EPA-600/8-81-020
September 1981
ASSESSMENT OF FUTURE ENVIRONMENTAL TRENDS AND PROBLEMS
INDUSTRIAL USE OF APPLIED GENETICS
AND BIOTECHNOLOGIES
by
Robert H. Zaugg
Jeff R, Swcrz
i eknekran Research, Inc.
1483 Chain Bridge Road
McLean, Virginia 22.0!
63-02-3638
iVtorrs A, Levin
Innovative Resecrc. Program
Office of Research and Development
U.S. Environmental Drotection Agency
40! M Street, S/A'.
Washington, D.C. 20460
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. . 2
EPA-finn/8-31-020 ~ ORD Report
3. RECIPIENT'S ACCESSION NO.
M 1 1095 S
4. TITLE AND SUBTITLE
Assessment of Future Environmental Trends and Problems
,-Industriai Use, of Apnl ie'df Genetics and
Biotechnologies
5. REPORT DATE
September 1981
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Swarz, J.R. and Zauge, R.
8. PERFORMING ORGANIZATION REPOFiT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Teknekron Research, Inc.
1403 Chain Bridge Road
McLean, VA 22101
10. PROGRAM ELEMENT NO.
CCHH1A
11. CONTRACT/GRANT NO.
68-02-3192
12. SPONSORING AGENCY NAME AND ADDRESS
US EPA, Office of Research and Development, Office of
Exploratory Research, 40T-;McStreet SW, Wash DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/60C/0ER
15. SUPPLE MENTARY NOTES
16. ABSTRACT
The proposed study is to be a technological assessment of genetic engineering
as it applies to commercial industries and its potential effects on the environ/rent.
This includes a detailed literature review and state of the art analysis of genetic
engineering, an analysis of how applied genetics will affect public health and public
welfare, its probable impact on the environment and environmental policies and an
analysis of knowledge gaps, including identification of inadequacies of analytical
methods and techniques. Additionally, the socioeconomic impact of genetic engineering
on commercial industry will be examined.
The approach will include a literature review of five key industrial sectors:
Pharmaceutical and Cosmetic, Industrial Chemical, Energy, Food Manufacturing and
Preservation, and Mining. Areas that will be examined at length include: Environ-
ment and Populations, Government Policy, and Technology.
The research will be carried out in three phases:
I Development of Data Base, . ,
il Potential Hazards of Genetic Engineering^
-.lib Analysis of Findings. > ; -
KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIF'ERS/OPEN ENO = D TERMS
c. cOSati Held/Group
Genetic Engineering
Envi ronment
Molecular Biology
ji8. DISTRIBUTION STATEMENT
Environrrental effects
of microorganisms
Unlimited
19. SECURITY CLASS (This Report)
None
21. NO. OF PAGES
20 SECURITY CLASS (Tins page,
N'one
22 PRICE
EPA Form 2220-1 (Rev. 4-77) previous ecition is oosolete
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ABSTRACT
This study reoresents a portion of an overafl EPA/CRD assessment of future
environmental trerds and problems. With the aim of providing the EPA with
information on emerging technologies, the focus of this reoort is the industrial
use of applied genetics. The following five industrial sectors ere examined:
pharmaceutical, chemicals, energy, mining, and po'lution control.
-oMowing a brief historical review of the important developments in ocsic
biological research that herclded the advent of modern biotechnology, the report
describes the variety of experimental and commercial techniques that are
encountered in this field. These various methods include recombinant DNA
technology, mutagenesis, cell fusion procedures, immobilized bioprocesses, and
fermentation technology.
!n a section entitled Interested Parties, the report lists *be numerous individuals
end organizations that are actively involved in the commericalizction of applied
genercs. Over ICO U.S. business firms and about 5C foreign concerns are
identified as having substantial commercial interest in biotechnology. This
sect!on also describes the ro'e of various U.S. government agencies in examining
progress in tnis field.
The buik of the report consists of an industry-by-indjstry analysis of current
R&D activities in biotechnology, an estimcte of future prospects, and an
assessment of potential environmental end health hczarcs cssociated with "hese
activities. Trends ere identified and, wherever oossioie, schedules for the
appecrance of new aop'ications are predicted.
The final section of rhe report summarizes *he findings and provides c number o*"
recommendations to "he EPA regarding future action 'n the fie1 d of apoliec
genetics.
This report is submitted in fulfillment of Contract N.o. 68-02-3638 by Tekne*ron
Research, Inc. under the spensorshio of the U.S. Environments Protection
Agency. This report covers the period October '930, to Febracry 28, i'?3l, and
the work was completed as of Vtarcn 31, '981.
iv Pages ii and iii are blank
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CONTENTS
ABSTRACT iv
FIGURES vii
TABLES viii
1. INTRODUCTION I
I.; Scope of the report I
1.2 Brief history 2
2. TECHNOLOGY OF APPLIED GENETICS 7
2.1 Recombinant DNA 7
2.2 Genetic alterations by non-
recombinant DNA methods 12
2.2.1 Induced mutations (mutageresis) 12
2.2.2 Cell fusion methods 15
2.2.3 Other gene-aitering techniques 19
2.3 Immobilized bioprocesses 21
2.4 Fermentation technology 24
2.5 Gene therapy 27
3. INTERESTED PARTIES 29
3.! Domestic activities 29
3.1.1 Universities 29
3. i .2 Commercial firms 32
3.1.3 Federal government -3
3.1.3.1 The National :nst:tutes of Health ...
3.1.3.2 Other Federcl agencies 43
3.1.3.3 Patent issues 51
3.2 Foreign activities 52
4. INDUSTRIAL APPLICATIONS, TRENDS, POTENTIAL HAZARDS 5?
4.1 Pharmaceutical :rcustry 59
-.1.1 Current cctivities 59
-.1.2 Future orospects 63
4.1.3 Potential hazards 71
4.2 Chemical industry 76
4.2.1 Current activities 76
4.2.2 Future prospects 86
4.2.3 Potential hazards 88
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CONTENTS (cont.)
4.3 Energy industry 94
4.3.1 Current activities 94
4.3.1, i Energy from biomass 94
4.3.1.2 Enhanced oil recovery 103
4.3.2 "uture prospects ! 04
4.3.3 Potential hazards 105
4.4 Mining industry 108
4.4.1 Current activities 108
4.4.2 Future prospects I i4
4.4.3 Potential Hazards 115
4.5 Pollution control inCustry 118
4.5.1 Current activities 118
4.5.1.1 Biodegradation of organic
substances 120
4.5.1.2 Denitrification and
desulfurization 123
4.5 J .3 Toxic metals 130
4.5.2 Future prospects 130
4.5 J Potential hazards 133
5. SUMMARY AND CONCLUSIONS 137
5.1 State of *he applied genetics inCustry 137
5.2 Overall assessment of risx 139
5.3 Recommencations to the EPA 141
BIBLIOGRAPHY 143
GLOSSARY ; 56
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FIGURES
Number Page
1-I Steps in the process of gene expression 6
2-1 Steos in conducting a typical recombinant
DMA experiment 13
2-2 Steps in the process of generating hybridomas i 7
2-3 -eatures of a contaired fermentor 26
3-1 Government agencies end reccmDinont
DNA activities 45
4-1 Alternative methods for insulin production 62
4-2 The construction of a bacterial plasmid coding for
The synthesis of human growth hormone 64
--3 Tine extraction of useful chemicals from clgce 82
4-4 Steos in a tyoical *ermentation process 92
4-5 Product recovery from a batch fermentation 93
4-6 Steos in the conversion of biomass to ethcnol ...... .97
4-7 The conversion of lignocelluiose into useful
chemical feecstocks 98
4-8 A single-tank digester for biogas production • 100
4-9 Leaching bacteria: organisms and ocsic
metabolism 109
4-SO The carbon cycle 1.9
4-1 I Degradation pathways of severci pnenolic compounds . . 122
4-12 The biological nitrogen cycle 127
4-13 Microbial conversion of dibenzothiophene 129
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TABLES
Nurrber Pcqe
3-1 A few academic scientists engaged in genetic
engineering research; commercial affilia-
tions (not inclusive) 31
3-2 U.S. companies engaged in applied genetics R&D .... 33
3-3 roreian companies or government cgencies
engaged in applied generics R&.D 55
4-1 Examples of pharmacologically GCtive natural
products isolated from microorganisms 69
4-2 Commercial uses of enzymes 78
4-3 Organic compounds obtainable by ferrren*ction 79
4-4 Examples of useful chemicals derived from olants . ... 83
4-5 Droducrion of amino acids from glucose 87
4-6 Some species of algae tha* oroduce hydrocarbons .... 102
4-7 Mi nereis readily leeched by bacterid action II!
4-8 Strategic minerals and U.S. dependence on
foreign sources i I 7
4-9 Microbial degradation of organic pollutants ! 21
4-10 Type reactions for transformation of chemicals
of environmental importcnce 12-
viii
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SECTION t
INTRODUCTION
I. I Scope of the report
The report is organized into f've sections. Sections I and 2 provide, respect-
:vely, historicci background for and a description of the techniques uti'ized by
the apolied genetics industry. Section 3, entitled Interested Parties, orovides a
listing of academic, government, and commercial concerns that have a stake in
the applied genetics industry. Both foreign and domestic concerns are included.
In Section k we present a detciled account of the various projects and activities
currently underway or planned withir the cpplied genet:cs industry, ond we also
identify and assess the environmental and health hazards posed by this new
industry. The information in Section k is presented according to industrial
sector. The following industries are examined:
• pharmaceuticals
« industrial chemicals
• energy
• mining
• pollution and waste management
Section 5 summarizes our general understanding of the sources anc mcqnitudes
of the potential adverse environmental and nealth effects of aooiied genetics.
Questions requiring further stucy are identified, as are possible areas of future
concern to the EPA.
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1.2 Brief history
The purooseful manipulation of neredi*ary information in olants and animals by
humcns, as well cs the expioitction of microbial orocesses, has occurred since
the time mankind formed societies. An understanding of the biological nature of
these processes has been ccquired only recently (i.e., during the past 100 years),
and their chemical basis has been unravelled even more recently (in the post 35
years). A variety of terms are now employed to encomoass this field of
knowledge, including applied genetics, biotechnology, bioengineering, and
genetic engineering. While recognizing *hat these general terms connote subtle
differences in scope, we will use them interchangeably in this reoort. However,
certain Dioengineering orocedures, such as recombinant DNA technology, sntcil
specific activities that require more careful definition. These techniques are
described in detail in Section 2 of the reoort.
Examples of genetic oractices and micropial processes tha* have ancient origin
include the following: c'cohol fermenration, cheese production, focc crop cnc
domestic cnimal breeding, crop rotction, and the use of human and animc. wastes
cs fertilizers. The utility of animal and plant breeding and se'eC'on was long
ago recognized as c controlled .method of generating improved strains of v:tc
food crops and hcrdier domesticated cnimcis. This ancient rea'izction ikely
arose from the observation that children fenced to oossess varous features
characteristic of each of the parents, although *he reasons for these similarities
were unkown. Alcohol and cheese fermentations were undertaken long before
the microbial basis for these processes wcs recognized. Likewise, occcsionai
plcnting of fields w!*h leguminous crops, such as soybecns, oeas and alfalfa,
proved to be a nelpful, often crucial, means of rep'erishing spent soi; before it
became known that bacteria were responsible for this outcome by virtue of their
cbility to convert atmosDher:c nitrogen into uscble, chemically reduced forms,
such as ammonia. This is the orocess of nitrogen fixation. Ana, 'ostly, ignorance
of the role of soil bacteria in recycling human end animal sclic wastes did not
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prevent ancient cultures from employing this rich source of nutrients to improve
crop production.
The biological basis of these various processes was recognized beginning in the
latter half of the nineteenth century. Two separate findings were essentia! to
the genesis of this understanding. First, during the years .'856 to '868, an
Austricn monk named Gregor Mendel demonstrated in his experiments with peas
that numerous observable traits, such as flower and seed colors, ere passed from
parent to offspring in.the form of discrete units of heredity and thct ecch ocrent
supplied independent traits. These revolutionary findings, which were ignored by
the scientific community until early in the twentieth century, orovlde the basis
for the gene theory of inheritance, which states that the multitude of traits that
constitute an individual organism are expressions of discrete hereditary units,
called genes. In higner organisms, these genes are located on chromosomes
within the nucleus of ecch eel'. In lower forms of life, such as bacteria, which
lack a defined nucleus, the chromosomes nevertheless consist of genes, in all
life forms, genes provide the information that determines the make-up of the
organism itself, cs wel! as- the means wnereby trcits are ex*endec to the rex*
generation.
The second fundamental discovery that led *o an understanding of the biological
nature of ancient endeavors in the rearm of coplied genetics was that of Louis
3asteur. In i860, he cemonstrated thct alcohol production *'rom :ermentab!e
substrates depended on the presence of viaoie microorganisms cahed yeasts.
This finding provided The initial example of a living microbe performing a
commercially useful process. Today's genetic engineering 'ncustry holds the
promise that many thousends of commercially useful products ana processes w>T
result from aop!'cations of recent discoveries :n biology that owe *heir heritage
in oart to the findings of Mendel and Pasteur.
The chemical basis of genetics was uncovered only recently. Although DNA
(deoxyribonucleic acid) wes located in cell nuclei in 1869, Its role cs the oecrer of
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genetic information was not revealed until by Oswald Avery and co-workers.
They demonstrated that pure DNA isolated from viru;ent pneumococci bacteria
was absorbed by a non-virulent pneumonia strcin and thereupon -transformed to
the virulent form. Further substantiation of the genetic function of DNA was
provided in 1952 by A.D. Hershey and M. Chase, who racioactively labelled both
protein and DNA constituents of bacteriophage viruses. (Sacteriophcge are
simple viruses that infect bacteria; they consist solely of a protein coat
surrounding a DNA core.) Infection of susceptible bacteria by these radiolabeled
viruses resulted in the finding that viral DNA is necessary end sufficent to
mediate the infection. Vira! protein is not required.
The cbove-rrentioned studies confirmed the role of DNA cs the bearer of genetic
information in living systems. It is r»ow well-established that DNA atone serves
this purpose in all forms of life, both plants and animals, both primitive end
advanced. The information contained within the chemical structure of DNA
determines to the fu'l extent the biological nature of the organism (i.e., its
cppearance and its life functions). (The only exception to the universality of
DNA as the genetic material is certain viruses, called retroviruses, that employ
ribonucleic acid, or RNA, in this role. Although they constitute an exceedingly
small proportion of the total biota on the planet, these viruses ere important
because they induce ma'igncnt tjmors in mamma's including, prcbaoly, humans.
Even so, retrovirus RNA is copied into DNA during *he process of infection.)
Knowledge of the chemical means whereby DNA maintains end replicates the
cell's store of genetic information evolved during the >950's and I9o0's. Many
scientific investigators contibuted during this t:me to this advenes in under-
standing, but several steps in particular bear mentioning. In !?53, James D.
Watson and Francis Crick proposed a double-nerical structure for DMA. This
model readily suggested a mechanism whereby DNA couid Pe faithfully repro-
duced. During the mid-1960's, Arthur
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code determines now the sequence of chemical constituents in DMA is translated
into a specific sequence of amino acids (via a nuc'eic acid intermeciate called
messenger RNA, or mRNA). Amino acids are the chemical building blocks of
oroteins which, in turn, provide structural integrity end mediate metabolic
acitivites within every cell of every orgenisrr. The steps in the octhway from
DNA to protein are diagrammed in Figure l-l.
This basic research in moleculcr biology and genetics paved the way for
developments during the I970's that have giver rise to *he techno'ogy of
recompinant DNA. These later achievements ard procecures wii) be detailed in
Section 2 of this report. It must be recognizee that the modern field of aop^'ec
genetics, with all its promise for future benefits to mankind (end :ts ootential
dangers), could not exist today but for the numerous accomplishments in basic
research in biology and biochemistry over the past severci decodes, only a few of
which are mentioned above.
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Figure 1-1
Steps in the process of gene expression
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SECTION 2
TECHNOLOGY OF APPLIED GENETICS
Applied genetics as prccticed by cncienf societies involved c minimum of humcn
intervention and consisted of little more than a'lowing ncture to take its course.
Thus, alcohol and cheese fermentation and the recycling of wastes, orocesses
that we now know to be mediated by microorganisms, were undertcken merely by
exposing the appropriate raw materials to the environment, whereupon c
transformation of the substrates took place. ControHec animcl and plant
breeding was imDlemented by piecing prospective pcrents in proximity to one
another. Ancient bioergineering technology, therefore, succeeded by virtue of
men's ability to manipulate crudely the biology of nis environment.
By contrast, the emergence of modern biotechnology as a scientific discip'ine
thct holds enormous potential for benefiting mankind stems from our recently
acquired ability to comorehend end mcnipulcte the chemistry of iving systems.
Thus, the currently popular notion that modern society is embarking on the "Age
of Biology'1 coulc be sightly repnrased to become the "Age of Biochemistry."
The modern technology of aoplied genetics encompasses a variety of procedures
and processes. Each of these will be dealt with separately in *he remainder of
this section.
2.1 Recombinant DNA techniques
Recombinant DNA technology refers to the ability to isolate frcgrr.ents of DNA
from seocrate sources and to splice them together chemicc.ly :n+o a functional
unit. The DNA fragments can derive from the seme organism or from d:fferen*
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organisms in the same species (techniques that have considerable future poten-
tial for gene therapy applications in humans), but the currently most promising
technique involves the joining of DNA segments from disparate species of
organism, such as bacteria and humans. This latter approach has been utilized,
for example, in recent efforts to mass-produce human interferon, c drug that
may combat viral diseases and ccncer.
A review of the recent developments in molecu'ar biology that hcve led to the
emergence of recombinant DNA technology ccn best be presented by considering
those specific laboratory procedures necessary to carry out such experiments.
There exist six distinct phcses in the orocess.
(1) Isolation and purification of DNA
Since DNA exists nature-ly as c long, fragile, chain-like structure, techniques for
gertly iso'ating extended sequences containing intact genes were neeced. Such
procedures, wh'ch include high-speed centrifugation and electrophoresis, were
developed durirg the early 1960's, largeiy by Julius Marmur and colleagues.
(2) Fragmentation of DNA into reassociable segments
This crucial step is mediated by a c;ass of bccterial enzymes, celled restriction
endonucleases, that introduce widely spcced breaks at specific sites in the DNA
chain. The nature of the cuts is such that the seccrated ends (so-ca:led "sticky
ends") can readily reassociate with one cnother, thereby regenerating the
original cleavage site. The rejoining can involve two DNA segments thct each
cerive from different sources, sc «ong as the DNA from ecch source was cMooed
into fragments by the seme restriction endonuc lease. Discovery of these
enzymes and elucidation of tneir physiological ro!e are !crgeiy crecited to
Werner Arber in Sw:tzerland and *o Dan Nathans and f—ami I ton Smith at Johns
Hopkins.
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(3) Sealing DNA fragments together
The rejoining of DMA fragments by way of their sticky ends reauires a further
step for the full stabilization of the recombined unit. Another enzyme, called
polynucleotide ligGse or simply ligase, performs this function. The ligase
enzymes were discovered independently by c number of investigctors, including
Malcolm Gefter at MIT and Arthur Kornberg at Stanford.
(4) Replication and maintenance of recombinant DNA molecules
Once DNA fragments have been cut-and-sp!iced together _in vitro, a suitable host
organism must be found into which the recombinant DNA can be stcbly incorpor-
ated end reproduced. The enteric bacterium, Eschericia col i, (or E. coli), was
The obvious first choice as c host since more is known cbout the genetics and
molecu'ar biology of this microbe than of any other organism. The reolication of
a DNA segment by E_. coli requires that the segment contain a specific short
sequence of DNA that serves as a signal to the enzymatic machinery inside the
cell. This signal, somet:mes celled the origin of replication, can be found on
certain small, self-rep'icating loops of DNA, called plasmids, that are commonly
found inside bacterial celts. (Plcsmids reproduce themsefves indeoendent'y of
the major chromosome 'n bacteria and they are readily transferred between
different bccteriai strains. !n eddition to other functions, piasmids are respons-
ible for the resistance to numerous antibiotics that has become a major medical
problem in recent years.) Thus, incorporation m vitro of the recombinant DNA
molecule into a bacterial plcsmic, followed by reintroduction of the hybrid
plasmid 'nto the bacteric! ceil, will permit stable reolication of *he recombinant
DNA.
Alternatively, :f *he recombinant DNA could be incoroorated into the major
chromosome of +he host bccterium, then it would be repl:ccted as pert of thc't
chromosome. This is possible through the use of a particular bacteriophage,
called lambda, that infects E.. colUpon infection, lamoca DNA becomes
incorporated :nto the bacterial chromosome wnere it replicctes ciong w;th 'he
host chromosome. Thus, attachment of the recombinant DNA molecule to
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lambda DMA prior to the infection of E. cofi wi«I similarly allow replication of
the recombinant DNA.
8oth plasmids and lambda bacteriophage ere termed vectors owing to this ability
to transfer recombinant DNA into suitable hosts for replication. A number of
scientists pioneered the effort to demonstrate the usefulness of vectors in
gaining expression of exogenous or foreign DNA in E. cofi, including Stcnley
Cohen and 3aul Berg at Stanford, and Herb Qoyer at the University of Californic,
San Francisco.
There exists c direct method of putting foreign DNA into host bGcteria witnout
the need for intact viruses or plcsmids. ^ure, naked DNA can be absorbed by
bacterial cells :n a process called transformation, "his is the procedure used by
Avery and co-workers in 1944 to "transform" r.on-virulen1, pneurrococcus strains
into virulent bacteric. Some bacterial strains, including E. coli, must undergo a
simple chemical pre-treatment with calcium salts in order to moke Them
amenable to DNA uptake.
(5) Selection of ceHs containing recombinant DNA
Since only a small percentage of potentia' host bacteria do in fact acquire
recombinant DNA by way of these procedures, it is necessary to perform a
selection step. Depending on the type of vector used, :t is possible to screen for
cntibiotic resistance (when the vector is a plasmid containing an antibiotic
resistance gene) or to screen for the presence of viable bacteriophage viruses
(when lambda is used as the vector). These selection methods give rise to clones
of bacterial hosts containing recombinant DNA;. tha* is, each bacterium in the
clone is derived from a single progenitor cell that mu/t'oiied repeatedly, with
exact copies of the ced's DNA having beer distributed inTo eccn dcugnter eel*.
The segment of recombircnt DMA contained therein is also replicated; thct is, it
has been c'oned.
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(6) Expression of recombinant DNA into gene products (proteins)
The recently acquired ability to incorporate exogenous DNA into bacteria, ard to
have that DNA replicated as though part of the bacteria! genetic complerrent, is
of considerable scientific interest. 3ut commercial applications of this new
technology demand that foreign genes implanted into bacteria be expressed into
the proteins encoded by that DNA. For example, in order to convert coli into
"factories" capable of producing human insulin, it is necessary both that the gene
for insulin is stably maintained in the bacteria and that the human DNA segment
is transcribed into messenger RNA, then translated into insulin (see Figure I.I).
