United States
Environmental Protection
Agency
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Industrial Environmental Research - ," -c*7 '
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-84-037 Sept. 1984
&ERA Project Summary
Genetic Engineering and the
Development of New Pollution
Control Technologies
James B. Johnston and Susan G. Robinson
This report relates genetic engineer-
ing to biological waste treatment, so
that opportunities for its improvement
can be identified and evaluated. It
compares the present (mid-1983) state
of development of gene manipulation
and natural limits to biodegradation.
It identifies a number of research
topics that are likely to contribute to
new pollution treatment techniques.
These topics include the basic mechan-
isms underlying microbial co-metabo-
lism and oligotrophy; molecular genet-
ics in filamentous fungi, in strict an-
aerobes and in archaebacteria; directed
evolution of enzymes and metabolic
pathways; and studies to advance under-
standing of dehalogenations by mi-
crobes.
This Project Summary was developed
by EPA's Industrial Environmental Re-
search Laboratory, Cincinnati, OH. to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
The objectives of this study were to
document the basis for developing new
pollution controls using genetic technol-
ogy, to describe the present state of such
development, and to recommend a re-
search policy for such an approach. The
study was performed by assembling three
review papers dealing with pollution
problems, gene manipulations, and natu-
ral limits to biodegradation. A panel of
experts representing disciplines that
might contribute to the development of
new treatment technologies assembled
in Urbana, Illinois, to discuss these pa-
pers. Their recommendations were
merged with the information gathered in
the background papers to provide a sound
basis for recommending policy.
It was generally concluded that the
spectacular recent advances in gene
manipulation afford a remarkable new
opportunity to design and create micro-
organisms with defined, desirable bio-
degradative capacities. But it was also
concluded that this ability represents only
one of the necessary conditions for the
creation of new and practical pollution
abatement processes. The other necessary
condition is to bring a population of
microorganisms having a suitable amount
of a relevant catabolic potential into
contact with a pollutant. This involves
either establishing a new population of
microorganisms in a polluted environ-
ment or distributing the genes for a
biodegradative pathway among the mem-
bers of an existing microf lora at a polluted
site.
Basic knowledge of these two process-
es is fragmentary compared to knowledge
of gene manipulation technologies. Con-
sequently, the primary recommendation
of the study is to support basic research
on organism establishment and gene
transmission in natural microbial popu-
lations. This recommendation states that
the establishment of microbes, or their
genes, in polluted matrices is as important
to practical treatment processes as the
creation of microbes with novel biode-
gradative capacities.
Release to the environment of engi-
neered microorganisms was identified as
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an issue of concern to the public and to
bodies such as the Recombinant DNA
Advisory Committee (RAC) that is charged
with the oversight of some of the new
genetic technologies. To allay fears and to
ensure a prudent and responsible de-
velopment of new biological pollution
treatments, it is recommended that exper-
iments involving the release of genetically
manipulated microorganisms be reviewed
and approved for environmental safety by
a scientifically qualified public body.
When possible, organisms destined for
release should be constructed without
the use of//? vitro recombinant DNA. This
should expedite approval of release, since
all techniques except the in vitro recombi-
nant DNA method are currently viewed as
essentially natural and innocuous.
Two conventional treatment problems
that might benefit from exploration from a
genetic perspective were the removal of
ammonia by low-aged wastewater sludg-
es and improvements in the flocculation
of these sludges. Unlike research on
organism establishment and gene trans-
mission in indigenous populations, no
priorities were assigned to these recom-
mendations.
The study recognized that the advance-
ment of waste treatment through genetic
manipulations will require the involve-
ment of scientists in a very broad range of
disciplines. Even in assembling the back-
ground papers for this study, communica-
tion among scientists was impeded by
differences in disciplinary perspectives,
in terminology, and, most importantly, by
a poor understanding of the fundamental
capabilities and limits of the essential
contributing disciplines. A number of
measures were recommended to improve
communication among the specialists in
the contributing sciences. These included
support for problem-focused symposia,
an occasional newsletter, the coordina-
tion of research support among govern-
ment agencies, and a listing of pollution
problems, research needs, and current
developments.
Results
There was general agreement among
the participants at the Urbana workshop
regarding some aspects of the develop-
ment of new biological pollution control
agents. It was generally held that this
approach offers a great deal of promise.
The following outline of conclusions and
recommendations provides a good sketch
of such promise and recommended re-
search.
Outline of Conclusions and
Recommendations
I. Release of Engineered Organisms
for Pollution Control
A. Concern about the release of
engineered organisms demands
the experiments involving their
release to the environment be
reviewed and sanctioned by a
scientifically qualified public
body.
B. Current regulations governing
the release of microbes altered
by in vitro recombinant DNA
methods are not a barrier to the
development of such organisms,
although release to the environ-
ment is regulated.
C. Development of new organisms
by in vivo recombination and
other techniques not now regu-
lated should be emphasized.
D. The possibility of exceptional
examples of potentially hazard-
ous strains arising during the
development of pollution control
agents by purely unregulated
manipulations cannot be ruled
out at present.
II. General Conclusions
A. Genetic engineering offers prom-
ise for new pollution treatments.
B. Single strains degrading a cate-
gorical collection of pollutants
(e.g., PCBs) will not be found.
C. Compound-specif ic and site-spe-
cif ic treatments must be empha-
sized initially.
