The committee has identified phenotypic handicapping and suicide sys-
tems as the primary methods that could be used for bioconfinement of
bacteria, fungi, and viruses. Although more field data are needed, it is
unlikely that the methods themselves would damage ecosystems and natural
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178 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
populations. However, because they have been studied only under labora-
tory conditions, three conceivable consequences can be foreseen that echo
the motivations for bioconfinement outlined above. All relate to the envi-
ronmental consequences of failure under natural conditions.
Although there are no supporting data, the possibility exists that release
of transgenic bacteria, fungi, and viruses can damage indigenous microbial
populations. For instance, although the effects of introduced viruses on
intended hosts can be gauged accurately through laboratory experiments,
the introduced viruses could attack nontarget hosts that are ill prepared to
defend themselves. Similarly, survival of bacteria in the wild could be under-
estimated from laboratory results. The second concern involves displace-
ment of resident species. Indigenous viruses could be less virulent than
introduced ones, creating the opportunity for engineered viruses to severely
reduce the population size of local hosts. Finally, genes from introduced
bacteria, fungi, and viruses could be transferred horizontally into resident
species. Because those genes could have unanticipated effects when they
migrate to new backgrounds, they could reduce fitness at one or more loci
through negative epistasis. Although selection should act to remove the
introduced genes from the local gene pool, it could take a long time for
dangerous alleles to be completely removed as a result of weakened selec-
tion as the genes become rarer in the population (Hartl and Clarke, 1997).
The committee believes that the ecological consequences of using fitness
reduction methods, such as phenotypic handicapping and suicide systems in
genetically engineered microbes, are likely to be minimal, because those
methods are designed to employ genotypes that are competitively inferior
indigenous strains. However, because the methods have not been evaluated
in the field, it is not possible to state with certainty that they will have the
desired effect in confinement.
Monitoring, Detection, and Culling: Needs, Feasibility, and Realities
The frequency of genetically engineered microbes in natural environ-
ments can be estimated rather straightforwardly if natural populations are
extensively sampled and screened with modern molecular techniques, espe-
cially if the engineered organisms contain easily detected phenotypic markers,
for example, that are visible on a selective agar medium. However, it is
virtually impossible to completely eliminate specific genotypes in natural
populations of microbes (Salyers and Whitt, 2002). This needs to be consid-
ered when deciding whether a genetically engineered microbe should be
released into the environment.
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VIRUSES, BACTERIA, AND OTHER MICROBES 179
Microalgae
Although this chapter focuses on bioconfinement of transgenic bacteria,
fungi, and viruses, the possibility of bioconfinement also should be evalu-
ated for genetically engineered microalgae.
Microalgae have already been successfully engineered (see review by
Minocha, 2003). The best results have been obtained with Chlamydomo-
nas reinhardtii, which has long served as a model system for physiological
and molecular studies (e.g, Cerruti et al., 1997; Dunahay, 1993). In particu-
lar, genetic engineering of C. reinhardtii could be useful for bioremediation
of heavy-metal pollution, a pervasive environmental problem because trace
metals cannot be decomposed but must be sequestered from the environ-
ment. Cai and colleagues (1999) demonstrated that the trace-metal-binding
properties of Chlamydomonas can be enhanced in transgenic genotypes
that express a foreign-metal-binding protein, without slowing their growth
rate relative to wild-type cells. In addition, stable nuclear transformation
has been achieved in the colonial green alga Volvox carteri (Hallman and
Sumper, 1994; Schiedlmeier et al., 1994). A few diatoms also have been
successfully transformed, including the widely studied model system
Phaeodactylum tricornutum (e.g., Apt et al., 1996). This is promising
because diatoms have commercial uses as feed in aquaculture and as poten-
tial sources of useful pharmaceuticals.
Most commercial-scale cultivation of microalgae is performed in large,
open outdoor ponds. Zaslavskaia and colleagues (2001) identified several
disadvantages of this approach, including invasion of ponds by contami-
nants and reduction in biomass production resulting from seasonal and
diurnal variations in temperature and light. Thus, improved efficiency and
reduced cost of micro-algal biomass production could be achieved if the
microbes were engineered to grow as heterotrophs in conventional micro-
bial fermenters (in the absence of light). Zaslavskaia and colleagues (2001)
introduced a gene that encodes a glucose transporter into the obligate
photosynthetic microalga P. tricornutum, allowing the diatom to thrive on
glucose in the absence of light. The approach seems promising because
fermentation technology eliminates contamination by microbes, which is
an important criterion for maintaining food industry standards.
Microalgae are biologically similar to bacteria (especially photosynthetic
bacteria) that grow in aquatic environments, and they have similar mechanisms
for horizontal gene transfer. Therefore, the same consequences and concerns
would apply to their bioconfinement. However, most transgenic microalgae
have been cultivated in closed-system indoor tanks and are not intended for
release into natural environments. Because of their similarity to bacteria,
phenotypic handicapping and suicide systems should provide effective bio-
confinement if necessary. The committee did not find any reports in the
literature of efforts to test the feasibility of those methods in microalgae.
Representative terms from entire chapter:
phenotypic handicapping