As mentioned above, gene replication (maintenance) is assured by the presence
of certain genetic signets. Similarly, the processes of transcription end
translation rely on signals thct inform the cell's enzymatic machinery where to
start and where to 4erminate ecch of these processes. A)! of these various
signals must be present at the appropriate locations in the DNA in order for gene
expression by recombinant DNA methodology to be successful.
Once a bacterial cell has been "tricked" into manufacturing c human or other
foreign protein, additional problems arise, "'"he bacterium may recognize insulin
as a "foreign" protein and may degrade it before it can be recovered. stcble,
¦""he foreign protein may simply cccumulate inside the bacterial cell,
necessitating its recovery by breaking open -he cells—a tedious and inefficient
orocess. Ideally, the foreign protein wi'l be excreted out of the host cell info the
growth medium from which it ccn be readily purified. Clever techniques are now
available to bring "his about, and improvements ere being made continuously.
One additional roadblock bears mentioning. Many human proteins possess
attachments that consist of sugar molecules. These glycoproteins are especially
common in b'ood serum; e.g., interferon is a glycoprotein, although insulin is not.
Bacteria do not possess the machinery to syrthesize or attacn sugars to proteins.
Although the precise function of the sugars in unclear, it is probable tnct they
serve a useful, pernaps crucial, role in maintaining the physiological activity of
the protein. Thus, considerable effort is underwey to develop microbial host
organisms that car attach sugars to proteins. Common brewer's yeast, or
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Sccchcromyces cerevisiae, is likely to be the preferred host ceM for this ourDOse.
Although it is a single-celled microbe, yeasts berong to the general clcss of
nigher organisms that include humans, namely eukaryotes. Eukcryotic organisms
are classified on the basis of their hcving a nuclear membrane surrounding the
genetic material within each cell. Bacteria end certain algae, on the other hand,
compose the class of organisms called prokaryotes (i.e., those lacking a defined
nuclear membrcne). Altnough researchers in recombinant DNA have oredomin-
ately utilized E_. coli as the host organism, there is no doubt that the future
commercial success of the technology hinges on the increasing use of eukcryctic
hosts such as yeasts and fungi.
A general scheme showing the steps involved in a recombinant DNA experiment
is diagrammed in Figure 2-1.
2.2 Genetic alterations induced by non-recombinant DNA procedures
A number of techniaues are currently utilized to induce gene*ic cirerations in
cells. The techncogy of recombinant DNA reoresents the most recently
developed and the most glamorous of these procedures, end it holds the powerful
advantage that the outcome of these alterations can be predicted and controlled
to a greater extent than with other techniques. Nevertne'ess, other gene-
alter irg orocedures ere available, several of which had beer in use for mcny
years orior to the aavent of recombinant DNA tecnnoloay. These alternative
methods will be described next.
2.2.1 Induced mutations (mutagenesis)
"he DNA of all living ceils is continuously undergoing s.ight changes in its
composition as a consequence of irterccting with its external environment.
These alterations, called mutations, ere thought to oe the driving force for the
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Figure 2-1
Generalized scheme depicting the steps in conducting a recombinant ONA experiment
E ~ colx plastnid
recombinant plasmid
plasaid ,oy
trans formation
chromosome
transformed E. col:
^replication^
?§Q) fo
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evolution of organisms into new species and, under natural conditions, they occur
at a low rate. Under experimental conditions, however, agents that induce
genetic mutations, called mutagens, can be administered in order to cccelerate
greatly the rate of mutation. This is the process of mutagenesis.
A variety of mutagens are used experimentally and commercialiy to induce
mutations. !n general, mutagenic agents operate by interfering with the normal
cellular processes involved in the repair of DNA. (Healthy cells maintain this
enzymat:c system for fixing mutations that arise from natural.'y occurring
sources.) Ultraviolet (UV) radiation and chemical cgents such as nitrosoguani-
dine and acridine ere the most commonly used mutagens.
The induction of mutations by methods such as these is highly non-specific; that
is, the experimenter cannot control the genetic site at which the mutction will
occur. Therefore, following the mutagenic step, it is necessary to conduct a
selection for those mutated organisms that oossess the desired traits. For this
reason, commercial mutcgenesis is feasible only with organisms that have a
relatively short generation time, such as microorganisms. Since a single
bacterial ceH will grow to a visible colony within a few days, it is possible to
observe the effect of the mutagenic procedure in short order. Moreover, many
thousands of such colonies can be screened simultaneously. Nevertheless, a
mutagenic procedure was recently described involving plant cells growing in
tissue culture. This advance suggests that genetic alterations in olants generally
wilt become feasibte by way of induced mutations.
Mutcgenesis methods have found widespread utility in the pharmcceutical indus-
try to enhance production of substances from microbia? sources. Particular
success has been cchieved with the antibiotic penicillin, which derives from c
strcin of mold, and qentamycin, which is orocuced by a bacterium of the
Streptomyces species. UnWke recombinant DNA methods, mutagenesis is incap-
able of encowing the microbe with orooerties that :t does not crready possess.
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That is, no new genetic material is introduced; rather, existing capabilities are
enhanced.
2-2.2 Celt fusion methods
A method whereby one cell type can be endowed with properties of another cell
involves fusing those two ceils together into a single unit. This procedure is now
commonplace in the experimental laboratory and has been applied to a variety of
eel's from microbes to men and from both plants and animais. The methodology
is relatively inexpensive and is essentially the same regardless of the eel1 type
involved. Two technical approaches are in genera! use.
(I) Monoclonal antibodies (hybridomas)
Mcmmals have evolved a comoiex intemcf system of defense agairst -oreign
'ntruders, such as bacteria and viruses. This immune system functions in part by
producing proteins called immunoglobulins, or antibodies, that specifically recog-
nize and eliminate these alien invaders. A typical immure response to a
bacterium, for example, consists of a great variety of different antibody
molecules, each capable of recognizing ard binding *o a specific antigen on the
surface of the microbe. Each of these distirct antibody types is manufactured
by a clone of antibody-producing cells. These ceils are called lymphocytes and
since numerous clones of lymphocytes are eacn reacting to the presence of the
bacterium, the response is termed polyclonal.
Antibody preparations (antisera) have ;ong been used to great advantage as
diagnostic agents. Immunodiagnostic assays currently comprise approximately
one-fourth of al! tests performed in the clinical laboratory. Such assays are
helpful in rapidly diagnosing bacterial or viral infections and in monitoring drug
or hormone levels in blood and urine.
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From a physiological standpoint, polyclonal antibody responses to antigens are
highly advantageous since they ensure that the individual will effective'y repel
foreign invaders. But to the clinical chemist, the diversity of antibodies can be
bothersome since closely related antigens may not be distinguishaole using these
conventional antisera. In 1975, a technique was described by Cesar Milstein in
Cambridge, England, that permits the generation of monoclonal antibodies; that
is, immunoglobulins derived from a single ceHular source or c single clone of
cells. Such antibody molecules are all chemically equivalent to one another.
The technique simply involves mixing antibody-producing celts (lymphocytes)
with cells from a type of tumor, celled a myeloma, that are themselves derivec
from lymphocytes. A fusing agent is added that partially dissoives the
membrane that surrounds both ceil types, thereby permitting contiguous cells to
merge together^ A common organic poiymer, polyethylene glycol, serves as c
satisfactory fusing agent. After removal of the fusing substcnce, the fused cells
are grown in tissue culture (see below) and desired crones are identified by c
suitcble selection procedure. Such a clone combines the qualities of the two
contributing cell tyoes: it secretes a specific, monoclonal antibody and it grows
continuously and raDidly owirg to its tumor-like properties. This duel capabHity
is refected in the term hybridoma, which is eppfied to a clone of cells secreting
monoclonal antibodies. A diagram of the steos involved in generating hvbrid-
omas is shown in Figure 2-2.
The initial demonstration of the hybridoma technique end subsecuent commer-
cialization of the orocess have involved ceils derved from laboratory mice. The
technology was recently extended "o the use oi human lymohocytes. This
advance will soon read to moncc-'onat antibodies for jn vivo diagnostic and
therapeutic uses.
(2) Protoplast fusions and plant tissue culture
The second general class of fusion techniques involves microorganisms or plant
celts, "hese cell types possess a rigid, orotective cell wall that surrouncs the
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Figure 2-2
Steps in the process of generating monoclonal antibodies
ANTIGEN
SPLEEN
f* ANTIGENIC
• determinant
LYMPHOCYTES MVES.OMA CELLS
'©
©O
r USE
1Q
HYBRID-
MYELOMA
CELi_S
\
CLONE
w
V
V
%
^ P
monoclonal antibodies
Source: Mil stein, C. (1580)
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cell membrane. (Animal eel's lack cell walls.) The wall from such eel's can be
readily removed using enzymes that specifically digest the cellulose-tike sub-
stance that comprises them. The spherical, membrcne-surrounded entitv thct
remains is calred a protoplast.
Using methods essentially equivalent to those described for hybridoma produc-
tion, protoDlasts can be fused together generating hybrid cells that exhibit
properties in common to both the contributing cell tyoes. Examples of the
application of fusion methods tc microorganisms include efforts to: (I) improve
the antibiotic y:eld from Streptomyces strains; (2) analyze the genetics cf
brewer's yeasts; and (3) develop hybrid strains of fungi from the Asperqil lus
family to enhance citric ccid production.
The application of protooicst technology to plcnts is a relatively recent develop-
ment, but one that promises :o revolutionize the food, agriculture, end forestry
industries, and to have considerabfe impccts on the energy, chemical, anc
Dharmaceuticci industries. Scientists are now able to regenerate full grown
oiants from singfe eel's or protoplasts. So fcr, only a few SDecies of plant have
been successfully cultured in this way, including tobccco, the Dougias fir, and
ccrrots. But rGpid advances in this field shou'd soon make avai'cble this
technology for most plants of commercial interest. This capability wi'l occasion
several advantages:
• mass production of clones of identical plcnts, each
having the improved qualities of the origincl Daren:;
• rapid growth of the plant in tissue cu'ture to the
seedling stage of development, thus shortening gen-
eration times:
• a ready-made _jn vitro system for conducting genetic
engineering in p;arts.
A great variety of plants produce chemical compounds thct are highly useful to
man. These compounds include drugs (such as digitalis, vitamins, s'eroids, ard
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anti-ccncer agents), rubber, and petroleum substitutes. The advent of plant ce'i
and protoplast tissue culture technology makes possible the large-scale ferment-
at;on of plant cells in much the same fashion that microorganisms are currently
grown in bulk. Useful plant products would be excreted into the growth medium
and readily isolated. Future processes of this type wiM obviate the necessity of
devoting large tracts of arable land to cultivation, and production costs should
plummet.
The-eelf fusion orocedures described in this section, both for plant and for animal
ce.'ls, depend greatly for their success on the technigues of _in vitro cell culture.
Scientists have known for some time how to explant eel's from particular
organisms or tissues and to keep them alive for limited durations under sterile
conditions in an incubator. Various nutrient media have been formulated and
growth conditions established for a wide variety of plant and animal cells. A
serious drawback to the large-scale commercial use of eel I culture technology is
its high cost, but future widespread industrial aDDlication, which seems likely,
will introduce economies of scale, and continuing refinements in the technigues
should lower costs.
2.2.3 Other gene-altering techniques
Several other methods exisr for establishing new genetic mater'a! in microbes
and in cells of higher plants and animals. Some species of bacteria possess c
natural abPitv to exchange DNA by way of a process carled conjugation.
Extrachromosomaf DNA, or pfasmids, is especiclly mobile and is transferred
between bacterial strains with considerable ease in some cases. (The ab;l:*y of
bacteria to develop resistance to many types of cnt:biotics is due To genetic
information 'ocated on plcsmids. Since these plasm ids move about so freely, c
number of bccteria' strains oathogenic to man have become relatively refrcctory
to antibiotic treatment.) -Masmids encoding distinct functions and resiclna in
different bccter:al strains can be combined into a single bacterium. Such c
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"superbug" was created by Chakrcbarty, who at that time was working at
General Electric. Plasnids from several strains of the species Pseudonnonas,
each capable of degrading a oarticular constituent of crude petroleum, were
combined into a single cell, enabling the strain that resulted to digest several
components of crude oil. This modified bacterial strcin became the subject of a
controversial pctent application, the litigation of which eventually reached the
Supreme Court (see Section 3.1.3.3).
An alternative to conjugation, which involves bacterium-to-bacterium transfer
of DNA, is the process of transduction, in which viruses serve cs transmitters of
genetic material. When a virus infects a celt, normal metabolic activities cease,
and processes are undertaken to mass-produce new virus particles. Part of this
process involves replicating virus DNA and packaging it into protein shells.
Occasionally, small portions of host cell DNA are carried along :nto the virus
sheds. After production of sufficient numbers of mature viruses, the host cell
bursts, releasing the virus particles to initiate another round of infection. Cells
infected in this second round will receive, in addition to virai DNA, the oortion
of DNA derived from the original host. Scientists have learned how to
manbuiate these processes so that soecific DNA secuences (genes) are trans-
ferred, thereby endowing the recipient celts with properties previously inherent
only to the initial hosts. The utility of transduction as c mecns of producing
genetic alterations is well established using bacteria and bacterial virjses
(bacteriophages). Recently, this general procedure has found application among
higher animals. Considerable experimental work is being devoted to oerforming
transduction in primctes (e.g., monkeys) using the virus SV ^0 ("SV" stands for
"simian virus"). The eventual success of these studies has profound implications
for genetic engineering in humans, with the prospects of curing genetic diseases,
such cs sickle cell anemia.
Genetic engineering in plants promises to be greatly stimulated in the years
ahead owing to the existence of a bacterium called Agrobccterium tumefaciens.
This microbe infects plant cefls, giving rise to a plant 'umor, called a crown gall.
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The agrobacter ium perpetrates this deed by transmitting to the plant cell c piece
of its own genetic material, called T-ONA, which is Dart of a piasmid, namely
the Ti-plasmid (for "tumor-inaucing" piasmid). Copies of the T-DNA are
incorporated permanently into the picnt genes, end the agrobacterium is no
longer needed. This instance of naturally occurring recombinant DMA provices a
potentially very powerful tool for introducing foreign genes into plants.
2.3 Immobilized bioprocesses
Several techniques have evolved in recent years that have managed, to some
extent, to exploit cellular biological processes on cn industrial scale. "These
methods generally consist of confining, or immobilizing, intact cells or cellular
enzymes within an irer* matrix, followed by passage of substrate materials
through this bioreactor. Chemical reactions (i.e., byconversions) then take place
that transform the substrate into more useful or less toxic products.
Enzymes are proteins that catalyze the chemiccl react'ons of living cells. Like
most proteins, they are reiativeiy jnstabie ana tend to lose their activity when
exposed to denaturing conditions, sucn cs hect, extremes of pH ard salr
concentration, the presence of surf ace-active agents (detergents) or heavy
metals, and so forth. Immobilization procedures generclly serve ro protecT
enzymes from denaTuration, thereby lengthening Their useful lifetimes. A ,crge
number of inert support materials have been *ested for various applications,
inciuCing naTural ana man-made polymers, such as cellulose, starch,
polyacrylamide. chirin, polyethylene, glass, end collagen. Enzymes can be eitner
linked securely to the surface of the polymer or entrapped within a porous
microcapsule. In order to maximize the reactive surface area, rhe support
matrix can be fashioned into riny oeads or hollow f'bers or semi-permeable
membranes pr;or to affixing the enzyme. In all cases, successful operation of
the oioreactor depends on maintaining a securely fixed, active orepcrar'on that,
nevertheless, permits free movement of the substrates and products.
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Current and potential applications of this technology are vast and will affect
many industrial sectors. A few examples include:
• Chemical industry
—alkene oxide production from corresponding glycols
—surfactant production from glycerices
—hydroxylation of carboxylic acids
—amino acid synthesis
• Energy industry
—hycrogen production from water using chloropicst enzyrr.es
—desulfurization of crude oi;
—biomass conversions into methanol or ethanoi
• Medical industry
—production of urocanic acid, a sunscreening agent
-inter-conversion cf various penicillin derivatives
—steroid derivitizations
— clinical analysis of blood and urine const:tuents (e.g.,
urea and glucose) by electrobiochemical reactions
— synthesis of the antibiotic Gramacidin
• Pollution control industry
—conversion of lignocellulosic wastes into useful products,
such as glucose
— biodegradction of toxic substances, such as PC3, kepone, di
oxin, DOT, phenols
—concentration of toxic heavy metcls in waste streams
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—air disinfection (e.g., for hospitals) using enzymes that
destroy viruses and bacteria
—conversions of whey (waste product from dairy industry) to
useful food products
— rotating biological discs for waste water treatment
-fixed-bed bioreactors for on-stream waste management
• Food end agriculture industry
— milk coagulation (the first step in cheese production) using
the enzyme rennet
—production of high-fructose syrups from starch and cellulose
for use as c sugcr substitute
—conversion of amino acid isomers to convert the nor-
nutritious D isomer into The L form
—clarification of fruit juices and wines
Future developments in this area are likely to include improved methods for
immobilizing live cells, especially microbes and plant ceils. The process of
microencapsulation promises to find considerable application here. Each
microcaosule ccn be thought of as a tiny living colony in which ceils divide ard
perform metabolic functions within the confines of the beac. Mecnwnile,
substrates pass Through the becds and are converted into procucts which flow out
of The system uncontaminated by cellular materici. Moreover, the biocaTalytic
system would be self-regenerating since the micro-colonies within ecch capsule
ere undergoing continuous turnover; thct is, dead cells ere alwcvs being replaced
by live cells. This form of reccTivation never occurs with immobilized enzyme
systems since isolated enzymes ere not cdDCble of self-re;uvenat;on and, upon
inactivGtion, must be replaced.
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Another likely development in the area of immooilized bioprocesses is the
increased use of enzymes isolated from thermophilic bacteria. These microbes
are remarkably insensitive to high temperatures, even up to 80° or 90° Celsius
(water boils at I00°C). Thermophiles can be recovered from hot springs or other
similar environments. They owe their heat resistance to having enzymes that
are extremely insensitive to heat denaturation. Thus, these enzymes are
considerably more stable than comparable enzymes from mesophilic orgcnisms
and are ideal for immobilization processes.
2.4 Fermentation technology
Commercialization of processes reliant on recombinant DNA or other modern
biotechnologies will frequently entail large-scale microbial fermentations. In-
dustrial fermentations have been carriea out with great efficiency for many
years and have made available at low cost such products as antibiotics, flavoring
and coloring agents, amino and organic acids, and vitamins. The expectation that
new drus, such as interferon and human insuiin, will soon be mass-produced
deoends to a large extent on the ability of the fermentation engineers *o adapt
the appropriate microorganisms for growth in quantities vastly greater than
those encounterec in the laboratory.
A standard aerobic fermentor consists of a c;osed, cylindrical vessel equipped
with a stirrer and internal baffles to provide agitction, heat exchangers to drain
off the considerable hect generated during fermentative growth of the microbial
culture, an cerator, and one or more inlets for media sampling and harvesting
and exhaust gas removal. A device for rapid steam sterilization of the vessel is
essential, as are controls designed to monitor and cdjust growth conditions, such
as tempercture, air flow, oressure, pH, and degree of foaming. Crucial to the
overall design is that no foreign microorganisms gcin access to the system; all
comoonents are sealed to prevent leakage and ccn oe steam-sterilized between
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batches. The fermenfor can be of any convenient volume, ranging up to about
100,000 gallons, in which the media alone would weigh more then 400 tons.
A fermentor designed by Eli Lilly for large-scale growth of recombinant DNA
organisms is shown in Figure 2-3. Incorporating design features such as exhaust
gas filtration and double agitctor seals, this reactor exceeds the safety and
containment specifications of typical fermentors and, as such, sets the standard
for fermentors designed for use witn recombinant DNA organisms.
The fermentor described cbove carries out a batch fermentation; tnct is, the
media and microorganisms are mixed, the microbes grow for a fixed period
(usually one to seven days depenaing on the organism and the conditions), then
rhe culture is harvested. After cleaning and sterilizing, the fermentor is ready
for another batch. Following completion of the batch, the fermentation product
must be isolated from the culture system. If the microbe excretes rhe cesirec
product into the growth medium, as is preferable, then the culture broth must be
processed following removal of the microbial population. On the other hand,
products that cccumulate within the microoe must be recovered by lysing the
microorganisms after ciscarcing or recycling the culture liquics.
Fermentction technology has advanced in at least two ways in recent years. It is
now possible to conduct continous fermentations in wnich growth mecia are
added slowly througn the growing phase of the microbial culture. At the same
time, smail port:ons of the culture are contirually removed from the fermentor
for processing. The possibility of inadvertent contamination would seem to be
greater for this continuous method, but efficiencies are greater since downtimes
oetween batches are eliminated.
A second advance :n this technology is called solid phase fermentation. In this
process, nutrient media are trickled through a reactor consisting of a solid
support matrix to which the microorganisms are steadfastly attached. Tne
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Figure 2-3
Features of a contained fermentor
Exhaust gcs
f;iter system
Source: E'i Lilly anc Cc.
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microbes continuously excrete the product of interest into the broth which
eventually emerges at the bottom of the fermentor. The broth is then processed
to isolcte the *ermentation product. .Clearly, solid Dhase fermentation methods
are not applicable to the mass-production of substances that accumulate inside
the microorganisms.
2.5 Gene therapy
Perhaps the most exciting topic in the field of applied genetics, and lixely the
most controversial, is the orobcbility that medical scientists will soon be aole to
perform genetic engineering in humans. Society in general views this prospect
with a mixture of hope and skepticism, and cssurcnces have been given that this
capability is many years away. But recent scientific developments suggest that
this future may be here quite soon.
Considerable attention was recently directed to the efforts of a team of UCLA
scientists, headed by Martin J. Cline, who traveled to Israel end Italy to conduct
experiments on human subjects. These experiments were deemed toe prelim-
inary to be performed in the United States. Those patients who were trected
suffer from a genetic blood disease, called beta-thalassemia, in which production
of one of the two protein components of hemoglobin is almost negligible. The
therapy attempted to insert copies of normal genes for hemoglobin into eel's of
the bone marrow, where hemoglobin is synthesized. The experiment is given
very little chance of success, but the mere fact that it was attempted, added to
the fact that a similar experiment succeeded in laboratory animals, hints
strongly that some primitive form of gene fherapy in humans will short'y be
possible.
In another recent develooment, scientists transplanted cell nuclei from ecrly
embryos of mice into fertilized eggs isolated from c different mouse strain.
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After severe! days in tissue culture, these new embryos were inserted into the
uterus of a third mouse. These embryos developed into normal infant mice that
were related genetically to the mouse that origncily donated the cell nuclei.