III. Recommended Research
A. Fields
i. Colonization of polluted
environments by intro-
duced microbes
ii. Gene transmission in the
environment
iii. Co-metabolism
iv. Oligotrophy
B. Areas
i. The genetics of catabolism
by archaebacteria
ii. The genetics of catabolism
by anaerobes
iii. The genetics of catabolism
by filamentous fungi
iv. The genetics of microbial
dehalogenation
v. Artificial evolution of en-
zymes and metabolic path-
ways
C. Projects
i. Flocculation in wastewater
sludges
ii. Ammonia removal by low-
age wastewater sludge
IV. Implementation
A. Knowledge from relevant fields
should be developed in parallel
and integrated through a news-
letter and problem-focused sym-
posia.
B. Research undertaken by the
USEPA and other government
agencies and organizations
should be coordinated.
C. A current listing of relevant
scientific developments, pollu-
tion problems, and research
needs should be compiled, dis-
tributed, and updated regularly.
Although some aspects of this new
pollution technique appear promising,
several cautionary notes were sounded.
An obvious caution was that pollution
problems must be clearly defined in terms
of their relevance to genetics and bio-
degradation before potential solutions
can be discussed. Seemingly self-evident,
this notion is violated by suggestions
such as the creation of "superbugs" to
reduce the BOD of an effluent or the
volume of sludge from an aeration basin.
Genetic engineering most often involves
the specific alteration of individual bio-
chemical pathways in particular organ-
isms. Individual strains with specialized
pathways can have but little effect on the
uncharacterized and presumably hetero-
geneous organic matter measured by the
BOD test or on the organic matter present
in wastewater sludge.
Similarly, a caution was expressed
about the prospects for developing single
organisms able to attack pollutants such
as PCBs or toxaphenes that are actually
collections of diverse substances. It was
pointed out that bacteria frequently attack
only specific members of a set of related
compounds. Thus, differences as small as
a single hydroxyl substitution, for example
in benzoic,p-hydroxybenzoic and salicylic
acids, are discerned by bacteria. Usually
such compounds are degraded by sepa-
rate strains using wholly separate path-
ways. A single bacterial strain will fre-
quently degrade compounds related as
intermediates in a catabolic pathway, for
example, naphthalene, salicylate, and
catechol, more readily than a set of
compounds with close chemical relation-
ships, for instance, the position isomers,
phthalic and terephthalic acid, or a set of
compounds with varying chemical substi-
tutions at one position, such as chloro-
benzene, phenol, aniline, nitrobenzene,
and toluene. This apparently stems from
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the generally high specificity of enzyme-
substrate interactions and from the need
to foster efficient degradation by integra-
ting the products of catabolism into the
central metabolism of an organism.
Apparently exceptions to this specificity
do exist. Some oxygenases occurring
early in a degradative sequence show a
more universal appetite than is usual for
typical enzymes of intermediary metabo-
lism, for example, benzoate oxygenases
that can accept chlorobenzoates as sub-
strates or naphthalene oxygenases that
can utilize methyl naphthalenes. Further,
it has been pointed out that many cata-
bolic pathways tend to converge on a few
common metabolites, for example, aro-
matic degradations converging on cate-
chol or gentisate. Nevertheless, these
exceptions to absolute specificity are not
a sufficient basis for designing an orga-
nism to attack so broad a categorical set
of compounds as the PCBs; it must be
anticipated that a large group of specifi-
cally developed bacterial strains, each
attacking one or only a few members of
the set, will be needed to achieve a
comprehensive attack on collections of
compounds like these.
Another caution noted was that com-
pounds rarely occur singly in nature.
Similarly, pollutants will most commonly
be found as components of a mixture of
compounds. The other compounds occur-
ring in conjunction with a target pollutant
might be either beneficial or detrimental
to the organism degrading the pollutant.
In addition, the physical conditions pre-
sent at the polluted site will individually
and perhaps synergistically alter the
functioning of any given engineered
microbe. Thus, a strain used at one site
would not necessarily work well at an-
other, even if both sites contain a common
target compound. This would be due to
the nature of the chemical mixture or the
environmental conditions present at each
site. Thus, at least initially, each pollution
problem should be addressed site-by-
site, pollutant-by-pollutant. Ultimately, it
may be possible, after achieving improve-
ments in treatments of some specific
pollutants, to identify groups of sites with
sufficiently similar characteristics that a
particular manipulated organism can be
used at all similar sites. The initial
experimental work, however, should be
done with a narrow focus to provide a
foundation for what will follow.
Conclusions and
Recommendations
Presently, the strengthening of re-
search in a few specific subjects appears
to offer the most expeditious means to
promote the genetic engineering ap-
proach to pollution control. The recom-
mended research outlined above varies in
scope from specific projects to broad
fields of study.
The establishment of introduced orga-
nisms in the environment and the trans-
mission of genetic material among indig-
enous microflora are the two steps basic
to every suggested improvement in pollu-
tion control through genetic engineering.
The current state of understanding of
these processes does not permit predic-
tion of the success of either and does not
provide the molecular biologist with
guides for the construction of successful
organisms. Study in these two fields was
assigned the highest priority.
James B. Johnston and Susan G. Robinson are with the University of Illinois,
Urbana, IL 61801.
William A. Cawley is the EPA Project Officer (see below).
The complete report, entitled "Genetic Engineering and the Development of Ne w
Pollution Control Technologies," (Order No. PB 84-148 972; Cost: $14.50,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
S GOVERNMENT PRINTING OFFICE, 1984—759-016/7815
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Environmental Protection
Agency
Center for Environmental Research
Information
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