This outcome has been hailed as the first instance of cloning in nammals. That
is, identical offspring were produced by taking cells, or ceH nuclei, from a singfe
individual and growing them up into complete, cdutt orgenisms. So fcr. it has not
been oossible to generate clones from adult donor ceils--only cells derived from
an early stage of development, such as the embryo, are suitcble. But this
stumbling b'ock may soon be overcome. If so, ond despite the claim by scientists
that these experiments are designed only to study gene expression :n mcmmcls,
then society will be faced with some very sticky ethical issues.
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SECTION 3
INTERESTED PARTIES
3.1 Domestic activities
Immense excitement has been generated in recent years by the advent of
recombinant DNA technology and the prospect that applied genetics wilt improve
the quality of life in many ways. This interest arose from findings made in basic
research labs at universities, which quickly burgeoned into a multi-million dollar
commercial industry. Activities on both fronts are expanding continuously.
Meanwhile, various government agencies have developed an interest in this area
owing, in part, to concerns for public safety arising from overly fast commerci-
alization of a technology whose safety has not been established absolutely. Thus,
all three sectors—universities, private industry, and government—are deeply
interested in the evo'ution of the cpplied genetics fierd. From a socioeconomic
viewpoint, applied genetics will provide the opportunity to cnatyze and improve
relationships between industries and universities on the one hcnd, and between
industries and government on the other.
The remainder of this section of the report will detail activates in each of these
three sectors in the United States, followed by c brief discussion of applied
genetics as practiced overseas.
3.1 Universities
Most fundamental advances in both the science and engineering aspects of
biotechnology have been made in university research labs. This fact will
continue to ho'd true for some time to come, although considerable expertise is
now being acquired by commercial firms engaged in cpplied genetics R&D.
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A few of the many academic scientists who have contributed to the foundation
of the applied genetics industry are listed in Tcbte 3-1. This list, by no means
exhaustive, includes many of those prominent academic scientists who have,
become affiliated with one or another genetic engineering firm. Several
companies were founded through the efforts and energies of university research-
ers who, nevertheless, maintained faculty status at their academic institutions.
This state of affairs has occasioned a certain degree of rivalry among university
scientists who now view their research as potentially lucrative. As a resu't, the
qualities of cooperation and intercommunication that once characterized aca-
demic research have been seriously compromised. This trend is likely to
continue for the foreseeable future with accompanying improvements in indus-
try-university relations at the expense of freedom of information flow within the
scientific community. The situation cou'd improve if private industries under-
take programs to support basic academic research on an unrestricted, ro-str:ngs-
attached basis. Commercial firms are being encouraged by Corcress •'o do so via
proposed tax credits and other investment incentives. Coroorate backing of
accdemic research hcs recently become especially desircoie since fec'erc'
sources of funds for basic biomedical research (i.e., NIH and NSF) have failed to
keep pace with growing demand.
University faculties are organizing to offer their services as technical experts in
applied genetics. Two examples are:
• Biolnformation Associates, Inc. - A group of MIT
professors from the biology, chemistry, and chemical
engineering departments who provide wide-rangirg con-
sulting services for basic and-applied research in gene+:c
engineering.
• Biotechnology Research Center - Established at Lehigh
University in Bethlehem, pennsylvania, this joint effort of
scientists and engineers provides education in biotech-
nology and conducts research 'n the areas of biomass
conversions, microbial desulfurization of coal, and im-
proved methods for waste treatment.
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Table 3-1
A few academic scientists engaged in genetic engineering
research; commercial affiliations (not inclusive)
Name
University
Affiliation
Bert O'Malley
Janes Sonner
Lerov Hood
Gerald Fink
Walter Gilbert
Philip Leder
Tom Maniatis
Matthew Meselson
Mark Ptashne
Dan Nathans
tiaTrilton Smith
David Baltimore
David Botstein
Arnold Demain
Philip Sharp
Paul Berg
Stanley Cohen
Ronald Davis
Roy Curtiss
Martin Cline
Winston Salser
John Baxter
Herbert Boysr
Anand Chakra'oar.y
David Jackson
Stanley Falkow
Winston Brill
T lino thy Hall
Howard Tenin
Baylor
Cal Tech
if ti
Cornell
Harvard
Johns Hopkins
rr
MIT
rr
rr
rr
Stanford
I r
II
Univ. of Alabama
UCLA
UCS7
Univ. of Illinois
'Jniv. cf Michigan
Univ. of Washington
Univ. cf Wisconsin
advisor to Collaborative Genetics
co-founder of 3iogen
founder of Genetics Institute
advisor to Monsanto
advisor to Cetus
advisor to Collaborative Genetics
advisor to Collaborative Genetics
advisor to Cetus
co-founder of Biogen
advisor to Cetus
advisor to Collaborative Genetics
co-founder of AMgen
co-founder of AMgen
co-
"cunder of Genentecn
on leave to Genex
advisor to Cetus
advisor to Cetus
advisor to Agrigenetics
Frank Ruddle
Yale
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Harvard University recent'/ developed, then rejected, a plGn to establish its own
genetic engineering corrtDany. This proposal evolved as a means to put the
considerable talent of the Harvard faculty to the purpose of genercting profits
for the university, rather than to serve the interests of outside commercial firms
(such as Biogen, co-founded by Harvard bio'ogist, Walter Giioert). The plan
succeeded only in generating controversy. Faculty members argued that profit
motives would add to the rivalry that already existed within the biology
deoartment and that traditional academic goals of education end research are
imcompatible with a profit-making orientction. Eventually, the plar was
abandoned, but Harvard biologist Mark Ptashne, who conceived the venture,
proceeded to establish his own firm, called Genetics Institute, Inc., loccted in
nearby Somerville, Massachusetts. Harvard owns approximately '0% of the
equity of this new firm.
3.1.2 Commercial firms
The excitemert generctec by the field of biotechnology, particu'arly recombi-
nant DNA and genetic engineering, has been felt most emphatically in the
private sector of the U.S. economy. We have identified over 100 companies
currently engaged in some aspect of modern applied genetics (see Table 3-2).
More firms are becoming involved every month. It is estimated that capital
investment :n eppfied genetics R&D reached $500 million in 1980. in five more
years, the va.'ue will be $5 billion, and in ten years, $25 bil'ion. Many investors
and business analysts anticipate that the decade of the I980's will occasion a
"biology boom" akin to the electronics explosion of the 1970's.
In general, capita! investment in biotechnology has occurred along two different
oaths. Initially-small, new companies specializing in genetic engineering were
created by young scientists/businessmen who combined keen foresight with a
propensity for financial risk-taking. These individuals anticipated the nuge
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Table 3-2
U.S. companies engaged in applied genetics R&D.
Name
GENERAL
fetus
I oral ion
Project s
Col laborat ors
(iOllPllt f'f h
Genex
Del.lu;siia Research
laboratories (Bill)
Berkeley. CA
San Francisco, CA
Rock vt lie, Ml)
Uethesda, Ml)
Co I lalMir.it i ve tienetics Walthaiii, I1A
t luotii ochem
Genetics Institute
AMgen (AppI ied Molec-
ular Genetics)
New York, NY
SomerviIle. MA
I os Angeles, CA
cthanol from biomass
alkcne ox ttiui/ f ructose
oil-related projects
interferon
si utile Cell prole irt
peptide hormones
interferon
In:111 iii
tjrowlli hormone
alpha-I-thymosin
commercial scale-up
i nterfrron
chemical processes
)ntcr fe run
research enzymes
inouoc I oria I an t i IkkI i es
hepatitis vaccine
etliannl from bimuass
mtprleion
industrial processes
qeneral biomedical
uenera I li i owe J i ea 1
general biomedical
National Distillers
Chevron
Aiiuico
Shel1 Oi1
Hoffmann-1 aRoche
Eli Lilly
A.G.Kabi (Sweden)
National Cancer Institute
Fluor
Lubrizol
MonSiilil o
flristol-hyer s
Koppeis
New York lllood Center
lireeri Cioss (Japan)
Dow Chemical
Nat ional Patent [level0|xnent
Harvard University
Abbott 1 aboratories
lessen
Synei <|en
Boulder, CO
general biomedical
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Table 3-2
U.S. companies engaged in applied genetics R&D.
Name
Iutalion
Collaborators
GENERAL (corit.)
UNA Science
Alpha therapeutic
Ai urns
Atlantic Antibodies
Becton 111 t: k ins on
Bioassay Systems
Bio-Response
Biotech Research
tiio- lectmica I
Re so u i c fs
Brain Research
Centocor
Clonal Research
Cy tocjen
Uamoii Biotech
Eastman Kodak
I lectro-NucI eonics
New York, NY
Los Angeles, CA
San fraricisco, CA
liar Harbor, Ml
Paiamus, NJ
Woburn, HA
Wilton, f.T
Rixkville, Ml)
Manitowoc, Wl
New Yor k , NY
Philadelphia, PA
Newpor t llench, CA
lil is on, N.I
Needham lie iijhts , HA
Rochester, NY
I'airfiold, N,1
provides venture capital
vaccines
instrument ation
di acpiost ics
ili aynos tics
interferon testing
tissue culture
di agnos t i cs
consul I in
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Table 3-2
U.S. companies engaged in applied genetics R&D.
Name
Location
Projects
to) labor iitui s
I. GlNERAl (tout.)
tneiyetics
Flow General
Genetic Systems
Hem Research
llybri tech
Inmiunotcch
IntelI (Genetics
Interferon Sciences
M.A. Bioproducts
Molecular Genetics
Neo-Bionics
New England UioLabs
Organon
University Patents
Vena Laboratories
Palo Alto, (.'A
Mcl ean, VA
San Francisco, ('.A
Itockville, HD
L aJo 11a, TA
I ampa, 11.
Stanford,CA
New York, NY
Walkersville, HI)
Minneapolis, UN
Monoclonal Antibodies Palo Alio, CA
Albuquerque, NM
Be veiIy , MA
He st Orange, N,l
Norwalk, CI
Fucson. A/
instrumental ion
interferon
tissue culture
ins t rumentat ion
inturlei on
tissue culture
monoclonal antibodies
diarjnost ics
tOiii|)iiter software for
(jene sequence una lysis
interferon
diaijnost ics
vaccInes
industrial processes
iiionoc 1cm.il antibodies
diagnostics
diagnostics
research en/ynies
di agnos t i cs
general binniedica 1
instrumentation
Hi 1 Cell Culture ('.enter
Applied Medical Devices, Inc.
Stanford University
National Patent Development
Orqannn
American C.yanamid
Microbiological Associates
(M.A. Rioproducts)
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I able 3-2
U.S. companies engaged in applied genetics K&f).
Name location Projects Collaborators
II. PHARMACEUTICAL
Abbott Labs
Nor th Chicago, 11,
urokinase
antibiotics
vi tamins
AHgen
Haxter 1ravenol
Deer field, II
diagnostics
Bristol Myers
New York, NY
inter feron
Genex
Hi I illy
Indianapolis, IN
i n;u1 in
growth licmiiOne
(ienentoch
University of California
G.l). Searle
Skokie. ll
interferon
subsidiary in High Uycomhe, U.K.
llof finann La Roche
Ihit ley, NJ
interferon
(lenentech
Me 1 oy 1 at>s
Sprint)field, VA
i nterfer on
(subsidiary of Revlon)
Merck
Haliway, N.J
interferon
antibiotics
Miles Labs
Flkhart, IN
hornu)nes
wastewater t re a t me n t s
(subsidiary of Oayer, A.G.)
Ortho
liaritan, N.I
iMoiioc I oiid 1 diil i bodies
(subsidiary of Johnson f> Johnson)
I'M/er
New York, NY
interferon
agri cul I in a 1 pi otitic Is
Richardson-Merrel
Wilton, t.l
general biomedical
(subsidiary of Dow f.lieiui < a 1)
Srhering Plough
Kenilworth. N.I
interferon
ISiogen (Switzerland)
Smithkllne
Philadelphia, PA
general biomedical
Southern Medical
Brandon . II
i liter feron
Key t.nei gy Enter pr ises , 1 anip.i
X Pharmaceutical
Sguibb Princeton, N.J general biomedical
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Table 3-2
U.S. companies engaged in applied genetics R&D.
Nome
Ioca tIon
Projects
Col 1 iibor «i tors
M. PHARMACEUTICAL (cont )
Synlex
Upjohn
Warner-I Hubert
Zuccon
I'alo Alto, CA
Kalamazoo, Ml
Morris Plains , N,l
Palo Alio, CA
general biomedical
Interferon-iruluc ing drugs
tumor diagnostic!;
steroids production
Perm State University
III. INDUSIRIAI ClirMlCAlS
Ayri-Dusiness Research Scottsdale, A/
Allied
American Cyanamid
Celanese
niamoiid Shamrock
Oow Chemical
Dul'ont
W I!. Grace
Monsanto
Morris town, N)
Wayne, N.I
New York, NY
Tucson, A/
Midland, M!
Mi liiiinyton, III
New York, NY
St . I oiii s , NO
petrochemicals from
desert plants
industri al/ayricul tural
chemi cals
general biomedical
methane I t orn petrochem-
ical wastes
oils and rubber from
desert plants
general biomedical
Industrial processes
interferon
general biomedical
chemical feedstocks
industrial chemicals
i ndustrial chemi calS
general biomedical
Arizona State University
Biol og lea Is (Canada)
Molecular Genetics
University ol Arizona
Collaborative ('.end i cs
New tngland Nuclear
(acquired by DtiPont)
Cal Tech
lllngon (Switzerland)
(ienex
Geiientec.h
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Table 3-2
U.S. companies engaged in applied genetics R&l).
Name
I or a (. i im
Projects
III. INDUS I RIAL f.HLMICAIS (conl.)
I'pimzo i I
RevI on
lUllull & Ileitis
Sliiuffpr Chemical
Union Carbide
Hons I on, IX
New York, NY
Phi I adp| phi a , I'A
Uestporl, n
Ni:w York, NY
|)(>tro<:lipniic.ils from plants
drut)s troni plants
a<]ri cul I m a I chemicalS
industrial chemicals
indus t r i a) clu.iiiicil s
U)
CD
IV. I.NtRGY
Arthur l). Lilt to
Ashland Oi1
Bio-lias
Dynatecli RSI)
Lcoenerqolics
Txxon UescNit ch
National Distillers
and Chemicals
Stic) I Oil
Standaid Oil of Cal-
ifornia (thevi oil)
Standard Oil of
Indiana (Amoco)
I utiri /o I
famhridyc, MA
Ashland, KY
Ar vada, CO
Camhr id<|p, MA
Vai iivi 1 It!, CA
f I oi ham I'ark, NJ
New York, NY
iloii'.ton, IX
San Iranrisco, CA
Chicayo, II
Wick I i I ie, Oil
h i o fup 1 s
elhanol i rom cornstarch
iiiclhanc from an una I waslcs
tup Is from ali|ap
b 101 up I s
(jpiiora I pnprijv -1 p i a led
(junera I eneryy- rel at ed
pctrochemica
is t i lulps
alternative sources ol
li(|uirl fuels
enhanced oil recoveiy
(jpnera 1 Pnori|y- rpl aled
pel locliemicaI subst i tutes
Col labora tors
Me lay l.uhs
Advanced (Jenpl it Systems
Oak Ridye National labs
001
I'ublicker Industries
Los Alamos national labs
fetus
Ce1 us
Co tus
Otul
Cipnent ech
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I able 3-2
U.S. companies erujoyed in upplied genetics R&D.
Name
I 0c.1L ton
Projects
foilahoratois
VD
IV. ENEItGV (umt.)
Native Plants
Sal t I ake Ci ly , Uf
Solar [ncrqy Itesearch Go I i t rie
Jacobs f nginoci lrK|
1'lii 1 a
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Table 3-2
U.S. companies engaged in applied genetics R&D.
Maine
l ocal ion
Piojec I :~>
Col laboratory,
V. I'Ol LOT ION CONTHOI (con I .)
Col yhac
SKI International
Syhron Ui r
treatments
nitrogen fixation
crop dovff Inpiiienl
ayritul lural rescii i li
olli.inol from cornstarch
fr'in. lose syrup
ayricu I tuia I research
food piodiictl
food products
animal husbandry
acjr i cul tin a I i fscnn h
subsidiary of Cytox Corp
ciijri cheniica Is
medic in.'i Is
food products
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commercia1 potential of modern biological techniques and managed to attract
venture capital to underwrite their business plans. The two pre-eminent
examples of this venture capita) apporach are:
• Cetus Corp., founded in 1971 by UC Berkeley physicist
Donald A. Glaser, biochemist Ronald E. CaDe (who also
earned an MBA degree from Harvard), and Peter J. Farley
(a medical doctor with an MBA from Stanford). Even
before the advent of recombinant DNA techniques, Cetus
funded its operations through contracts with larger com-
mercial firms, especially pharmaceutical houses. Current
backers of Cetus include major oil companies, such as
Amoco, Chevron, and Shell.
• Genentech, Inc., founded in 1976 by Robert A. Swanson
(who holds degrees in chemistry and business management
from MIT) and UCSF biochemist Herbert W. Boyer. The
firm was established express'y to commercial ize on DNA
technology and was initially underwritten by venture
caDitcl, chiefly from Kleiner & Perkins in California,
Wilmington Securities in Delaware, and Lubrizoi Entercri-
ses in Ohio. Genentech currently operates with ccpital
derived from specific contracts with 'arge firms, such as
Hoffmann-LaRoche and Eli Lilly, and with capital derived
from a recent public sale of stock.
The contribution of smell, innovative companies such as these to the emergence
of the applied genetics industry has been summarized by Nelson M. Schneider, a
drug-industry investment analyst for E.F. Hutton:
All major new technologies have been promoted and
fostered by small companies. The small guys have the
opportunity only because the bigger guys ignore it. The
big companies can't see the forest for the trees. They
choose not to participate because of their own ingrown
bureaucrccies.
Thus, small companies such as Cetus and Genentech, and the dozen or more
similar young firms that have sprung up recently, have the flexibility and the
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expertise to take advantage of scientific advances in the cpplied genetics field.
But today's smalt companies are determined to grow. Says Peter Farley,
president of Cetus:
It's biology's turn now. We actually saw it coming, and we
were determined right from the outset to become a major
company. It's not a get-rich-quick scheme. We expect to
be around fifty years from now as a major company.
It remains to be determined whether Cetus, as a major corporation, can continue
to "see the forest for the trees."
The second principal business strategy for investing in biotechnology has been for
large, technically oriented companies to undertake independent, !n-house R&D
programs. Virtually every major U.S. pharmaceutical firm has engaged in or
made plans to initiate recombinant DNA research. The high level of interest
among firms in this industry stems from the obvious applications of the new
technology to the manufacturing of new or improved drugs. Some drug firms
have undertaken collaborative research ventures with smell genetic engineering
firms directed towards the development of specific products. Examples include
arrangements between Genentech and Hoffmann-LaRoche to mcke interferon,
Genentech and Eli Lilly to make human insulin, and Gerex and 9r:stol-Myers to
make interferon.
Large corporations representing other industrial sectors are also investing
heavily in aoplied genetics. DuPont, the world's largest chemical producer, has
undertaken a sizeable commitment to R&D in the biosciences. Likewise, several
major oil comDanies, such as Amoco and Phillips, have established in-house
programs in biotechnology. These large, wealthy companies are hiring high-
quality scientists and bioengineers for the purpose of developing biological
solutions for problems such as alternative sources of energy and petrochemical
feedstocks. Collaborative agreements with small firms exist here too, such as
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contracts between Cetus and severaf oil companies, including Amoco, Chevron,
and Shell, to conduct R&D on energy-relcted projects.
While smaM genetic engineering firms will continue to conduct laboratory-scale
R&D, commercial scale-up of biotechnological processes will require capital
investment that only large firms can undertake. Thus, the relative importance
of large companies, with respect to the growth of the applied genetics industry,
will increase at the expense of the smaller firms. A trend can be anticipated
paralleling that which occurred in the semiconductor industry during the 1970's;
namely, larger firms will acquire through merger (or drive out of business) the
many small, specialized genetic engineering companies that have emerged.
The recent efforts of Cetus and Genentech to raise large sums of money by
offering shares of stock to the public reveal the difficulty that small companies
face when undertaking capital-intensive projects. A short time ago, the
corporate mancgement of both companies expressed desires to avoid going public
with their stock until the mid-1980's at the earliest. However, several fcctors
served to alter their plans: (I) commitments to pursue costly in-house programs
of commercial scale-up; (2) the failure to attract additional financing from >arge
corporate backers (such as Chevron and Amoco in the case of Cetus); and (3) the
absence *o date of saleable products derived from R&D investments. ?ub'ic
ownership may compel these companies to lose some of their flexibility end
farsightedness that provides them with the competitive edge over large, bureau-
cratic corporations.
3.1.3 Federal government
The involvement of the federal government in applied genetics stems from a
concern, first expressed by research scientists in the mid->970's, that the
application of recombinant DNA techniques could produce new organisms that
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might escape from the laboratory and endanger the human population and the
environment. Thus, the role of the government in biotechnology has so far been
limited to considering the practice of recombinant DNA methods in academic
and commercial settings.
3.1.3.1 The National Institutes of Health
In the United States, the National Institutes of Hearth (NIH) control the
administration of all federally supported recombinant DNA research and of all
such activities carried out by commercial firms in voluntary compliance with
NIH Guidelines for Research Involving Recombinant DNA Molecules. Other
government agencies also involved in the Dotentia! regulation or control of
commercial activities are the Food and Drug Administration (FDA), the Occupa-
tional Safety and Health Administration (OSHA), the Notional institute for
Occupational Safety and Health (NIOSH), end the Environmental Protection
i
Agency (EPA). Figure 3-1 depicts the organizational relationships between the
various government agencies and any company involved in recombinant DNA
activities.
The purpose of the NIH guidelines for recorroinart DNA research is to specify
proper practices for constructing and handling recombinant DNA molecules and
for handling organisms and viruses containing such molecules. Compliance with
the guidelines is mandatory for ail institutions engaging in such researcn anc
receiving federal support, "he guidelines were first pub!ished in the - ederol
Register in the summer of 1976. Since then •'hey have been amended consider-
ably end now reflect a more confident and relaxed attitude about potential risks
inherent in these activities. The most recent version of the guidelines aopeared
in the November 21, 1980, issue of the Federal Register.
The director of the NIH is responsible for the estaolishment, implementation,
and final interpretation of the guidelines. Pursuant to the guidelines, the
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Figure 3-1
Government agencies involved in recombinant DNA activities
I )pfior linehl
of Labor
ionul
Sale!y and
Heal Ih
Administration
(OSHA)
Ul
Center for
Dispose:
Control
(COO
Safely
Recoil 11 net »do I i ins
MoIhvxiI
Inst i lute ol
Occupational
Safely oml
Heollh
Dcpnr liiiiitl of
I leollh (Hid
I lumon Services
I cclinical
Aisislance
Dirci lor,
Nulionul
Institutes «f
I knllli
(Nil I)
Recombinant
UNA
Advisoi y
Coinmi I lee
(K AC)
Nrilionnl
Institute of
Allergy and
Infectious
Disrobes
(NIAIIj)
Administrative
Assistance
1
Food and
Or tig
Adtitinislr lieu I ton
(.ompuoy
TfF j^o. n
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Director has established the Recombinant DNA Advisory Committee (RAC) and
the Office of Recombinant DNA Activities (ORDA) to proviae technical and
administrative assistance in the fulfillment of these responsibilities.
The RAC was established to provide technical and scientific assistance to the
Director of NIH. Consequently, its membership must collectively reflect
expertise in scientific fields relevant to recombinant DNA technology and
biological safety. Additionally, at least 20 percent of its members must be
knowledgeable about applicable law, standards of professional conduct and
practice, the environment, public and occupational healtn, and related fields.
The RAC meets four times a year and advises the NIH Director on changing the
containment levels specified for various types of experiments covered under the
guidelines, assigning containment levels to experiments not covered by the
guidelines, and recommending new host-vector systems. The recombinant DNA
field has expanded rapidly over the past few years, especially with the increasing
involvement of private industry. Consequently, the RAC has been compelled to
assess large-scale fermentation procedures, to examine confidential industrial
data, and to review occupational safeTy and health standards.
However, the RAC has little expertise in industrial engineering. Furthermore, as
cn advisory committee to a non-regulatory agency, it has no authority to require
compliance with its advice. The RAC has recently decided to limit fs
assessment of industrial facilities to an excmination only of the biological
characteristics of the operation (45 FR 77379). Consequently, private
commercial firms will no longer be requested to suomit to the NIH the details of
their physical plants, medical surveillance programs, environmental monitoring
schemes, or emergency plans.
The Office of Recombinant DNA Activities (ORDA) is responsible for providing
maximum access to information on every aspect of the recombinant DNA field.
As the focal point for all information on such activities, it provides tecnnical and
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administrative advice to any institution, agency, or individual within or outside
the NIH. In cddition, it serves as executive secretary to the RAC, publishes the
Recombinant DNA Technical 3u!letin, and reviews and aporoves IBC membership
tists (see below). ORDA's responsibilities also include the scheduling and
announcing of RAC meetings and the publishing of revised guidelines in the
Federal Register. ORDA also distributes information regarding po^cy decisions
relevant to recombinant DNA research, announcements of training courses
dealing with experimental and safety issues, up-dating of cpproved host-vector
systems and other experimental protocols, and publishes a bibliography of newly
released articles on recombinant DNA.
The NIH guidelines were amended in January 1980 to include a section dealing
with voluntary cop-id'iance by private commercial firms engaging in recombinant
DNA activities. This action was taken as a compromise to proposed mandatory
controls put forward by the FDA. A scheme of voluntary compliance encourages
private companies to follow the same administrative and technical orocedures
that are required of any federaf'y supported institution. However, all items of
information provided to the RAC or to ORDA by .complying companies ere
protected as trade secrets, thus prohibiting subseauent disclosure under the
Freedom of Information Act. In addition, companies that comply voluntarily are
asked to register alt projects involving recombinant DNA technology.
In Aoril '980, the NIH published a set of guidelines dealing with large-scale
apolications of recombinant DNA methodology. These guidelines detail the
physical containment requirements for the production of recombinant DNA
organisms in volumes exceeding ten liters.
Any company engaged in recombinant DNA activities and in voluntary com-
pliance with the NIH guidelines must establish in Institutional 3!osafety Commit-
tee (IBC). Each firm's IBC must include ct least five members, with c minimum
of two (but.no fewer than 20 percent) having no affiliation with the company.
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The IBC members collectively must have expertise in recombinant DNA research
and be capable of assessing the safety of such experiments and the risks to
workers and the community. The non-affiliated members are to represent the
best interests of the surrounding community with respect to health end the
protection of the environment. Officials of state or local oublic health or
environmental protection agencies, members of other local government organiz-
ations or persons active in community medical, occupational health, or environ-
mental affairs are ail eligible to serve on an IBC.
The primary responsibility of the IBC is to review aH recombinant DNA
experiments conducted by the company to ensure compliance with the NIH
guidelines. The IBC review must include cn assessment of the containment
levels utilized as well as cn evaluation of the facilities, procedures, training, and
exDertise of the oersonne! conducting the experiments. The IBC must a'so adoDt
emergency oians covering accidental spills end contamination resulting from
recombinant DNA research.
In addition to an IBC, any firm or institution engaging in recombinant DNA R&D
invo'ving high levels of physical containment (P3 or P4) must appoint a biological
Safety Officer (BSO). The 3SO is a member of the IBC and is responsible for
conducting periodic inspections of lab facilities, reoorting to the IBC any
significant violations of the guide'ines, developing emergency plans, and inter-
acting with the orincipal investigator (PI) in areas of lab security, technical and
safety procedures, and adherence to the guidelines. •
3.1.3.2 Other Federaf agencies
Recombinant DNA technology is rapidly moving out of the exclusive domain of
university research laboratories and into industrial laboratories and large-scale
production facilities. Concurrently, government agencies other than the NIH are
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becoming increasingly involved with issues concerning environmental monitoring,
worker safety and health, and product quality.
The Food and Drug Administration has been invoivec from the very beginning
with industrial scale-up of recombinant DNA technology. This interest stems
from the fact that the first commercial products emerging from this new
technology are likely to oe intended for human use; namely, insulin, human
growth hormone, and 'nterferon.
New drugs intended for human use must se certified by the FDA through the
approval of two company-submitted forms: (I) a notice of Claimed investigation
Exemption for a New Drug (IND); and (2) a New Drug Application (NIDA).
Together these forms supply the FDA with proprietary information on drug
composition, resuits of human and anima! testing, end manufacturing procedures.
As of June 1980, the position of the FDA was that drugs produced by recombinant
DNA technolgoy could not be marketed unaer existing INDs or NDAs as simply
changes in manufacturing technique.
Submission of an IND informs the FDA thct a company nas tested a potential
new drug and that it will be testing it further. Required by the form is a
statement of the methods, facilities, ana controls used for the manufacturing,
processing, and packaging of the new drug to establish and mcintain appropriate
standards of identity, strength, quality, and purity.
The NDA is a request for approval to market the drug. Although intended
primarily to provide information on the results of clinical testing, the NDA also
contains detailed information on the manufacturing of the drug. It covers all the
information that the sponsor ;
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within the Department of Labor that is charged with developing, and promulga-
ting standards, formulating and enforcing appropriate regulations to maintain
safe ana healthful conditions in the workplace. OSHA has announced that it will
develop a recombinant DNA regulatory policy over the next two years. This
could prove to be a difficult task since hazards associated with this technology
have remained speculative. Two recent events will further contribute to the
difficulty that OSHA will encounter in efforts to regulate recombinant DNA
technology. In a recent Supreme Court decision, reduced standards for exposure
to benzene were disallowed owing to a Sack of evidence that the existing
exposure levels were dangerously nigh. Similarly, there exists no firm evidence
of risk resulting from contact with recombinant DNA organisms (at cny level of
exposure). Secondly, OSHA has no authority to preview the technical details
that a company intends to use in the large-scale production of recombinant DNA
organisms. Until now, OSHA has obtained this information from the RAC, but.
as mentioned cbove, the RAC no longer intends to gather this information.
The National Institute for Occupational Safety and Health (NIOSH) was
established by the Occupational Safety and Health Act of 1970. NIOSH is a
component of tne Center for Disease Control under the Public Health Service
and is authorized to conduct research and recommend workplace standards to
OSHA. NIOSH is interested in the following areas relevant to recomoinart DNA:
• Process operations with attendant potential for worker
exposure;
• Engineering controls, such as pnysic'al containment design,
ventilation, exhaust gas filtration, waste product control,
etc;
• Validation procedures pertaining to sterilizction of equip-
ment, physical containment, and process termination;
• Work prcctices, emergency and accicent procedures, med-
ical surveillance, environmental monitoring, ard employee
training and education.
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3.1.3.3 Patent issues
The issue of patent protection for products and processes evolving from
recombinant DNA R&D is both controversial and very important to commercial
firms engaged in these activities. Two events have had a sizeable impact so far.
One established the legal precedent that man-mode microorgcnisrr.s are not
excluded from patent protection; the second extended oatent coverage to the
inventors of certain basic laboratory procedures.
On June 16, >980, the U.S. Supreme Court ruled in a narrow 5-^ decision that the
Generai Electric Corp. should not be denied patent protection on an "oil-eating"
microorganism developed by Dr. A. M. Chakrcbarty. The decision hi.nged on
whether a microorganism is unpatentable subject matter simply beccuse it is
alive. The Court found that the principal criteria upor which an invention is
deemed Datentabie (namely, that it be new, useful, and non-obvious) were in no
way infringed by the fact that the invention is alive. The dissenting minority
argued that those who originally framed the patent statuses never intended that
pctent protection be afforded to living things. The Court admittec to c lack of
competence in evaluating the potential dangers or benefits of this new technol-
ogy, and they further declared that any binding policy regarding patentability of
living organisms must originate in Congress.
On December 2, I960, Stanford University and the University of California were
jointly awarded a patent dealing wth gene cloning techniques used in recomoi-
nant DNA experiments. The techniques, developed oy Stanley Cohen at Stanford
and Herb Boyer at UCSF, have become the bcsis for virtually all recombinant
DNA experimentation to date. The two universities have declared that tney will
license the technology to any company that wishes to employ the techniques and
they will collect royalties on its use. They have further stated that a condition
for use of the technology will be adherence to the N1H guidelines. However, the
patent applies only to the use of recombinant DNA technology within the borcers
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of the United States and would not cover overseas operations by U.S.-based
companies, or by foreign firms. !t is highly unlikely that this-patent could
withstand a legal challenge if it is deemea to inhibit the commercial develop-
ment of recombinant DNA technology. Moreover, litigants will argue that
numerous refinements to the basic techniques hcve been made so that the
original inventors are no longer entitled to patent protection.
3.2 Foreign activities
The overseas practice of applied genetics has proceeded in a fashion similar to
its evolution in the United States. Much of the basic biologiccl research that
gave rise to this new industry occurred In foreign laboratories, particularly in
Western Europe. As in'the United States, there has emerged in several countries
a variety of small new genetic engineering companies. Likewise, established
corporations are engaging in aDplied genetics R&D. We are aware of over forty
foreign companies, large and smell, that have invested in biotechnology (see
Taole 3-3).
In contrast to U.S. cctivities, however, some foreign governments have suopiied
considerable financial backing to fledgling genetic engineering companies. ror
example, the British government, in concert with four London investment firms,
has established Cell tech. This nationally owned venture came into being only
after extensive hand-wringing on The part of government planners, but Ceiltecn
can now hope to commercialize significant scientific achievements of 3ritish
researchers, several of whom have already lost the opportunity to capitalize on
their findings owing to a lack of public interest. (cor example, the monoclonal
antibody technique was discovered in England, but the scientists involved failed
to patent the process within the necessary time limits.)
With regard to appiied genetics in France, the government there has fostered a
healthy relationship between jniversities and industries, thereby facilitating the
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transfer of basic biotechnology from academic tcbs into the commercial sector.
The French government is committed to spend about $25 million over the next
five years in support of biotechnology. Several French government science and
research agencies hcve cooperated in support of a new business venture, caMed
G3, which will concentrate on biomedical applications of genetic engineering.
In Japan, where government-industry cooperation is legendary, over a dozen
established chemical and pharmaceutical firms are cctiveiv pursuing genetic
engineering programs with government support. The Japanese are considered
world leaders in certain areas of biotechnology, particularly fermentation
techniques. They are far ahead of the rest of the world with regard to the
quantity and diversity of products, such as antibiotics, vitamins, and food
additives, that can be readily manufactured by fermentation procedures.
Most of the commercial development of genetic engineering in Canada has
preceeded via private investment. A small new firm, BioLogicals in Toronto,
recently signed a multi-million dollar agreement with Ailied Chemical (a U.S.
firm) to conduct apDlied research into the uses of genetic engineering for the
production of industrial and agricultural chemica's. Connaught Laboratories,
formerly associated with the University of Toronto and the site where the
hormone insulin was first isolated in 1921, has been largely taken over by the
Canadian government. The firm is now engcged in an ambitious revitalization
program that includes large-scale investment in genetic engineering.
In Israel, biotechnology is being applied to meet national needs in the areas of
agriculture, industrial chemicals, and waste management. Considerable effort is
being expended to investigate various types of photosynthetic algae as potential
sources of single cell protein and useful biochemicals. Geneticclly engineering
salt tolerance into algae, thus allowing the microbes to thrive in brackish ponds,
has received soecial attention.
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For the most part, government regluation (or pseudo-regulation) of recombinant
DNA activities in foreign countries has followed c path similar to that in the
United States. Actual legislation dealing with this area exists only in the United
Kingdom, where the GMAG (Genetic Manipulation Advisory Group) reviews
experimental protocols much as does The RAC in this country. In Britian,
however, emphasis has been placed solely on physical containment of recombi-
nant DNA organisms, rather than on both physical and biological containment, as
in the United States. Other Western European nations have generally followed
the modei set by GMAG.
The Japanese government has followed the U.S. lead in establishing voluntary
guidelines for recombinant DNA resecrcn. The trend in Jcpan, as in all nations,
has been to continually revise downward the restrictions imposed by the
guidelines as information accumulates indicating biohazards inherent to recombi-
nant DNA techniques are no greater than the risks associated with microbiologi-
cal methods in general. Governments in all nations are fearful that unnecessary
regulction of genetic engineering may adversely effect the commercial potential
that this new technology offers.
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Table 3-3
Foreign companies or government agencies engage*! in applied genetics R&D
Name
Pro jet lb
Col labora tors
CANADA
BioLoyicals
Connauiflit Labs
Inco
I evochem Industries
National Research C.ounc
of Canada
Sybron Biochemical
indus tri a l/a(|ri ml t ura 1
chemicals
vaccines
industrial chemicals
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Table 3-3
Foreign companies or government agencies engaged in applied genetics R&D
Name
Projects
Col lahnrafnrs
FRANCE
f.himie Industrie I le
11 (-Ai)ui taine
Genet icd
G3 (Gioupement Ue
Genie Genetique)
Sanui i
1 ransgene
industrial chemicals
energy from bioniass
amino acids
nitrogen fixation
phariiiaceut ica Is
lii-pat i t is vaccine
honnones
interferon
pliannaceuticdls
hioeiwrqy
(subsidiary of Rhone-Poulenc, France)
Institut Pasteur
National Institute for Health and
Medical Research (INSERM)
National tenter for Scientific
Research (f.MRS)
National institute for Agricultural
Research (INRA)
lnstit.iit Pasteur
Paribus (France)
MUST GERMANY
Doehringer-Mannheim
Noechsl
enzymes
synthetic peptides
hormones
Massachusetts General Hospital (U.S.)
Seller i rig
pliarmaceu t i ( al S
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Table 3-3
Foreign companies or government agencies engaged in applied genetics R&D
Name
Projects
Col laboratory
SWUZERLAND
B ioyen
Cilxi-Geigy
llo f finrtiut - L aRoche
Sanduz
interferon
industrial chemicals
miniiKj
phannaceut ical 5
agricultural chemicals
interferon
bioloyicdl insecticides
Schering-Plough (U.S.)
Monsanto (U.S.)
lnco (Canada)
Grand Metropolitan foods (U.K.)
Roche Institute (U.S.)
University of California
OTHER EUROPEAN
A1 fa-Laval (Sweden)
Kabi (Sweden)
Novo Industri (Denmark)
fiis t-Brocades (Ne titer 1 ands)
Ni^o (Netherlands)
etlianol from biomass
human qrowt li hormone
human insulin
industrial chemicals
amino acids
etlianol from liiomass
(ienenlrch (U.S.)
Dutch Institute for Dairy Research
lSRAfl
Koor I ood
Yeda N&ll
slni|le cell protein
inte» feion
Ueizmann Institute (Israel)
Arcs Co. (Switzerland)
UNA Science (U.S.)
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Table 3-3
Foreign companies or government agencies engaged in applied genetics R&D
Name
Projects
Co 1I allocators
JAPAN
Oi
On
Ajinomota
Asahi Chemical
Kyowd lldkko Koyyo
Mitsubishi Chemicals
Sumi (Ohio Chemicals
Uainichiseika Chemicdls
Hi k 1
Japanese rermentation
Research Institute
.Japanese Science and
lechnoloqy Agency
Mitsubishi Kakoki Kaishha
Ringon Dinlogical Research
Shiuiiogi
Green Cross
Takeda Chemical
loray Industries
I!
honiiones
vacc ines
amino acids
flavoring agents
industrial chemicals
microbial air deodorants
wastewater treatment
bacteri a 1 concentrat ion
of heavy metals
general microbial
biotechnology
wastewater treatment
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SECTION 4
industrial applications, trends, potential hazards
This section contains en industry-by-industry analysis of biotechnology. Each
industrial sector will be examined for:
• Current activities in applied genetics. Some speculation
may be required inasmuch as certain information is held
as proprietary by private industries.
• Future prospects for the application of biotechnology
within each industry.
• Assessment of potential hazards of applied genetics both
as practiced currently within each industry end as possible
future uses unfold.
The following commercial sectors wilt oe examined: (I) pharmaceuticals,
(2) industrial chemicals, 13) energy, (4) mining, and (5) pollution control.
4.1 Pharmaceutical industry
4.1.1 Current activities
The largest efforts to date towards commercial application of modern oiological
techniques have taken place in the pharmaceutical industry. The manufacture of
new or improved drugs and vaccines surely wiil be the first commercial fall-out
from recombinant DNA technology. Scientists recognized very early in the
development of these techniques the immediate potential T'or mess-producing
human biologicais, such as normones and serum proteins, for eventual use as
therapeutic agents. If available at all, such agents traditionally have been
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isolated from animal sources, a practice frequently leading to shortages in supply
or to variation in quality from batch to batch. Moreover, biologies aerivec from
animals generally differ slightly in structure from the human form of the analo-
gous compound, thus providing a less than optimal human therapeutic.
The production by modern biotechnological methods of specific pharmaceuticals
will now be considered.
Interferon is a protein synthesized by most cells of higher organisms in response
to virus infections. Its production and secretion in miniscule amounts by
infected cells serves to "interfere" with the spread of the infection to healthy
cells. Thus, the administration of interferon as a drug promises to be a potent
anti-viral therapy. In addition, interferon has been shown to act as an anti-
tumor agent for certain types of cancer. Its potential as a cancer drug is now
under thorough investigation at several clinical centers in the United States,
notably the M.D. Anderson Hospital and Tumor Institute in Houston.
The severe shortage of purified human interferon hes hampered adequate testing
of its therapeutic value, but scientists have succeeded in applying recombinant
DNA techniques to create bacterial interferon "factories" thct promise to
increase greatly the supply of the drug, while reducing enormously its current
cost of several thousand dollars per dose. A predicted market of $3 oillion per
year has lured numerous commercial firms, both in the United States and
overseas, to invest huge sums in interferon production anc testing. Some
companies, are pursuing tissue culture methods, rather than recombinant DNA
techniques, to obtain usable quantities of interferon. The therapeutic and
commercial values of interferon w:ll likely be revealed within the next year or
two.
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Insulin, a hormone Tiade in the pancreas, aids in the metabolism of sugar. It is
composed of two small polypeptides, the A and B chains, which are composed of
21 and 30 amino acids, respectively. In the pancreas, proinsulin is made as a
precursor to insulin. Prior to secretion, the proinsulin is converted to insulin by
the enzymatic removal from the middle of the molecule of a stretch of 35 amino
acids called the C chain.
There are currently several strategies for producing insulin by recombinant DNA
technology. Here we discuss two of them. In one, the genes for the A and B
chains are chemically synthesized separately and inserted into separate plasmids
as fusion proteins joined to the lac operon enzyme, beta-galactosidase. The gene
to be cloned is a combination of the gene for the A or B chain and the gene for
the enzyme, joined by the triplet codon for the amino acid methionine. The
plasmid is then cloned in a bacterial host. Since neither the A nor 3 chain
contains methionine, it can be efficiently removed from the fusion protein after
the orotein is extracted from the host. Removal is accomplished by treating the
fusion protein with cyanogen bromide, which cleaves at the methionine juncture.
The A and B chains are bound together as insulin by two disulfide bonds. After
extraction from the enzyme proteins, they can be joined in the laboratory by
using an air oxidation technique involving S-sulfonated derivatives and an excess
of A chain. This methodology is 50 to 80 percent efficient in mcking the
complete insulin molecule.
The second method utilizes only one organism to produce a fusion protein
containing proinsulin. As in the first method, the extraction is made with
cyanogen bromide. The isolated molecule is then treated with enzymes to
remove the C chain, and the active insulin is recovered.
Figure 4-1 shows schematically the synthesis of proinsulin and insulin by recombi-
nant DNA methodology.
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Figure 4-1
Alternative methods for insulin production in E. coli
Pancreas
Preoroireutin 13 enzymancaily processed and foWsd to lorn
Pancreas
Proinsulin is enzymaticaHy processed to insulin
CCOH^"
human insulin
Method A
Method B
C. coll
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Human growth hormone (hGH) or somatotropin is produced in the pituitary gland
and mediates growth and stature, particularly in children. The hormone
traditionally has been extracted from the oituitaries of human cadavers (animal
substitutes are not suitable) and is used in the treatment of dwarfism in children.
Recombinant DNA technology offers the prospect of sharply increased supplies
of the scarce hormone, leading to speculation that hGH will be useful in the
treatment of a variety of disorders including uicers, burns, bone fractures, and
bone deterioration (osteoporosis, a common ailment of the elderly). Moreover,
hGH may stimulate growth in a group of children (numbering close to a million in
the United States) who are abnormally small despite having seemingly normal
levels of circulating growth hormone. Clinical trials of "recombinant hGH" have
just been initiated.
Human growth hormone has been sequenced in its entirety. The synthesis of an
expression plasmid for bacterial hGH synthesis involved cloning a synthetic DNA
fragment coding for the first 2k amino acids separately from a clone coding for
the remaining 167 amino acids. The nonconjugable Dlasmid pBR322, which codes
for resistance to the antibiotics ampicillin and tetracycline, was used as vector
for both clonings. The combined hybrid gene for the entire 19 ! amino acids was
fused to the gene for beta-galactosiriase in the lac operon and then inserted into
a new expression plasmid subsequently designated pHGH|Q7. Figure'4-2 shows
schematically the stages involved in constructing the final expression plasmid
coding for the complete amino acid sequence of hGH.
The synthetic DNA segment coding for the 24 amino acids was constructed from
16 chemically synthesized lengths- of DNA. These fragments were joined
together using the enzyme Ik ligase. The resulting 8^-ocsepair fragment was
cesigned to nave sticky ends by adding additional nucleotides at each end end
then treating it with the restriction endonuclease enzymes Eco Rl and Hind 111 .
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Figure 4-2
Construction of a bacterial plasmid coding for
the synthesis of human growth hormone
Cloning of Synthetic Fragment
Synthetic oliganuc eo'iaes
Sco Pi
Ao / / \ \
; C3P322 >
v-" \ ^
U -» Lj -2
i iigase • *4 iigase i T4 iigase
! ~i iigase
ec -i Hina II
cA o.p.
i T4 . ;ssa
5:d ai
/^SV^
AO3 / / '\\ \ TcJ
: CHGH3
2. Cloning of cDNA Fragment
*S\ I
;8R322 ' i
X
°ST
Hunan srtutary
n^NA
I Reverse
' :ranscr:otase
Terminal transferase 0:5 :^NA
, • aGTP
sf^\
: -te in
Assembly of HGH Gene
£C3 31
?st 1
"fse:llx.Vs^
i3Mci shGHoU
*a» II
Pst I
\ Eco ft'. nae in
£cc A! * *,7® l-l ."ae >lt
H-nd lii . !!
51 nuclease U
rco Ri ^
] T-i icase
| ;rans-
i 'orrranon
t-GHlC7 ; / «
Source: Miozzari, G. (198C)
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Eco Rl and Hind II! were also used to open the vector, plasmid pBR322. The
synthetic fragment was inserted into the plasmid and subsequently cloned. Then
the plasmid with the correct DNA fragment was isolated from a clonea colony
and designated pHGH3.
The 501-basepair cDNA fragment coding for the remaining 167 amino-acids was
prepared from pituitary mRNA, treated with the restriction endonuclease
enzyme Hqe III, and tailed with chemically synthesized segments of cytosine (C)
nucleotides. The plasmid was treated with Pst I and joined to synthesized
segments of guanine (G) residues. The vector and the fragment, rendered
complementary under these conditions, were then joined together. Insertion anc
cloning followed.
In order to clone the complete gene, the two fragments were isolated from their
plasmids and then joined together. The shorter, synthetic piece was cieaved
from its plasmid with Eco Rl and Hind III and then treated with Hae 111 to
produce an Eco R I sticky end at one end of the fragment and a Hae ill blunt end
at the other. The larger cDNA fragment was then cleaved with Hae 111 and
Xma I to produce a Hae III blunt end and an Xma I sticky end. The complete
gene was made by joining the two Hae III blunt ends of the fragments with TA
ligase. Simultaneously, the Xma I end of the larger fragment was blunted with
Sma I. This made that end suitable for insertion into a new plasmid (pGH6), as
shown in the figure. This plasmid had been previously cloned with a copy of the
lac operon. It was opened with Eco Rl and Hind 111 and treated with SI nuclease,
thus leaving the plasmid with one Eco Rl sticky end and one blunt end. The
complete hGH gene was then fused to the lac operon, which permitted the
expression of the hGH gene in the presence of lactose.
A number of other human peptides have been synthesized using recomoinant
DNA techniques. These include:
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• Somatostatin, a short fourteen amino acid peptide hor-
mone secreted by the hypothalamus, was the first human
substance produced in bacteria. It may have therapeutic
potential in the treatment of diabetes:
• Thymosin, a thymus hormone, regulates the development
of a portion of the immune system. As c potential drug,
it may influence the aging process and have application in
cancer therapy;
• Beta-endorphin is a naturally occurring opiate that mimics
the action of morphine. It has considerable therapeutic
potential as a safe, non-narcotic pain-killer;
• Urokinase, a kidney enzyme, dissolves blood clots. It has
potential as a drug to reduce the likelihood of heart
attacks and strokes.
A second major pharmaceutical area in which recombinant DNA techniques are
finding considerable application is in the development of new vaccines. Conven-
tional vaccinations against viral diseases involve immunizing with inactivated
virus particles, which stimulates the host's immune system to defend against a
subsequent exposure to a live, active virus infection. The use of entire viruses as
the immunizing agent, however, entails the risks that either the vaccine may
elicit the disease (owing to incomplete incctivation), or that the vaccine will be
ineffective cs a result of denaturation of the virus during inactivation.
Medical scientists have acquired an understanding of the molecular basis of
vaccination, so it has become possible to isolate the specific proteins from the
outer surface of viruses that are responsible for stimulating an immune response.
Injection of these proteins alone is sufficient to generate adequate immunity to
the viruses, but the proteins are totally non-pathogenic; that is, they do not
mediate an infection. Using recombinant DNA techniques, it has been possible
to clone the viral DNA that directs the synthesis of these proteins and to gain
expression of The genes in bacteria so that the proteins are manufactured. Such
research has focused on efforts to generate vaccines to immunize against:
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• Hepatitis,.a serious liver disease that has reached epidem-
ic proportions in some parts of the world;
• Influenza, the many forms of which have made reliable
vaccines unobtainable using conventional techniques;
• Foot-and-mouth disease, a life-threatening disease among
domesticated livestock.
In addition, vaccines are under development to combat certain pathogenic
bacteria and the diseases that they cause, including:
• Gonococcus, which causes venereal disease;
• Pathogenic E. coli, which give rise to digestive ailments
such as severe diarrhea, of life-threatening concern in
infant children;
• Oral bacteria, which are responsible for tooth decay.
The discussion of applied genetics in the pharmaceutical industry has so far
centered on the uses of recombinant DNA technology. A variety of other
biotechnologies are finding application in this industry as well, including:
¦ Monoclonal antibodies for use as diagnostic agents for
viral and parasitic diseases, such a hepatitis and malaria;
• New antibiotics generated by combining the synthetic
capabilities of different cntibiotic-Droducing strains;
• Bacterial production of chemical intermediates for use in
drug synthesis, such as glutathione, a liver drug interme-
diate;
• The production of human serum oroteins by tissue culture
of cells derived from fusions between human embryonic
cells and mouse liver tumor cells;
• The production of vitamin 312 by bacteria, which should
prove more economic than its isolation from fungi, as
currently practiced;
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• Chemicai modifications by microbes of drug intermedi-
ates, as in the synthesis of various antibiotics (streptomy-
cin, penicillin, and gentamycin), and the transformation of
steroids towards the manufacture of contraceptives;
• The use of higher plants and sea creatures for the produc-
tion of steroids, antibiotics, atropine, digitalis, etc;
« A great variety of pharmacologically active agents can be
isolated from naturally occurring microorganisms; a part-
ial list is shown in Table 4-1.
4.1.2 Future prospects
It is the biomedical field where applied genetics will likely make the most
dramatic, and most controversial, future impacts. The prospect of genetic
engineering in humans raises deeply personal ethical questions that are not of
concern to applications of biotechnology to other commercial sectors. As is the
case with other new technologies, however, specific developments are cifficult
to predict; often the most significant applications are not even conceived of
several years in advance. Nevertheless, certain trends are apparent that will
direct the course of commercial activity in the biomedical area over the next
few years at least.
• Interferon is not a single substance, but exists in multiple
forms (numbering at least eight so far). The physiological
role of each of these interferons has yet to be unraveled,
but a better understanding of this biological system will
lead to a wide variety of new drugs for treating specific
viral diseases and some cancers. A note of pessimism:
patients on long-term interferon therapy will probably
develop resistance to the drug, much as chronic use of
some antibiotics and antimalarials has reduced the ef-
fectiveness of these drugs. Thus, continual development
of new chemical forms of interferon will be needed.
• There will be a resurgence in the search for natural drug-
like substances produced by plants and sea crectures. A
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Table 4-1
Examples of pharmacologically active natural products
isolated from microorganisms
Activity
Product
Producing strain
anticoagulant
antidepressant
anthelmentic
antilipidemic
antipernicious anemia
coronary vasodilator
detoxicant
DNA transformation
inhibitor
esterogenic
food pigment
herbic ide
hypotensive
inmune enhancer
insecticide
miticide
plant hormone
salivation inducer
serotonin antagonist
Phialocin
1,3-Diphenethylurea
Avermect in
Ascofuranone
Vitamin B
Naematolin
Detoxin
Antraformin
Zearalenone
Kcnascin
Herbicidin
Fusaric acid
N-acetylmuramyl
tripeptide
Piericidin
Tetranactin
Gibberellic acid
Slatranine
H02135
Phialocsvhala repens
Sirepiomyces sp.
Stvevzomjces aveTmitilns
Asaochyia vicice
Streptorryces grn.se us
'•Jaerrcctaloma fasoisv. lave
Streptomyoes caespitosus
Sireptonvy aes s p.
Gibbevella z&ae
Monasaus sp.
Streptorryces 3aeononensic
Fusarin/n sp.
Bacillus cereus
Streptorvjces rnobaraensis
Streptorryces aureus
Jibberella fucikuroi
P'nizocvon-La Zegyrrrlnioola
Streptorryces gviseus
Source: Woodruff, H.B. (1980) Science, 208:1228.
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variety of powerful drugs (e.g., digitalis, morphine, vin-
cristine/vinblastine cancer drugs, and many steroids) were
first isolated from plants. Modern drugs based on these
compounds are now chemically synthesized. There exist
numerous natural products that may serve gs useful drugs
but which occur in such limited quantities or which are so
difficult to synthesize that commercial development is
unlikely. Applied genetics will soon permit mass produc-
tion of these substances by genetic manipulation of the
organisms that produce them.
Monoclonal antibodies ("hybridomas") have so far been
produced only in mice. Mouse antibodies are inappropri-
ate for use as human therapeutic cgents, but recent
developments have extended the hybridoma technique to
oermit the production of human antibodies. Such antibod-
ies will have multiple drug uses: as antidotes for acute
bacterial or viral infections; as agents for localizing and
treating inaccessible tumors: as an adjunct to tissue
transplantation to prolong graft survival; as safe contra-
ceptive agents. Although the array of potential applica-
tions of hybridoma technology is considerably smaller
than that of recombinant DNA methodology, drugs based
on monoclonal antibodies will appear on the market in
greater variety and with shorter delays than will the
products of gene-splicing tecnniques.
Recombinant DNA methods include the ability to transfer
human genetic material into bacteria. This capability
depends on certain bacterial vectors, usually plasmids or
viruses, that carry the foreign DNA into the nost microbe.
Similarly, transfer of human DNA into other human cells
or tissues is feasible through use of appropriate vectors
thct mediate the exchange. Such vectors, namely mam-
malian viruses, are under development; their availability
will facilitate genetic engineering in humans. More
serious technical questions stand in the way of eventual
medical application, however. For example, which human
genes should be transferred in order to treat which
disease, end how ere those genes isolated? What steps are
required to establish those new genes in the recipient
individual? Apart from techniccl obstacles, unresolved
political and ethical issues pertaining to genetic experi-
mentation in humans are certain to forestall widespread
application of this technology for years to come.
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4.1.3 Potential hazards
The range of potential health and environmental hazards posed by appliea
genetics will vary depending on the industrial setting. In the pharmaceutical and
chemical industries, for example, processes involving genetically engineered
microorganisms are likely to be contcined within closed reactors or fermentors.
Many applications of biotechnology in the mining and pollution control industries,
on the other hand, foresee deliberate release of microbes into specific open
environments. These two general modes of oDeration clearly impose different
risks on (I) the health of the workers involved and of the surrounding community
and on (2) the local ecology.
The application of biotechnology in the pharmaceutical industry gives rise to
potential hazards at several levels of activity:
• The research laboratory, where scientists and technical
personnel engage in the initial stages of development of
new drugs or therapeutic regimens. A potential risk
arising from the creation of new microbial strains via
recombinant DNA techniques, for example, will be first
experienced by laboratory personnel. The specific
hazards involved are mitigated, however, ay the high level
of personnel training in general laboratory safety ana by
the relatively small quantities of materiel encountered in
the laboratory setting.
• The production facility, where large-scale manufacturing,
product isolation, and oackaging processes are under-
taken. The drug industry has amassed considerable exper-
ience in the safe operation of huge fermentation facili-
ties. There remains the potential risk, however, of
exposing the workplace (and to a lesser extent, the
surrounding community) to aerosols containing viable
microorganisms. Although their health is monitored quite
closely, production workers are less able than are highly
trained iab personnel to recognize the symptoms of
microbial infection. Certainly, individuals residing in the
surrounding community are generally unaualified to
appreciate the risks posed by these activities.
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• The end users, including medical personnel ana patients,
of drugs manufactured through applied genetics. Risks
here are minimized by (I) enforcement of existing
government regulations pertaining to the introduction of
new drugs and biologicals and by (2) strict control of
product quality by the manufacturer.
Microbiology laboratories have been examined since the turn of the century as
sources of bacterial and viral infections. A recent survey (see R. M, Pike, 1976)
summarizes nearly 4000 lab-associated infections dating back to the early 1950's.
The most common bacterial and viral disecses reported among lab workers were
brucellosis, typhoid, tularemia, and hepatitis. However, fewer than 20% of these
infections could be associated with a known laboratory accident of any kind.
(The lab practices most frequently giving rise to infections are mouth oipetting
and the use of needles and syringes.) Although the incidence of such infections
among lab workers is 5- to 10-fold higher than their frequency in *he general
population, the local community surrounding a microbiology laD appears to be at
no greater risk than the population at-large, s-or example, despite 109 lab-
associated infections at the Center for Disease Control during the period 1947-
1973, no secondary cases were reported in family members or community
contacts. In sum, these data suggest that, while workers in microbiology labs are
exposed to increased health hazaras, the risk to the surrounding community is
minimal.
As mentioned previously, applied genetics, esoecially recombinant DNA technol-
ogy, has received more commercial promotion in the pharmaceutical industry
than in other commercial sectors. For this reason, assessments to dcte of the
potential risks arising from this new technology have been made in the context
of laboratory and industrial practices pertinent to the pharmaceutical sector.
A number of risk assessments have been conducted attemoting to evaluate the
safety of using E. coli KI2 as a host bacterium for the manufacture of human
proteins via recombinant DNA techniques. Three conferences dealing with this
issue have been held: (I) at Falmouth, Massachusetts, in June 1977; (2) at Ascot,
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tngland, in January 1978; and (3) at Pasadena, California, in April 1980. The
viewpoints expressed at these sessions are summarized as follows:
• The natural fragility of KI2 would make it very difficult,
if not impossible, for it to colonize the huncn gut, or to
be communiccted between individuals.
• The transfer into <12 of genes encoding the manufacture
of virulent proteins (toxins) would not produce a fuily
pathogenic <12 strain, and the insertion into <12 of DNA
from human viruses would present fewer risks than the
same viruses existing freely in nature.
• The ingestion of a
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Bacillus subtil is, and brewer's /east, Soccharomyces cerevisiae. As with EE. coli,
more is known of the genetics and molecular biology of these organisms than of
any other microbes. Apprehensions regarding risks inherent in the use of tnese
microbes have been far less than for E. coli K12. Neither subtilis nor 5^
cerevisiae cause serious infections in humans; only easily treated minor eye
infections are attributed occasionally to B. subtilis.
Thus, these three microorganisms will likely underlie most commercial
recombinant DNA activities within the pharmaceutical industry for the
foreseeable future. Each of the three microbes has certain tecnnical advantages
and disadvantages that recommend its use on a commercial scale. The choice of
which organism to use in a particular application will be made largely on
economic grounds.
The NIH has recently approved the use of varous species of Streptomyces as
host orgcnisms in recombinant DNA experiments (see '45 FR, 5053',). These
microorganisms are especially important in the drug industry owing to their
ability to manufacture the aminoglycoside class of antibiotics, including
streptomycin, erythromycin, and tetracycline. The application of recombinant
DNA technology to these microbial strains promises to generate improvements in
product yield and, perhaps, to new and useful types of antibiotics.
NIOSH and NIH have examined the issue of worker safety in the pharmaceutical
industry within the context of recombinant DNA activities. The NIH 'has
proposed recommendations for large-scale fermentation of recombinant DNA
organisms (analogous to the PI to P4 designations for laboratory
experimentation). Commercial firms are expected to comply voluntarily with
these recommendations. So far, two U.S. firms, Eli Lilly and Genentech, have
been granted NIH approval to proceed with scale-up operations.
In the spring of 1980, a NIOSH team conducted walk-through surveys of both
these facilities. Eli Lilly operates a state-of-the-art commercial fermentation
plant. All operations are closely monitored for leakage or contamination of
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biological material. Equipment is designed to minimize the formation of
aerosols and to initiate sterilization procedures in the event of an accidental
spill. Programs to ensure worker safety and health are in place, including
medical surveillance, periodic safety inspections, monitoring employee work
practices, and the provision of safety equipment and protective clothing.
Similar programs have yet to be instituted at Genentech, a firm that was
founded in 1976 and has !00 yecrs less experience than Lilly in large-scale
fermentation operations. NIOSH, therefore, has recommended that Genentech
plan immediately to implement similar safety and health protocols.
In summary, the pharmaceutical industry as a whole appears to be well equipped
to deal with the various experimental and engineering safety issues that are
posed by the advent of recombinant DNA technology. This industry historically
has been involved in the "business of biology," and there exists a long tradition of
safety associated with their operations. Moreover, a firmly established regula-
tory apparatus (largely housed in the FDA) already exists that closely monitors
activities and screens new products originating from this industry. One must
conclude that new products and processes stemming from various applications of
genetic engineering in the pharmaceutical field will encounter the same careful
scrutiny that has been devoted to conventional activities.
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4.2 Chemical industry
4.2.1 Current activities
While not attracting public attention to the extent that interferon has, the
chemical industry has been influenced by recent advances in biotechnology.
Moreover, the industry may be on the verge of a technical revolution in which
biological processes and renewable resources will rapidly replace the physical-
chemical transformations of petroleum feedstocks upon which the industry is
currently based. This section will attempt to outline some of the applications of
biotechnology that are now in use end which serve as prototypes for the kinds of
bioprocesses that may soon pervade this industry.
One chemical process utilizing biotechnology that has received some attention is
under development by Cetus in conjunction with Chevron Oil. The process
entails oxidation of alkenes to the corresponding alkene oxides. These end-
products are utilized in enormous quantities for plastics manufacture; for
instcnce, ethylene oxide and propylene oxide are the raw materials for the
production of polyethylene and polypropylene, respectively. The Cetus/Chevron
bioprocess consists of three enzyme-catalyzed steps, as follows:
glucose + ^
> fructose + HLO,
propylene
+, bromoisopropanol
bromoisopropanol — . ¦¦ » propylene oxide + KBr
enzyme 1 = glucose oxidase
enzyme 2 = chioroperoxidase
enzyme 3 = halohydrin epoxidase
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The process is now undergoing pilot plant scale-up. The plan calls for designing
an immobilized enzyme bioreactor in which the three enzymes are stably linked
to an inert mctrix. A continuous flow process ensues in which starting materials
are percolated through the reactor, and products (fructose and alkene oxide) are
recovered at the reactor outlet. It remains to be seen if this process is
economically competitive with conventional alkene oxidations. Moreover, since
the alkene starting material will generally be obtained from petroleum feed-
stocks, the process fails to overcome the dependence on dwindling and ever-
more-costly oil supplies.
The general use of microbial enzymes in industrial orocesses (Taole4-2) is
rapidly becoming a big business. One estimate places tie 1985 market in enzyme
technology at $500 million. The food industry historically has been the orimary
user of enzynne-based processes, and will continue in this role as demand
increases for sweeteners Cerived from cornstarch and from other less conven-
tional forms of biomass. 3ut rising demand for gasohol will lead to further uses
for enzymes in ethanol oroduction. Three general classes of enzymes are finding
increasing commercial use:
• Amylases break down polysaccharides, such as cellulose,
and mediate biomass conversions;
• Proteases break down proteins and ere used commoniy in
the food industry, for examole as meat renderizers;
• A miscellaneous group, which includes oxidases and
isornerases capaole of performing specific chemical trans-
formations of substrates, may soon find considerable
utility in the chemical industry.
These industrial processes utilize microbes gs sources of biological catalysts
(enzymes) that, in turn, convert organic starting materials into products. A
large variety of microorganisms directly synthesize simple organic chemicals
when grown on carbohydrcte substrates (see Table 4-3). Since many of these
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Table k-2
Commercial uses of enzymes
mzyme
Uses
proteases:
alcalase
bromelain
papain
pepsin
trypsin, ficin, and
streptodornase
rennin
carbohydrases:
aicyiase
amyloglucos lease
cellulase and
hemicellulase
glucose iscmerase
invertase
lactase
pectinase
C 3. L ci ciS 6
detergent additive to remove protein stains
neat tenderizer
stabilise chill-proof beer; meat tenderizer
digestive aid in precooked foods
wound debridement
cheesemaking
digestive aid in precooked foods
production of dextrose from starch
preparation of liquid coffee concentrates
and conversion of cellulose to sugar
production of high-fructose syrups
prevention of sugar granulation
prevention of lactose crystals in ica cream
clarification of wine and fruit juices
peroxide removal in cheesemaking
lipase
lipoxygenase
flavor production in cheese
bread whitening
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Table k-3
Organic compounds obtainable by microbial fermentation
Compound
Structure
acetone
acetic acid
acrylic acid
butanol
citric acid
ethano1
ethylene glycol
furfural
0
ch3cch3
ch3cooh
CH?=CHC00K
ch3ch2ch7ce,oh
HOOC-£- (CH0COCH) ry
OH
ch3ch9oh
hoch?ch9oh
CHO
gluconic acid
COOH
I
(CHOH),
I *
r-ij nu
giyceroj
HCCH 0 C H CH 0 OH
21 2
OH
isopropanol
rdvCHCP*
3I 3
OH
itaconic acid
COOH
I
C=CH,
I
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Table 4-3 (cont.)
Compound
Structure
keto-giuconic acid
COOH
I
C=C
I
(CKOE).
I
CH OH
lactic acid
COOH
I
CHOE
I
CH.
malic acid
CCOH
I
CHOH
CH^COOE
metnano
propionic acid
tartaric acid
CH30H
CK3CH0COOH
COOH
(CHOH)
I
COOH
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compounds are toxic at relatively low concentrations, considerable research
effort is being expended to generate microorganisms that tolerate higher doses
of these organics. Also, modern fermentation technologies, such as continuous
flow and solid state processes, will be useful here since metabolic products never
accumulate to poisonous levels. In addition, the use of unconventional substrates
for microbial fermentation, such as cellulose and lignin wastes, is rapidly
becoming feasible. These technical advances may soon make economic biopro-
duction of these and many other organic compounds possible.
A number of microbial species growing on carbohydrate are able to synthesize
surfactants or detergents. These compounds are typically long-chain fatty acids;
current commercial production of surfactants requires petroleum feedstocks.
The British sugar producer, Tate & Lyle, is now engaged in pilot-scale develop-
ment of this process. Similar microorganisms are exploited for the production of
polysaccharides for use in the food industry and the production of chemical
flocculants, or precipitating agents, for use in sewage disposal.
Photosynthetic algae offer the prospect of direct conversion of sunlight into
useful organic chemicals. A considerable variety of end-products may be
obtained from marine algae as shown in Figure ^-3.
t
Whereas certain microorganisms can syntnesize various simple organic com-
pounds, some higher plants have acquired the ability to manufacture rather
complex molecules. As shown in Table 'a-'a, these substances include rubber and
petroleum substitutes, insecticides, steroids, anc other drug precursors.
The potential applications of genetic engineering in the chemical industry lie
largely in the area of organics production. Many organic chemical feecstccks
can be produced utilizing fermentation technology (see Table k-3). In principle,
the efficiency of microbes in any fermentative process can be improved by using
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Figure 4-3
The extraction of useful chemicals from algae
Source: Sanderson, J.E., etal. (1979)
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Table 4-4
Examples of useful chemicals derived from plants
Plane Substances Uses
gopher plant
(Euphorbia)
jojoba
(Simmondsia)
buffalo gourd
(¦Cuourbita)
guayule
(Fartkenium)
scorpion flower
(.Fhacelia)
milkweed
(Asalepias)
juniper
(Pinaceae)
Varthemia candicans
¦Jotrovha
meadowfoam
(Limianihes)
money plant
(Lunaria)
bladderpod
(Lesquerella)
thistle
(Chamaspsuoe)
kinkaoil irenweed
(Vemonia)
hartleaf Christinasbush
(4 tdhomea)
latex
sterols
long-chain esters
starch
xinoleic acic
latex
latex
chromenes
latex
silk-like fiber
terpenoids
cadinene
sesquiterpene lactones
vegetable oils
fatty acids
fatty acids
hydroxy fatty acids
hydroxy fatty acids
epcxy fatty acids
epoxy fatty acids
rubber, petroleum
substitutes, drugs
surfactants, emulsifiers,
waxes, lubricants, pre-
servatives, cosnetics
sweeteners
edible oils
rubber.
rubber
insecticides
rubber, chemical feed-
stocks, textiles
antimicrobials
insecticides
antimicrobials
surfactants
surfactants
surfactants
lubricants
lubricants
ointments
lubricants
dlas t ic s
coatings
coatings
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recombinant DNA techniques or other biotechnologies. The extent to which
biological processes will supplant chemical processes in the chemical industry
will surely be a function of economics; the future cost of petroleum will be
particularly influential. Though the entire chemiccl industry uses only 7 percent
of the petroleum supply in the United States, this industry is neavily dependent
upon this resource.
Fermentation technology is not new to the chemical industry. Prior to World
War II (before the introduction of cheap oil), scores of chemicals were manufac-
tured by fermentation processes. For example, only 36 percent of total ethanol
production during the mid-1940's was based on petroleum sources; the remainder
was made biologically. However, ten years later, almost 60 percent of the
ethanol production was derived from oil. Fumaric acid was also manufactured on
a commercial scale by fermentation. Production by this route ceased when a
more economical synthesis from benzene was developed. In general, once a
chemical process using petroleum was aeveloDed, it quickly replaced the existing
fermentction process.
In spite of this history, a few chemicals are now produced by fermentation,
notably citric acid, lactic acid, and various amino acids. These processes have
all been improved over the years using applied genetics (e.g., microbial mutagen-
esis), but recombinant DNA technology has yet to have an impact in this crec.
Citric acid is the most important acidulant in the food industry, representing
55-65 percent of The acidulant market. This acid also has pharmaceutical and
chemical processing applications. Citric acid is produced commercially using the
fungal organism, Aspergillus niqer. The efficiency of this mold has been
dramatically improved using mutagenic techniques. A four-folc increase in
product yield has been obtained.
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The bacterium Lactobacillus is used in the commercial production of lactic acid
Large quantities of this product are obtained using such raw materials as
sucrose, glucose, and lactose (from cheese whey). Most of the problems in the
manufacture of lactic acid exist in product recovery, not in the fermentative
process itself. Thus far, biotechnology has been applied very little to improve
this industrial process.
World production of amino acids is currently dominated by Japan; there is very
little domestic U.S. production. The bulk of amino acids, production is destined
for research aopiications and to nutritional or biomedical preoarations. Three
amino acids are particularly useful: glutamic acid for the production of
monosodiurr, glutamate (MSG), a flavor enhancer; lysine and methionine as
animal feed additives.
Glutamic acid production provided the first instance in which biotechnological
methods were applied to enhancing amino acid production. The method involves
the manipulation of microbial growth conditions and isolating mutant strains.
Glutamate is produced in the presence of ammonia by a species of
Corynebacterium. Growth of this particular species ciso requires the addition of
biotin to the growth medium. In the presence of low concentrations of biotin,
bacterial cell membranes become leaky to small molecules, thereby permitting
glutamate to diffuse out of the ceil. But at high biotin levels, the membranes
are normal and prevent glutamate secretion. Furthermore, the biosynthesis of
glutamate is reduced in the presence of high biotin levets through a feedback
inhibition mechanism.
Lysine is produced both by chemical and fermentation processes. This represents
one example where the chemical production method has not totally replaced the
biological procedure. Due primarily to the lower direct operating costs incurred
by fermentation procedures, about 80 percent of the lysine production world-
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wide in 1980 was via microbial means. The United States imported about 7,000
metric tons of lysine in 1979.
Recent announcements made by Bethesda Research Laboratories (3RL) indicate
that recombinant DNA technology has been used to isolate some of the genes
required in the synthesis of the amino acid oroline. BRL is currently seexings
ways to exploit this discovery on an industrial scale.
Table 4-5 lists those amino acids that are produced microbiologicalty and the
bacterial species used in their manufacture.
4.2.2 Future prospects
The ease with which applied genetics has been integrated into the-pharmaceut-
ical sector is a result of that industry's predisposition towards-the biological
sciences. On the other had, the chemical industry depends largely on the
technical disciplines of physical and organic chemistry end chemical engineering
for its commercial foundation. Recent decades have seen remarkable advances
in the mass production of industrial chemicals that have benefited society in
numerous ways. Agricultural chemicals have improved food production,
synthetic fibers have revolutionized the clothing industry, plastics influence our
lives in countless ways, and so forth. But traditionally the chemical industry has
not involved itself with biological processes. Only within the past few years
nave chemical firms, such as Allied, Dow, DuPont end Monsanto, undertaken
programs to examine Diotechnology as a way of doing business in the future.
A variety of issues relevant to the future of biotechnology in the chemical
industry can be adduced.
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Table 4-5
Fermentative production of amino acids from glucose
Amino acid
Yield
(gm/1)
Microorganism
DL-Alar.ine
40
CorynebacteiKivv geZatilnasum
L-Arginine
29
Br,evibaotevi:.m fZ avian
L-Citrulline
30
Bfevibactevium fZavvjn
L-Hiscidine
10
Brsv-ibactei^ivjv. flavian
L-Homoserine
15
Corunebacteviim gZutam-Lc>an
1-Isoleucine
15
Brsvibacievivjn flavu/v
L-Leucine
28
Brevibactevivjn I a ate fs merit ion
L-Lysine
32
3r&vibactevium fZavvm
44
Co vu neb acte ri um glu tanri ciar
L-Ornichine
26
Covunekaoieviicr, gZutcanicvun
L-Phenylalanine
2
Brevi-bacteriivr. fZavim
6
BaciZZus s^bviZis
L-Proline
29
Erevibaaterium ft avian
L-Threonine
18
Bvevibacteriwn flav-m '
L-Trypcophan
2
Brevibaaieriwr, fiavum
1-Valine
23
Bvevibac~svivan Za.oicfevmeniurn
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• It is unlikely that biological processes will be applied in
the near future to the large-scale manufacture of most
commodity chemicais; i.e., bulk chemicals whose produc-
tion capacity is measured in the millions of pounds
annually. Although many of these products are derivec
from ever-more-costly petroleum feedstocks, bioproces-
ses will be unable to compete economically with tradi-
tional synthetic routes for 10-20 years or more. There
exist obvious exceptions to this general conclusion, sucn
as ethanol and some short-chain organic acids (see Table
4-3), but even these substances will be more cheaply
produced by conventional methods for some time to come.
• A significant role for applied genetics in the chemical
industry will be in the manufacture of high-priced special-
ty chemicals or in synthesizing new chemicals that have
no practical alternative route. Enzymes will be employed
as highly specific catalysts for performing discrete chem-
ical steps in a synthetic route. Microorganisms that
express the desired enzyme activity may be used directly.
Microbes will be sought that carry out chemical trans-
formations otherwise requiring large inputs of energy,
such as hydrogenations, amidations, etc.
• The economics favoring the use of bioprocesses in the
chemical industry will depend substantially on process
design and engineering characteristics, rather than on the
biotechnology involved. This is true for the chemical
industry to a much greater extent than for the pharma-
ceutical industry. Thus, practical cpplications of biotech-
nology in this industrial sector will appear slowly end only
following extensive anclysis of the relevant biochemical
engineering factors.
4.2.3 Potential hazards
The near-term role of applied genetics in the chemical industry predicts that
bioprocesses will be developed, that .perform chemical transformations on
specific feedstocks to manufacture specialty products. Conseauently, the
industry will be compelled to engage in large-scale microbial fermentations in
order to obtain the necessary reagents (either the organisms themselves or the
enzymes they synthesize) to perform these chemical reactions. Such
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fermentations, and subsequent product isolation procedures, will proceed in a
manner entirely analogous to similar operations in the pharmaceutical industry.
There exist differences, however, that may be of concern from an environmental
or safety and health standpoint:
• The species of microorganisms likely to be utilized in the
chemical industry differ from those in the drug industry.
For example, various species of Pseudomonas,
Acinetobacter, and Flgvobacteria may find application in
mediating chemical processes because these organisms
naturally possess enzyme systems capable of catclyzing
chemical reactions involving organic substrates (such as
petroleum products) that are of interest to the chemical
industry. Many of these microbes are opportunistic
pathogens in man; that is, they infest skin lesions or cause
severe infections in individuals who are already weakenea
by a pre-existing ailment.
• The chemical industry is unaccustomed to the application
of biological processes as a business enterprise.
Commercial-scale fermentations are alien to this
industry. Chemical firms interested in adopting one or
another bioprocesses may choose to purcnase the
technology, or to obtain the service through outside
contract, rather than develop in-house capabilities.
• The chemical industry has a poorer record than the
pharmaceutical sector in areas relcted to worker safety
and environmental protection. This discrepancy may
reflect the grossly aifferent commercial operations
performed by these two industries rather than neglect.
Nevertheless, one might be apprehensive of The
introduction of a new technology into an industry where,
historically, hazards have surfaced only after serious
harm was done to workers or the environmenr.
In the long run, the replacement of conventional chemical processing steps with
biological processes should serve to reduce the level of overcil risks. The
microbes or enzymes that mediate the bioorocess will be susceptible to
inactivation by high concentrations of many organic feedstocks. Thus, feed
streams will have to be diluted with non-toxic substances to obtain
concentrations that permit survival of the biological systems :nvolved. As a
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consequence of this dilution, the feed streams will become less toxic to workers
who run the processes end to an environment that may encounter the stream in
the event of a spiil.
All currently envisioned applications of biotechnology in the chemical industry
anticipate the use of closed bioreactor systems for performing discrete chemical
reactions or for growing large volumes of microorganisms for use as biocatalysts
or as sources of substitute feedstocks. Experience accumulated in the
pharmaceutical sector indicates that routine operation of such systems poses
minimal environmental hazard. A typical fermentation operation is depicted in
Figure 4-4. Each step in the process, including douDle-seaied stirring rotors,
positive pressure inside the vessel with loss-of-pressure alarms to warn of a
breach in containment, and pre-sterilization of all added materials, including air,
anti-foaming agents, acid, and base for pH control, is conducted to ensure
sterility and containment. Furthermore, since the air vented from the fermentor
generates aerosols containing microorganisms, this exit gas should also be
sterilized. Although not performed routinely, this can be accomplished by
passing the air through high-efficiency particulate cir (HEPA) filters, or by
exposing the gas stream to radiation, electrical dischcrge, or germicidal sprays.
The fermentation process shown in Figure 4-4 involves sterilization of the
reactor contents prior to sample work-up; that is, the microbes are killed before
they are discharged from the vessel. The chemical industry might employ such a
procedure in order to isolate an enzyme that the microbes have accumulated
intracellular^ or excreted into the medium. Figure 6.-5 dicgrams a process flow,
including feed streams end waste streams, for isolation of an intracellular
enzyme. The wastes from these processes consist of highly vcriabie liquid
streams containing high levels of suspended solids. These wastes typicclly have
elevated chemical end biochemical oxygen demands (COD and 30D), as well as
significant nitrogen ana phosphate loadings. The pH is generally in the
acceptable range, pH 5 to 9.
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The application of biological processes in the chemical industry is in a very early
developmental state. The ability of microorganisms, or their products, to
mediate chemical transformations of organic substrates on a commercial scale
has yet to be demonstrated. It seems probable that processes based on microbial
systems will rely on activites that occur naturally among populations of
microorganisms. Thus, genetic engineering to endow the microbe with new
characteristics will find limited application for the foreseeable future; that is,
for the next five to ten years.
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Figure 4-4
Steps in a typical fermentation process
• • Mec'urn itdw* 2. Srsr 'lzcrion j. 'noc*-.ct;cj"
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Figure 4-5
Product recovery from a typical batch fermentation
Typical
sc stream
3rocX'C* Mass
500 liters cells
*•00 3 procwct
: yO
Soiven? wosh
•i Cen-r
ifugs
3C0
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4.3 Energy industry
4.3.1 Current activities
The potential applications of biotechnology in the energy field are vast.
Proponents anticipate that c sizeable proportion of future world energy needs
will be met through biological processes. Genetic engineering of microbes and
higher plants will undoubtedly have significant impact on the development of
future bioenergy systems, although activities to date nave shown little evidence
of this practice. Current activities in this industry will be considered within two
general areas: energy from biomass and enhanced oil recovery.
4.3.1.1 Energy from biomass
Biomass resources encompass cil the storage repositories of solcr energy. This
includes photosynthetic organisms of all types, organisms that feed on photosyn-
thetic biomass, end animal wastes. Biomass is a renewable energy source, a
quality that distinguishes it from fossil fuels, which are aiso derived from
biomass, but which require eons of time to develop. The energy content of the
carbohydrates generated annually in higher plants alone has been estimated to be
ten times the globcl energy consumption, "["he inclusion of marine biomcss, such
cs phytoplankton, might increase this factor another ten-fold. Clearly, tapping
this vast.energy supply must be consiaered c top priority in the years ahead.
Biomass fuel sources have five major advantages over fossil fuels:
• They are renewable:
• They do not contribute to carbon dioxide pollution be-
cause, at a steady state, carbon dioxide is incorporated
into plant material and removed from the atmosphere at
the same rate that it is put into the atmosphere by
combustion;
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• The rate of carbon dioxide fixation into usable plant
material by photosynthesis is fifty times greater than our
current rate of fossil fuel consumption;
• Biomass ootential is more evenly distributed geographi-
cally than are fossil fuel reserves; and,
• The potential market in biomass is huge allowing R&D
costs to be amortized over a large number of production
units.
Biological systems useful in the conversion of biomass to liquid fuel have not
been intensely developed. Current commercial practice is founded on the
production of alcohol for distilled beverages. Corn is the main feedstock and the
yeast Saccharomyces cerevisiae is the principal fermentation organism. It is
clear that cerevisiae can be made to convert carbohydrates by fermentation
to ethanol with a much higher efficiency than is currently achieved. This higher
yield can be approached in two ways: (1) a greater mess of ethanol cGn be
produced per mass of carbohydrate consumed, and (2) a product with a higher
percentage of ethcnol can be produced. The overall efficiency of the process
can be improved by exploring mixed bacterial-yeast fermentation systems cind by
adapting the whole fermentation process to a continuous flow mode.
Ethanol for use as fuel, either alone or mixed with gasoline to make gasonol, ccn
be produced by microbial fermentation of sugars. Two sources of sugars cbound.
First, starch (for which fermentation technology is well advanced) is available
from edible plant products, such as corn, wheat, potatoes, sugar cane, sugar
beets, and cassava. Second, cellulose, from which conversion to ethanol is
difficult, is abundant in municipcl/agricultural wastes and forests. Utilizing
starch as the feedstock for ethanol production- entails a diversion of crop land
that could- otherwise contribute to the food supply. This disadvantage has not
deterred the government of Brazil from investing $5 billion during tne past
decade on facilities to mcnufacture eThanol from cassava, sugar, and molasses.
Ultimately, all of Brazil's motor vehicles will be run on ethanol. It is estimated
that 2% of the nation's land will be devoted to this enterprise.
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Ethanol represents one of the most promising alternative fuels to OPEC oil. it
can be burned in conventional automobile engines without modification as a 20%
alconol/80% gasoline mixture. Relatively minor engine and fuel system adjust-
ments are required to convert gasoline engines to 100% ethanol use. The Ford
Motor Co. of Brazil currently sells a conversion kit for about $250 that will
convert a standard auto engine to permit use of 100% ethanol. Alconol can be
efficiently handled as a fuel by existing petroleum distribution networks.
Ethanol production by yeast may be greatly enhanced using molecular cioning
techniques. The biochemical pathway unique to ethanol metabolism is relatively
simple. Pyruvate is converted by the enzyme pyruvate decarboxylase to
acetaldehyde and carbon dioxide. Acetaldehyde is convertec to ethanol by the
enzyme alcohol dehydrogenase. The gene for alcohol dehydrogenase has been
cloned in several laboratories and it aopears possible to increase the efficiency
of the fermentation process by increasing the level of alconol dehydrogenase in
the cell using genetic engineering techniques. The other enzyme in the process,
pyruvate decarboxylase, should also be amenable to genetic engineering.
In the long run, ethanol production from cellulosic wastes will be oreferable to
using foodstuffs as the raw material. Typical cellulosic materials consist of 50%
cellulose (a glucose polymer), 25% hemiceilulose (a polymer of xylose, a f;ve-
carbon sugar), and 25% lignin (a complex phenolic polymer). This semi-
crystalline lignocellulase is broken down with difficulty into fermentable
constituents, by ccid treatment or by the enzyme cellulose. This expensive
initiai phase of cellulose preparation is where process improvements are most
needed. Figure 4-6 provides a general scheme for ethanol production utilizing
either cellulosic or starch feedstocks, anc Figure 4-7 provides an overview of the
variety of petrochemical feedstocks that can be ootained from cellulosic starting
materials.
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Figure k-6
Steps in the conversion of biomass to ethanol and by-products
Inputs Processes Outputs
Source: King, S.3. (1979)
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Figure 4-7
The conversion of lignocellulose into useful chemical feedstocks
CH, = CH,
Ethylene
(CsH13Os)n Hydrolysis ^8Hi2°s Fermen- ^ C2H«OH
Cellulose Glucose tation Etnanos
x
Butadiene
CH - CH = CHj
HOCH2 - C = CH - CN = C — CHO
Hydroxymethyl- » Leyulmic
Fur'ural acid
CH.COCH.CH.COGh
J c Z
(C5H804Jn
C5H1305).
Hemiceiluiose
c5h12o5
Mannose.
=ermentation
C5H:c05
Xyiose .
c:h5oh
Etnanoi
Acid
CH 3 CH — CH = C - CHO
Furfural
Lignin
Hydrolysis
Hydrogenation
Pvrciysis
Phenoiic
mixture
c5h5oh_
Phenol
C£H6
3enzsne
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Methane generation by anaerobic digestion of biomass provides another route
whereby renewable resources are utilized for energy proauction. Animal feedlot
wastes and municipal sewage are most often cited as providing the raw materials
for this process, although forest residues and food crop biomass are also suitable.
The process produces:
• Biogas, consisting of approximately 60% methane and 40%
carbon dioxide;
• Residual solids, containing vegetable proteins, which have
potential value as feed additives or fertilizers; and,
• Spent process water, laden with nutrients, which is suit-
able for growing algae or as a fertilizer.
Since the process occurs in closed digesters to exclude oxygen, the waste
matericls used as feedstock are prevented from spoiling the environment or
giving rise to pathogenic organisms. Anaerobic reactors are classified into three
types depending on the operating temperature (i.e., the optimal temperature for
growth of the particular microbial strain involved): (!) psychophilic (under 20°C),
(2) mesophilic (20° to 45°C), and (3) thermophilic (45° to 65°C). A typical
digester used for sewage treatment is depicted in Figure 4-8.
Most applications of this technology involve small, community-scale operations,
Biogas generators associated with large animal feedlots or municipal sewage
treatment facilities might readily supply the energy neeas of the local popula-
tion. Simple anaerobic digesters of this type are common in the Peoole's
Republic of China, Korea, Taiwan, and India. But large-scale oDerations may be
feasible. A study commissioned by the U.S. Science and Education Administra-
tion of the USDA found that economical biogas production could be achieved
witn feedlots averaging 1,000-2,000 head of cattle in size. Others have proposed
a large, centralized facility that could produce 50 million cubic feet of methane
per day using biomass crops as feedstock. Also, the mass-cultivation of water
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Figure 4-8
A single-tank anaerobic digester of biogas production
Oigastar gas
Source: U.S. Environmental Protection Agency (1979)
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hyacinths on sewage lagoons for use as a fermentation substrate has been
proposed. Current R&D efforts in this area tend to emphasize aspects of process
design and engineering rather than microbiology. Nevertheless, genetic engin-
eering may have a significant role in future developments of biogas production.
Hydrocarbons are synthesized and accumulated by a wide variety of bacteria,
algae, and yeasts (see Table 4-6). These microbes generally extract carbon
dioxide from air and utilize energy derived from photosynthesis to reduce
chemically and oolymerize CC^ into long-chain lipids. Some microbes utilize
carbohydrates such as glucose as carbon sources. As much as ^0-50% of the dry
weight of certain oil-bearing microorganisms can consist of reduced hydrocarbon
materials suitable as substitute fuels. Many higher plant species produce a sap
or fruiting body that is high in hydrocarbon content. Most familiar are vegetable
oils, such as sunflower, cottonseed, linseed, palm, etc., some of which ere under
investigation as diesel fuel additives. A variety of less familiar tropical plants
and trees is also unaer examination as hydrocarbon producers.
The production of hydrogen gas from water has oeen demonstrated in laboratory
studies—its commercial-scale feasibility remains to be shown. The system
utilizes units of photosynthetic activity, called chloroplasts, isolated from green
plants, such as lettuce or spinach. A biophotolysis reaction is established in
which energy from sunlight splits water (H^O) into molecular hydrogen (h^) and
oxygen (C^). Successful operation of the system requires a means of removing
oxygen to prevent reaction with hydrogen to regenerate water. Rather than
isolating chloroplasts from higher plants, it may be preferable to use intcct,
photosynthetic algae or bacteria. The future use of hycrogen as c fuel offers the
promise of a non-polluting, inexhaustible energy source. However, numerous
technical obstacles remain before this prospect will be realized.
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Table 4-6
Some species of aigae that produce hydrocarbons
Species
Lipid content
(% dry wt.)
3idd.ulph.ia audita
12.2
Crlcarydomonas qpp lanate
32.8
Chi ore 11 a pyrenoidosa
14.4
Chlorella vulgaris
28.8
IZcnallanthus salina
40.8
'lannochloris so.
20.2
Nitzsakia galea
22.2
Gocystis pclyrr.orpha
12.6
Outocooc^js sp.
27.C
Skeletonerva CGsiaium
23.8
Source: Shifrin, N.3. and Chisholm, S.W. (1980)
in "Algae Biomass", p. 633, Elsevier Press.
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4.3.1.2 Enhanced oil recovery
A second major area of the energy field in which applied genetics will have an
impact is not the creation of new sources of energy but enhanced recovery from
existing energy supplies. Primary and secondary oil recovery techniques manage
to extract only about one-half of a known oil reservoir. An estimated 200 billion
barrels of oil in the continental United States remain out of reach with
conventional recovery techniques. A variety of microbial-based tertiary recov-
ery methods has been proposed as a means to tap this vast resource. These
include:
• The injection of oil-degrading bacteria into an oil field
would reduce the oil's viscosity, or convert oil to natural
gas;
• The injection of microbes to re-pressurize a spent oil well
by synthesizing carbon dioxide or other gaseous metabo-
lite;
• The injection of microbes that manufacture and secrete
chemical surfactants that would act to mobilize tightly
bound oil.
In addition to these potential applications in existing oil fields, microbial
processes have been promoted for use in extracting tar and oil (bitumen) lodged
in tar sands. Also, a bacterial process is under development at the University of
Southern California that would release kerogen (a petroleum material) from oil
shale. This process could generate a barrel of oil per ton of western oil shcle
without extensive ore crushing, retorting, or environmental damage that attend
strictly physical recovery methods. Finally, analysis of subsoil microbial popula-
tions may assist in locating previously unknown oil and gas fields below. This
microbiological method of prospecting for petroleum is stiil in an early stage of
commercial development, as are all of the microbial recovery methocs described
above.
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k.3.2 Future prospects
The range of potential uses of applied genetics in the energy industry appears to
be far wider than in the chemicals sector. However, most of these possibilities
lie far in the future, at least with regard to large-scale commercial application.
Development of systems for ethanol production from biomass for use in gasohol
are proceeding apace, especially in petroleum-poor areas like Brazil, but the
economics of this process and the energy savings incurred will remain unfavor-
able, probably for the remainder of the 1980's. Nevertheless, several long-range
projects can be envisioned that may one day provide significant sources of
energy.
• Mass production of hydrocarbon substances from various
species of higher plants, including those listed in
Table b-k, can become economically feasible when either
(I) plant cells are manipulated to grow in massive cultiva-
tors, akin to microbial fermentors, in which excreted
hydrocarbons are continuously collected, or (2) the genet-
ic information that enables the plant cell ro synthesize
hydrocarbons is transferred to microorganisms which, in
turn, manufacture and excrete the fuel-like substances.
The biotechnical and engineering obstacles that stand in
the way of such a project are formidable.
• A biological solar battery will someday replace the panels
of silicon solar cells that find specialized uses today. The
biological battery will operate via a direct conversion of
sunlight into electricity (i.e., a current of electrons) that
is generated during photosynthesis. Although all green
plants engage in photosynthesis and are, therefore, suit-
able sources of materials for constructing a biological
battery, a primitive, purple photosynthetic bacterium,
called Rhodospirillum rubrum, may be exploited as the
living solar cell. Alternatively, the pnotsynthetic blue-
green algae, which utilize carbon dioxide and nitrogen
directly from air may serve this purpose.
• Ethanol production may become more efficient through
use of microorganisms other than common yeasts (e.g.,
Saccharomyces cerevisiae, or brewer's yeast). A bacterial
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species, called Zymomonas mobilis, carries out alcoholic
fermentation two to three times faster than yeasts. This
bacterium, now employed to make tequila, is under inves-
tigation by researchers at the USOA's Northern Regional
Research Lab in Peoria, Illinois.
• Acidophilic, iron-oxidizing Thiobacilli bacteria (commonly
used in mineral leaching operations, see Section 4.4.1) may
prove useful in oil shale or coal conversion processes. The
bacteria will mobilize the inorganic mineral content of
the shale or coal without affecting the hydrocarbon
content of the material. The porous zones that this
process generates m situ may assist in subsequent retort-
ing or gasification scnemes.
4.3.3 Potential hazards
The application of biological processes to the energy industry is ct a very early
stage of development. Other than the fermentation of ethanol from cornstarch
for use in gasohol production, no commercial-scale bioprocess will have an
impact on the energy sector for at least five years. The oroduction of biofueis
(e.g., ethanol, methane, vegetable hydrocarbons) from unconventional feedstocks
has progressed only to the pilot scale, whereas biologicci hydrogen production is
little more than a laboratory curiosity at present. Likewise, field tests have so
far failed to demonstrate the general feasibility of using microbial systems for
enhanced oil recovery. Thus, potential environmental hazards resulting from the
use of applied genetics in energy production are highly speculative.
Nevertheless, several comments are appropriate and some areas of potential
concern can be identified.
• The production in the United States of sufficient ethanol
to have a significant impact on domestic fuel supplies will
require the diversion of enormous quantities of food
crops, particularly corn. According to one estimate, a 4
billion gallon-per-year ethanol program could result in a
10 to 20% shortfall in corn supplies by 1990. This would
severely limit the availability of grain for livestock feed
or exports, thereby driving up food prices. Four billion
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gallons represent less than 5% of current annual fuel
consumption. Clearly, useful alternative biomass feed-
stocks for ethanol production are sorely needed.
The prospect that cellulosic materials may serve as
suitable feedstocks for biofuet production forewarns of
large-scale deforestation, particularly in areas lacking
alternative sources of biomass. Huge tracts of prime
forest land in certain parts of the world have already been
cleared for purposes of agriculture or fuel use. The
wholesale conversion of wood biomass into ethanol
threatens to exacerbate this trend.
Processes designed to convert lignocellulosic materials
into substrates suitable for ethanol fermentation entail an
initial hydrolysis step (see Figure 4-6). Hydrolysis can be
accomplished either chemically, using strong mineral
acids, or biologically with enzymes. The latter approach
is preferable from a safety and environmental point of
view but is less likely to be implemented in the near term.
Thus, commercial processes generating large quantities of
acid wastes can be anticipated.
As mentioned previously, the utilization of wastes and
municipal sewage as raw materials for biogas generation
promises to lessen the environmental burden imposed by
these pollutants. Hazards may arise, however. If large
centralized biogas facilities are planned, then one faces
risks associated with the transport of the raw wastes to
the site from various points of origin. A program to
establish numerous local biogas generating stations may
encounter variations in operating characteristics or in the
level of personnel training that could mitigate against
long-term safe operation of any particular facility.
The species of microorganisms likely to be utilized in
enhanced oil recovery schemes -- Pseudomonas and
Acinetobacter, for example — ere the same as those
mentioned previously in the context of biotransformations
of organic substances in the chemical industry. As
already discussed, these microbes are potentially serious
pathogens in man.
It is probable that the near-term, use of microorganisms to
mediate bioprocesses in the energy industry will exploit
naturally occurring microbes. Thus, as is true for the
chemical industry, the impact of genetic engineering
(especially recombinant DNA techniques) will be minimal.
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• Finally, established energy companies are not accustomed
to dealing with biological systems as a means of producing
energy. (Most oil companies, however, do maintain some
expertise in microbiology to assist in prospecting.) These
firms will be compelled to strengthen their technical
competence in areas related to biology as commercial
orospects for bioenergy brighten. Hopefully, they will
devote adequate attention to environmental hazards that
may emerge from these new areas of business.
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4.4 Mining industry
4.4.1 Current activities
The impact of biotechnology on the mining industry is currently quite limited in
scope, consisting of two general areas:
• The accumulation of metals by organisms, either by
binding at. ceil surfaces or by intracellular uptake of
metals; and,
• Biochemical transformations of metals, including
solubilization or precipitation, oxidation/reduction pro-
cesses, and the interconversion of inorganic and organic
metal compounds.
The various bioprocesses subtended under these categories are all carried out by
a relatively small number of bacterial species. Figure 4-9 lists these organisms
and summarizes the means by which these microbes extract energy from
chemically reduced inorganic compounds (such as ferrous iron or sulfur com-
pounds) and employ either inorganic (CC^) or organic carbon sources.
The microbial orocess whereby metals are solubilized from their ores is called
bacterial leaching. The operation consists of percolating acidified water through
heaps or dumos of low-grade ore that may contain up to four billion tons of rock.
Bacterial action within the dump oxidizes mineral sulfide, producing sulfuric
acid, and soluDilizes the metal. The solution, or leachate, is collected and
processed to recover the dissolved metal. The residucl liquid, containing sulfuric
acid and ferrous/ferric iron, is recycled to the dump. This somewhat crude pro-
cess has been used in mining operations since Roman times. Currently, its
greatest use occurs in copper and uranium mining operations. Approximately
12% of U.S. copper production stems from dump leaching of this ""ype.
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Figure 4-9
Leaching bacteria: organisms arid basic metabolism
Organisms
Thiobacillus ferrooxidans
Lap to spirillum ferrccxidans
Sv. ifolobus
Thermophil ic thiobaciHi
I. fervooxidccr.s
Thermophilic thi obaci11i
Sv.lfolobv.s
Mixed cultures.
I. thiooxiaans
T. fervooxidcrAs
T. thevrnosulfidocxidzns
Sv.lfo lobus
Some heterotrophs
Other thiobaciili
I
Fe
I'
Fe
t-
2+
3+
C0? and/or
Organic substances
\
\
\
Metal sulfides ^ \
(FeS2 or CuFeS2)
Energy Vs.
I'
Metal + sulfate
t-
Elemental sulfur
Soluble inorganic
sulfur compounds
Cellular carbon
biosynthesi s
Source: Bull, A.T., etal. (1979)
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The feasibility of large-scale dump leaching was first demonstrated in 1750 in
Rio Tinto, Spain. The technology is still practiced widely in mining operations
throughout the western United States, including mines owned by Kennecott
Copper at Bingham Canyon, Utah and Santa Rita, New Mexico, as well as the
Butte, Montana mines operated by Anaconda Copper.
Despite the long history of mineral leaching, the role of microorganisms as
mediators of the process was not recognized until the mid-1950's. The principal
microbes involved in copper extraction are Thiobacillus ferrooxidans and Thio-
bocillus thiooxidans. 3oth species are rod-shaped, aerobic bacteria that thrive in
an acid environment (pH 1.5 to 3.0) and use carbon dioxide as a carbon source.
They function within a temperature range of 18° to 40°C (64° to 104°F).
The bacteria require, in addition to water and oxygen, a reduced iron or sulfur
energy source, as seen in the following equations (unbalanced):
Fe"sO, + O, + H-SO, ~ Feii^SO,)-, + Ho0
*4 i. Z 4 Z 4 J Z
SQ -i- 0_ + H_0 ¦- ¦» hLSO,
8 2 2 2 4
H^S + 0^ ~ H2~^4
Ferric iron (Fe'") ana sulfuric acid (H^SO^) generated by these bacterial
reactions are very effective chemical solubilizers for numerous minerals, includ-
ing those listed in Table 4-7. In addition to those shown, other minerals, sucn as
uranium oxides, that co-exist with iron or sulfur-containing ores, ere readily
leached. These reactions occur at rates approximately 500,000-fold faster than
the oxidation of iron end sulfur by air in the absence of bacteria. Both of the
Thiobacillus species are found in great abundance in leaching operations--as
many as 10^ organisms per gram of ore. Indeed, the high concentration of mi-
crobes in leachate solutions poses difficulties during subsequent mineral extrac-
tion and isolation.
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Table 4-7
Minerals readily leached by bacterial action
Minfitdl
Formula
pyrite
FeS7
chalcopyrite
CuFeS 9
c'nalcocite
CU2S
covellite
CuS
arsenopyrite
AsFeS
molybdenite
McS9
stibnite
Sb^S^
pentlandice
¦ NiF2S2
zincblende
ZnS
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Bacterial, leaching is also utilized to recover uranium from low-grade ores, mine
tailings, and other ores that are rich in pyrite. The following reaction pertains
to this process:
U02 + Fe2 (S0^3
UO,SO,. + Fe SO,
L 4 4
This solubilization process has been used in scavenger operations in mined-out
and low-grade stopes in the Elliot Lake region of Ontario. The ores of northern
Ontario are amenable to bacterial leaching due to the presence of large amounts
of pyrite. whereas the uranium deposits in the U.S. Rocky Mountains and
southern Texas contain insufficient pyrite to allow successful leaching opera-
tions.
Other bacterial species have been implicated in mineral leaching, including some
members of the Sulfolobus genus. These bacteria are obligate thermophiles,
requiring a temperature range of 45° to 80°C (HO to I75°F), cs well as an acidic
environment. LeptospiriHum ferrooxidans is another iron-oxidizing acidophile
that has been shown to release pyrite more efficiently than T. ferrooxidans when
grown in mixed cultures with sulfur-oxidizing bacteria.
All organisms, including microbes, can accumulate certain metal ions that are
essential for metabolic activity. Iron, magnesium, zinc, manganese, copper,
cobalt, nickel, moybdenum, and vanadium are required by various organisms,
albeit frequently in trace quantities only. Nevertheless, certain microbes have
evolved highly efficient means of permitting the selective concentration of
metals far in excess of the local concentration. Toxic metals, such as cadmium,
lead, silver, and thallium, can be accumulated even though these substances have
no metabolic function. Apart from the intracellular uptake of metals, positively
charged metal ions can be removed from solution by adsorption onto the
negatively charged surface of the microoe.
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Microorganisms can also be utilized in the restoration of wastewaters from
mining and milling operations. One successful operation uses algae to remove
both soluble and particualte lead from the mill tailings of several mining
ventures in Missouri.
These operations consist of settling ponds and a series of shallow meanders in
which the algae are encouraged to grow. Chemical analysis has shown that the
algae accumulate heavy metals from the effluent released from the settling
pond. Algae species that have been identified to function effectively in these
types of operations include: Cladophora, Rhizoclonium, Hydrodictyon, Spiroqyra,
¦ Potamoqeton, ana Oscillatoria.
At present the potential to use genetic engineering to improve the performance
of these, or other, algae species is remote, and may not be possible for several
years. On the other hand, genetic engineering techniques could certainly be used
to improve Thiobacilius ferrooxidans.
As explained above, T. ferrooxidans derives its energy from the oxidction of
ferrous ion, metal sulfides, and soluble sulfur compounds in an acidic medium.
The ferric ion generated in the form of ferric sulfate is then able to react
chemically with several ore minerals and oxidize them. The ferric ion is then
regenerated by the microorganisms. Apparently, one of the primary rate-
limiting steps in the leaching of metal ores is the ferrous-to-ferric reoxidation.
Ferric ion competitively inhibits the rate of ferrous ion oxidation. Thus, as the
concentration of ferric ion increases, its production is slowed.
Since ferric ion has no other metabolic effect on T. ferrooxidans except to slow
its own production, it should be straightforward to isolate a suitable mutant
strain that is not affected by ferric ion concentration.
Thiobacilli are able to develop considerable resistance to the very high concen-
trations of the metals being leached, but the microbe is inhibited by some metals
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such as silver, mercury, and cadmium, at quite low concentrations. Metabolic
resistance to heavy metals is frequently conferred by the presence of certain
bacterial plasmids. Experiments could be undertaken to isolate appropriate
plasmids from other bacteria and to introduce them into Thiobacilli using
recombinant DNA or conventional genetic technologies.
Improved bacterial growth and mineral leaching activity have resulted when
Thiobacilli are grown in conjunction with the nitrogen-fixing bacterium, Beijer-
inckia lacticoqenes. This latter bacterial species probably supplies Thiobacilli
with essential nitrogenous nutrients. Since B. lacticoqenes is less able to
withstand the highly acidic environment required by ThiobaciHi. it may prove
worthwhile to introduce the nitrogen fixation genes (nif genes) directly into
Thiobaci 11 i. Alternatively, njf genes from Azotobacter or Klebsiel la (two free-
living nitrogen fixers) can be utilized since these species share several structural
and biochemical features with Thiobacilli.
4.4.2 Future prospects
Of the five industrial sectors considered in this report, the mining industry has
demonstrated the least interest in applying biotechnology to its ODerations. The
types of bioDrocesses that do pertain to mining are rather limited in scope, but
technical advances, leading to increased interest on the part of the industry, can
be envisioned.
• All known strains of leaching bacteria are aerobic; that is,
they require oxygen. However, essentially oxygen-free
conditions exist in the center of huge slag heaps of low-
grade ore. Thus, the engineering of anaerobic strains of
Thiobacillus would be received with great enthusiasm by
the mining industry. The technical feasibility of this
proposal is uncertain. Likewise, development of improved
thermophilic leaching bacteria would be very useful,
owing to the heat generated within ore dumDS.
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• The United States relies on imports for the vast majority
of certain mineral resources, including chromium, titan-
ium, and manganese (see Table 4-8). Recycling of these
materials is of increasing importance. The development
of efficient microbiological systems for extracting these
metals from industrial effluents and other waste reposi-
tories would constitute a major industrial and political
tour de force.
• Very little basic information is available regarding the
biochemistry and genetics of leaching bacteria. Conse-
quently, genetic engineering, especially recombinant
DNA, will have little impact on developments in this area
for at least five years. The properties and commercial
suitabilities of existing, naturally occurring leaching bac-
teria will undergo thorough examination first.
4.4.3 Potential hazards
The limited scope of biotechnology in the mining industry confines the range of
environmental concerns that demand consideration. However, all foreseeable
applications of biological processes in this industry involve microbial systems
operating in relatively open environments, such as slag heaps or tailings ponds.
Consequently, there are risks that microorganisms or their metabolic products
will inadvertently contaminate the local ecology. Specific areas of concern
include the following.
• Bacterial leaching operations generate large quantities of
sulfuric acid which, if poorly contained, could seriously
contribute to the aciaification of U.S. fresn water
supplies.
• Thiobacilli and related bacterial species are not known to
be pathogenic in man or animals; indeed, their peculiar
metabolic characteristics suggest that they should be
quite innocuous from c public health standpoint.
However, increased use of such microbes on an industrial
scale (resulting in greater exposure to human populations)
may select for bacterial strains that have acquired the
ability to infect humans.
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The use of bacteria to concentrate metals from dilute
waste streams or settling pond entails the risk that metals
will accumulate in the food chain. Even though metal
ions, such as mercury and silver, are highly toxic to
bacteria, it is through microbial action that mercury, for
example, is transformed into organic compounds that are
responsible for mercury toxicity in higher forms of life.
In other words, metals released into the environment are
metabolized by naturally occurring bacteria. The key for
safe commercialization of this bioprocess will be
adequate containment of the operation to prevent
dissemination of toxic metals into the general
environment.
As with other industrial sectors examined in this report,
the mining industry has very little experience with
biological processes. This lack of familiarity could result
in a failure to recognize impending environmental hazards
or in an eagerness to carry out biological processes before
their safety has been firmly established.
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Table 4-8
Strategic minerals and U.S. dependence on foreign sources
Mineral
Uses
Percentage
imported
Sources
bauxite
aluminum
9
Jamaica, Guinea,
Surinam
chromium
ferroalloys
91
South Africa, USSR
cobalt
superalloys
93
Zaire, Belgium,
Zambia
colun'oium
ferroalloys
100
Brazil
manganese
steel
9/
Gabon, South Africa
nickel
steel
73
Canada
p la t i nuTH
catalysts
87
South Africa, USSR
rutile
pigments
100
Australia
tantalum
electronics
components
97
Thailand
titanium
aerospace
components
47
Japan, USSR
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4.5 Pollution control industry
4JS.I Current activities
The organic matter invested in all living things, whether plant or animal, is
eventually recycled back into the environment as CC^. The process whereby
organic carbon is converted into inorganic carbon is called mineralization.
Representing a major portion of the overall carbon cycle (see Figure 4-10),
mineralization is almost always a consequence of microbial action. That is,
bacterial decomposers are ultimately responsbile for the degradation of all
organic carbon-containing substances in the biosphere. For example, bacteria of
the Pseudomonas species metabolize simple alkane compounds, such as octane,
by means of an enzymatic oxidation pathway that converts the alkane (R-CH^)
into the corresponding carboxyiic acid (R-COOH). The acid is then consumed as
an energy source by the bacterium via further oxidation to carbon dioxide.
Therefore, it is hardly surprising that microbiology plays an important role in
pollution control and waste management, particularly in the case of organic
pollutants. Moreover, inorganic pollutants, such as nitrogen-containing substan-
ces and toxic metals, are often treatable using biological systems. Bioiogical
waste management has been practiced by mankind iiteraily for thousands of
years, but modern advances in applied genetics may revolutionize the pollution
control industry to the extent that bioprocesses may soon replace many currently
employed chemicai/physical systems. Current activities within this industry fall
under three general headings:
• Biodegradation of organic substances, such as petroleum
products, pesticides, herbicides, industrial solvents, and
lignin wastes;
• Biological denitrification and desuifurization processes;
and,
• Removal or concentration of toxic heavy metals.
Each of these areas will be examined in the following sections.
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Figure 4-iO
The carbon cycle
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4.5.1.1 Biodegradation of organic substances
Most current activity, and that which has the most potential for biological waste
treatment, exploits the capacity of microbes to degrade toxic chemical pollu-
tants. A great variety of naturally occurring microorganisms, largely isolated
from soil or aquatic environments, are known to utilize hazardous organic
substances as carbon and energy sources. Table k-3 provides a sample of the
biodegradative processes that are currently in use or under investigation. Figure
4-1 I shows the degradative pathways of several specific pollutants.
Efforts to improve on nature by applications of genetic engineering in this area
have been minimal to date, but such activities are certain to increase in the near
future. Several efforts bear mentioning. Among the first highly oublicized uses
of genetic engineering was that of Chakrabarty, then at General Electric. He
combined the qualities of several strains of the bacterium Pseudomonas, each of
which could degrade a single hydrocarbon component of crude oil, into a single
bacterial strain. This "man-made" bacterial culture proved superior to a mixed
microbial culture composed of each of the contributing strains in breaking down
crude petroleum. The "oil-eating" microbes feed on the crude petroleum,
converting the hydrocarbon compounds into cellular constituents (biomass) and
carbon dioxide, however, the petroleum constituents that ere converted by this
microbial strain (namely camphor, naphthclene, and short-chain alkanes) are not
the major environmental concerns arising from oil spills. These volatile
hydrocarbons are either vaporized or readily degraded by natural bacterial
action. Of greater impact are the various asphcltenes that constitute the heavy,
non-volatile fraction of crude oil. These compounds are extremely refractory to
microbial degradation. Despite the publicity that attended Chakrabarty's
efforts, GE has not pursued this project beyond the laboratory stage of
development.
Scientists at the Battelle Memorial Institute in Columbus, Ohio, are engagea in
genetic engineering of microbes that efficiently degrade the chlorinated
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Table 4-9
Microbial degradation of various organic pollutants
Pollutant
Microbes involved
I. petroleum hydrocarbons
II. pesticides/herbicides
cyclodiene type
(e.g., aldrin, dieldrin)
organophosphorus type
(e.g., parathion, malathion)
2,4-D
DDT
kepone
piperonylic acid
III. other chemicals
bis(2-ethylhexyl)phthalate
dime thy initrosainine
ethylbenzene
pentachlorophenol
IV. lignocellulosic wastes
municipal sewage
pulp mill lignins
(various phenols)
200+ species of bacteria, yeasts,
and fungi; e.g., Acinetcbacter,
Avthrobastsv, Mycobacteria,
A.ctinormyoetes, and Pseidomonas
among bacteria; Cladosporiurr. and
Saolecobasidiim among yeasts
Zylericn xylestrix (fungus)
Pseudomonas
Pseudomonasj Arihrobaoter
Psnio-illiim (fungus)
Pseudomonas
Pseudomonas
Serratia marasosns (bacteria)
photosynthecic bacteria
llooardia tariaricans (bacteria)
Pseudomonas
Pseudomonas
Thermonospora (a thermophilic
bacterium)
yeasts: Aspergillus
Trickosporon
bacteria: Artkrobaater
Chromobacter
Pseudomonas
Xccntkomonas
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Figure 11
Degrcxkition pathways of several phenolic compounds by Pseudomonas putida
HjC.
.OH
°i
CHO
Y" COCH
OH
H.C CCOH
COCH
OH
J
co
-CH,
COOil
o
00 K
COCH
^^OH
"^H-0
'^Sv»-CHSC
COH
:ooh
^OH
hn.
CHjCHOHCH; COCOOH
i
CH j CHO-
OH, COCOOH
Source: Bull, A.T., etal . (1979)
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herbicide, 2,4-D. Likewise, SRI International has undertaken a program to
compile a list of common toxic chemicals that are amenable to microbial
biodegradation and to isolate and engineer improved strains that might have
commercial value.
In general, chlorinated organics are more recalcitrant to biodegradation than are
non-chlorinated substances. Thus, persistent pollutants such as DDT, PCBs, etc.,
represent a more serious challenge to pollution control engineers who are hopeful
of applying biological treatment systems to waste management. Microbes exist
that can perform chemical transformations of these recalcitrant substances (see
Table 4-10), but microorganisms have not yet been isolated that can utilize these
compounds as carbon or energy sources. Indeed, it is this lack of direct
metabolism by microbes that explains the environmental persistence of com-
pounds such as these. Future success in developing biodegradative systems for
pollutants of this type may depend on locating microbial communities consisting
of several species of microorganisms which function cooperatively to decompose
recalcitrant compounds.
4.5.1.2 Denitrification and desulfurization
The various oxidized forms of nitrogen (NC)^) and sulfur (50^) present serious
environmental concerns owing to the ease with which they are converted to
strong acids (e.g., nitric and sulfuric) upon exposure to water. The acidiciation
of Ickes and gound water poses a serious threat to the maintenance of aquatic
life and fresh water supplies. Although large amounts of nitrogen (and lesser
quantities of sulfur) are nutritional requirements for life, the large-scale burning
of sulfur and nitrogen-containing fossil fuels and the release of certain industrial
wastes have loaded the environment with toxic levels of these inorganic
substances. Traditional schemes for reducing the emissions of these pollutants
have been largely physical/chemical in design. Biological processes are under
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Table 4-10
Type reactions for transformation of
chemicals of environmental importance
Reaction type
Reaction
Example
dehalogenat ion
deamination
decarboxylation
RCK2C1—^RCH20H
ArCl—*¦ ArOH
ArF—*• ArOH
ArCl—s-ArH
Ar2CHCH9Cl >-Ar0C=CK2
Ar7CHCHCl2—»-Ar2C=CHCl
Ar,CHCCl3 ^Ar7CHCHCI2
Ar2CHCCl3-
~Ar2C=CCl2
RCC13 -RC00H
HetCl »-HetOH
ArNH,
¦ArOH
ArCOOH—»ArE
Ar„CHC00H—>-Ar.,CH.1
- RCEO
RCH-j—»¦ RC00H
ArK-
ArOH
—*-RCH(0H)R'
R(R')CHR"—~ R(R')CH0H(RM)
RCH2R'
R(R')(R")CCH-
R(R')(R")CCH2OH
ArO(CH-) CHoCHoC00H—»¦
Cm n Cm im
ArO(CE0) COCK + CH3C00H
RCK=CHR'—~RCH(0)CHR1
Bromacil
diisopropyInaphthalene
pentachlorobenzol
Benthiocarb, Dicamba
Carbofuran, DDT
Bux insecticide
Denmert
o)-(2,4-dichlorophenoxy)-
alkatioic acids
Heptachlor
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Table 4-10 (cont.)
Reaction type
Reaction
Example
N-oxidation
R(R')NR"—*¦ R(R')N(0) R"
Trideicorph
S-oxidation
RSR'—~ RS(0)R' or RS(02>R'
Aldicarb
=S to =0
(AlkO) ?P(S)R—*¦ (Alk0)2P(0)R
Parathion
RC(S)R'—>-RC (0) R'
ethylenethiourea
sulfoxide reduction
RS (0)R1—~¦ RSR'
Phorate
triple bond reduction
RC=CH—fRCH=CH2
Buturon
double bond reduction
Ar9C-CH?—»-Ar0CHCH3
DDT
Ar2C=CHCl—^Ar?CHCH2Cl
DDT
double bond hydration
A'r2C=CH9—~ Ar9CHCH20E
DDT
nitro metabolism
RNO,—~ ROH
Nitrofen
rno2—~ rnh2
Sunithion
oxlme metabolism
RCH=N0H—~ RC =N—~ RC (0) NH n
or RCOOH '
Aldicarb, Bromoxynil,
Dic.hlobenil
'Abbreviations: R = organic moiety
.Ar = aromatic
AIk = alkyl
Het = heterocycle
Source: Alexander, M. (1981) Science, 211:134.
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investigation, however, and several systems have demonstrated feasibility in
laboratory-scale applications.
The biological nitrogen cycle entails three phases involving various oxidation
states of nitrogen (see Figure k-12). Atmospheric nitrogen is relatively
inert chemically and must be "fixed" into usable forms such as nitrate and nitrite
(oxidized nitrogen) or ammonia (reduced nitrogen). Meanwhile, fixed nitrogen is
recycled back into the atmosphere by anaerobic processes. All these steps are
ccrried out by various species of bacteria, according to the chemical reactions
shown in Figure ^-12. Of these three phases, the third (denitrification) is the
least understood, but it is this process that promises to alleviate pollution
problems stemming from excess nitrate and ammonia.
Pollution by sulfur-containing compounds presents a more serious problem than
pollution by nitrogenous substances because of sulfur's greater toxicity to living
organisms and its greater prevalence in fossil fuels and industrial waste streams.
Inorganic sulfur compounds, such as sulfate (S07) and hydrogen sulfide (HLS), can
4 £.
be metabolized by certain microbial species, as shown in the following reactions:
50= ! ~ H2S - 5° ¦
I. Desulfovibrio desulfuricans
II. Chlorobium thiosulfatophilum or Chromatium vinosum
Laboratory-scale systems utilizing these microbial populations arrangea serially
are under investigation for potential use in trecting high-sulfur effluents, such as
those arising from coal and gypsum mining and general metallurgical operations.
Fossil fuels may contain up to 7% sulfur by weight. This sulfur is generally in
one of three forms:
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Figure k-12
Microbial components of the biological nitrogen cycle
II
I. Nitrogen fixation
Ekizcbiian: + H ~ NH^
II. Hitrification
flitvosomonas: NH^ + ~ NO-?
Nitvobactsr: NO2 + 0^ ~ NO^
HI. Penitrification
Pseudomonas.: NH^ + NO^ —* ^0 or
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• Organic sulfur, in which sulfur is covalently linked to
carbon either directly as R-S-S-R or R-S-R, or bound as a
sulfate, R-O-SO^;
• Pyritic sulfur in the form of iron pyrite, Fe$2; and,
• Inorganic sulfate, SOJ
Of these, organic sulfur predominates in crude petroleum, whereas pyritic sulfur
and sulfate are found largely in coal. In all cases, the combustion of untreated
crude oil or coal releases to the atmosphere huge quantities of sulfur dioxide gas
(SO2) and particulate sulfates. These sulfur compounds are intrinsically toxic
and, moreover, combine with water to form sulfuric acid. The removal of sulfur
compounds from fossil fuels prior to combustion has been deemed a desirable
adjunct to, or possible replacement for, costly scrubbers now widely used to
control stack emissions.
Biological desulfurization is still in the experimental stage, but several microbial
systems are under investigation. Pyritic sulfur can be leached from mined coal
using Thiobacillus ferrooxidans and Thiobacillus thiooxidans—the same bacterial
species employed for mineral leaching in the mining industry. Also, a thermo-
philic microbe, Sufolobus acidocalderius, has been isolated. All these organisms
operate under acidic conditions (pH I to 3) and convert sulfides to sulfuric acid.
Thus, the pyritic sulfur content of the fossil fuel is transformed into a water
soluble compound that can be readily washed away. However, the acid that is
generated represents a pollutant in its own right that must be dealt with.
Organic sulfur exists in crude petroleum largely as linear mercaptans (R-SH) or
as aromatic thiophenes. Microbial systems for converting thiophenes into water
soluble compounds are under development. The biochemistry involved in this
transformation is shown in Figure 4-13. The principal drawback of this orocess
lies in the loss of carbon atoms (and, therefore, of Btu content) resulting from
the removal of sulfur-containing organics.
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Figure 4-13
Pathway of microbial conversion of dibenzothiophene
into water soluble compounds
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4.5.1.3 Toxic metals
Biological concentration of heavy metal ions from a dilute waste stream involves
processes essentially identical to those described for the mining industry in
Section 4.4.1. The emphasis for pollution control, of course, is isolation and
disposal of toxic metals, whereas ultimate recovery of the metals is of concern
to the mining industry. The incorporation of metals from an industrial effluent
into biological sediments (i.e., activated sludge) has proven to be a satisfactory
application of biotechnology to pollution control. The immobilization of metals
by these sediments may be the result of (I) direct intracellular uptake, (2)
adsorption to cell surfaces, or (3) sequestration in a microbialiy produced
exopolysaccharide matrix. In addition to bacteria, other organisms are used to
concentrate metals from dilute waste streams. Settling ponds containing
photosynthetic algae or rapidly growing aquatic vegetation, such as water
hyacinths, are also fulfilling this purpose.
4.5.2 Future prospects
The greatest R&D effort involving near-term applications of biotechnology to
pollution control will be in developing improved microbial strains for decontami-
nation of polluted waste waters and for in situ detoxification of contaminated
soils and sediments. There exist considerable gaps in our basic knowledge of the
types of microorganisms capable of degrading toxic chemicals. In particular,
anaerobic bccteria and filamentous fungi represent two diverse classes of
microbe for which considerable potential exists for biological pollution control,
but little is known of their general properties.
Bacteria ere classified into several groups based on the effect that oxygen
has on their growth and metabolism:
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Obligate aerobes require oxygen for growth. An example
is the tubercle bacillus, the causative agent of tuber-
culosis.
• Obligate anaerobes survive only in the absence of oxygen.
Examples include Clostridia (various species of which
cause botulism, tetanus, and gangrene), bacteroides
(intestinal bacteria that ferment glucose to form organic
acids; e.g., formic, acetic, propionic, butyric, lactic, and
succinic), denitrifiers that reduce nitrate to nitrogen gas,
sulfate reducers that produce hydrogen disulfide (a source
of pollution in anoxic ponds and streams), and methane
producers that form marsh gas.
• Facultative • organsims, such as many enteric bacteria
(e.g., E. coli), ccn thrive with or without oxygen by
shifting to different metabolic processes in each case.
Anaerobic bacteria are are particularly relevant to pollution control practices
because of their prevalence in sub-soil. Thus, bacteria of this type will
encounter toxic chemicals or petroleum wastes that have been spilled, as well as
herbicides end insecticides that have been applied to the ground. A subgroup of
anaerobic bacteria, called microaerophilic, can tolerate or even prefer low
oxygen pressures (but much less than in air). These conditions prevail just
beneath the surface of the soil. Thus, microaerophiles, about which very little
basic information is known, should receive considerable attention for possible
future use as in situ decontaminating agents. Likewise, anaerobic bacteria that
thrive in underwater sediments, such as anoxic settling ponds or in the bottom.of
the kepone-laden James River, will be the subjects of more intense research in
the years ahead.
Fungi are classified into three groups: (I) single-celled yeasts, (2) multicellular
filamentous colonies, or molds, and (3) mushrooms. The filamentous fungi
include some well known types, such as Neursopora, Penicillium, and Aspergillus,
as well as lesser known cquctic watermolas and soil fungi. The genetics and
biochemistry of fungi are mucn less well understood than are bacteria. However,
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it is certain that, like bacteria, fungi serve crucial roles in recycling organic
matter throughout the biosphere. The contribution made by fungi to the
decontamination of polluted soils and streams is becoming better appreciated,
and research into the application of fungi to waste management shouid receive
greater attention in the years ahead.
The following list outlines some aspects of applied genetics and waste manage-
ment that will be under development.
• Cataloging the tyDes of chemical transformations per-
formed by microorganisms and the microbes involved.
• Isolating and characterizing the genetic material and
enzymes responsible for the observed transforming act-
ivity.
• Conducting genetic engineering on organisms that occur
naturally in a particular environment (e.g., river bed
sediment) to confer the ability to degrade a pollutant that
is not normally present in that environment (e.g., kepone).
Successful decontamination of polluted sites by jn situ
biotreatment requires that the engineered microbe will
compete favorably with existing microflora.
• Developing biotreatment systems for dealing in situ, with
specific wastes under a given set of conditions. For
example, a chemical spill at a particular site may require
a different microbe depending on the ambient tempera-
ture, or on the presence of certain nutrients. Exogenous
nutrients such as glucose may have to be supplied.
• Designing bioreactors for on-line waste stream treatment.
Systems for immobilizing microbes are under develoment.
Monitoring and controlling the concentration of toxic
substances in the waste stream are vital since excessive
doses of most pollutants are deadly even to microbes that
thrive on low concentrations of these chemicals. Thus,
the design and engineering of systems for diluting con-
centrated wastes prior to biotreatment may be a greater
technical challenge than is the development of microbial
populations capable of performing the biodegradction.
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4.5.3 Potential hazards
Biological processes are currently in wide use throughout the pollution control
industry, but so far modern applied genetics or genetic engineering has had
negligible impact. The potential exists, however, that these new biotechnologies
will drastically alter or replace conventional physical/chemical waste
management processes. Nevertheless, considerable basic information regarding
the relevant biological systems must be acquired before genetic engineering can
be implemented to improve on naturally occurring organisms. Very little is now
known of the biochemistry, metabolism, genetics, or natural ecology of the
microbial species that mediate biodegradative processes. Indeed, the mere
identification of potentially useful microorganisms is far from complete. Thus,
the impact of applied genetics may not be felt in this industry for five years or
more.
Nevertheless, increasing utilization of natural bioprocesses in pollution control
entails certain potential hazards that are noteworthy and that mcy forewarn of
future risks evolving from the application of genetic engineering in this industry.
Chief among these concerns is the generation of biological aerosols. These are
tiny droplets of water or dust particles containing active microbial material.
They remain suspended in the atmosphere to be transported by air currents to
distances of several miles from their origin. Many industrial processes have the
potential to create hazardous aerosols that contain pathogenic microorganisms.
Among these are:
« Agricultural practices. Stockyards and poultry feedlots
generate contaminated dust aerosols that mcy elicit very
serious health problems, such as anthrax. The
increasingly common practice of applying partially
treated or untreated municipal sewage to crop lands has
led to improved crop yielcs and has provided an
alternative to the direct discharge of sewage into lakes
and streams. But this practice gives rise to potentially
harmful aerosols and to increased risk of ground water
contamination. Waste water from food processing plants
has also been utilized in land application programs.
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• Textile mills. The processing of wool and animal hair
produces dust aerosols that are known to contain
pathogens, such as the causative agents of anthrax and Q
fever.
• Abattoirs and rendering plants. The slaughtering and
processing of livestock is a serious source of infectious
aerosols, occasionally causing epidemics among
employees. The condition is exacerbated by livestock
farmers or ranchers who frequently rush their stock to
market at the first sign of disease among members of the
herd.
• Sewage treatment plants are probably the most numerous
and varied sources of pathogenic aerosols. The bubbling
of air through an activated sludge facility and the
splashing of sewage water over the rock oed of a trickling
filter operation both generate numerous aerosolized
particles. Approximately one-half of these droplets are in
the size range (I to 5 microns) that are carried downwind
for considerable distances and which are readily inhaled
and deposited in the human lung. The magnitude of the
potential hazard posed by sewage treatment plants is
related to the abundance of these facilities, their
proximity to residential areas, the great variety of
microbial species found in sewage, and the nigh frequency
of aerosolization from these facilities which are in
operation all day throughout the year.
Thus, the threat to public health posed by infectious aerosols is considerable.
Moreover, many laboratory-associated infections also aDpear to result from the
production of aerosols, rather than from more obvious lab incidents, such as
pipetting by mouth, needle and syringe accidents, or simole spills. Clearly,
future efforts to minimize risks associated with any microbiological process,
including those involving recombinant DNA organisms, should focus on methods
of controlling aerosols.
The biodegradation of organic pollutants by indigenous microorganisms is chiefly
responsible for the eventual recycling of most environmental wastes. Organic
pollutants fall into three general categories:
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• Completely biodegradable, for which there exist microbes
capable of mineralizing the substrate. Examples include
relatively simple hydrocarbons and aromatics, such as
phenols,
• Totally recalcitrant, for which there exist no known
microorganisms capable of chemically transforming the
substrate, or if so, at a very slow rate. Synthetic plastics,
such as polyethylene or polyvinyl chloride, appear to be
in this category, as do some polychlorinated aromatic
hydrocarbons and pesticides.
• Co-metabolized compounds are transformed to some ex-
tent by microbes that utilize other substances as sources
of energy and biomass. Pollutants in this category, such
as DDT, aldrin and heptachlor, are degraded slowly.
Microbes have yet to be isolated that can use compounds
of this type as nutrients.
The impact of applied genetics in this area will be minimal until more is learned
of the types of microorganisms involved. The suitability of naturally occurring
microbes for waste cleanup will be examined initially. Particular attention will
be paid to jn situ decontamination processes. However, several factors mitigate
against widespread success in this area.
• The concentration of toxic chemicals at the site of a spill
or dump site is often too high to permit survival of any
microbe capable of degrading the pollutant.
• Concentrations of toxic chemicals sufficiently low to
permit survival (generally less than 1,000 opm) may be too
low to sustain bacterial growth. Thus, additional
nutrients must be supplied.
• Some toxic compounds, particularly those that are co-
metabolized, are partially degraded into substances that
are slightly less toxic than the parent compound but which
are more readily mobilized (that is, dispersed throughout
the local food chain). This is the fate of most polychlori-
nated organics.
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Finally, there exist natural microbial systems capable of concentrating inorganic
metal ions from dilute waste streams. This accumulation appears to be
associated with chemical transformations of the metal into organic forms. The
methylations of mercury and arsenic, for example, are known to occur as a result
of microbial action in aquatic sediments. These organic derivatives are more
toxic than the corresponding inorganic substances, and they are more readily
taken up from the sediments by aquatic animals. Thus, commercial use of
microbial systems to remove heavy metal ions from waste waters must be
monitored for the release of even more toxic organic derivatives.
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SECTION 5
SUMMARY AND RECOMMENDATIONS
5.1 State of the applied genetics industry
All indications are that the U.S. economy is on the verge of a "biology boom."
Excitement over the commercial potential of genetic engineering has been very
high, as exemplified by the considerable media attention to this area, as well as
the enthusiasm shown by investors. Public expectations are also very high that
applied genetics will quickly and effectively solve many societal problems, such
as cancer, the energy crisis, our polluted environment, and the world food
shortage. The next several years will be crucial to the future development of
the biotechnology industry. If few (or none) of the expected benefits from
applied genetics are realizea in the short run, public enthusiasm for this modern
technology may dissipate. Further commercial development will be hampered by
a lack of investment capital, and adverse publicity will deter innovative
entrepreneurs from entering the field with ideas that could lead to short-term
success.
One aspect of applied genetics, recombinant DNA technology, has received the
bulk of public attention. These experimental techniques involve the cutting and
splicing of genes and the subsequent joining together of DNA from different
organisms. This new technology offers the prospect of treating previously
incurable genetic diseases, such as sickle cell anemia and hemophilia, and of
understanding and eventually curing human cancer. On the other hand, the
practice of recombinant DNA nas produced the specter of inadvertent creation
of new and threatening life forms or of deliberate manipulation of human genes
for mischievous purposes. 8ut the risks inherent in recombinant DNA tecnnology
are surely much smaller than originally feared (see below). As with other
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technological advances, recombinant DNA will be applied where it will yield
substantial commercial pay-off. Only the pharmaceutical industry is likely to
realize near-term returns on investments in this new technology. Other
industrial sectors will thoroughly investigate naturally occurring biological
systems, including microorganisms and higher plants, for potential commerciali-
zation, prior to making significant investments in recombinant DNA technology.
Although many non-pharmaceutical firms have initiated in-house programs in
recombinant DNA research, it may soon become apparent that this is a case of
putting "the cart before the horse." Two reasons for this conclusion are:
• Considerable basic scientific information must be
acquired in many areas pertaining to the species of
microorganisms and higher plants that will be of
commercial interest to non-pharmaceutical firms. For
example, the use of genetic engineering to endow
microbes with useful characteristics, such as the ability
to fix atmospheric nitrogen, salt and drought tolerance,
and anaerobiosis, must await a better understanding of
the biochemical and genetic basis of these traits.
• Technical advances in recombinant DNA methodology will
largely be made in academic laboratories, and they will
occur at a faster rate than will commercial developments.
Thus, the scientific feasiblity of a particular genetic
engineering operation will precede by several years, per-
haps, its commercial application. The chief factor contri-
buting to this time lag is the considerable disparity
between laboratory and industrial settings for the per-
formance of oiological processes. For example, an engin-
eered microbial strain might thoroughly degrade kepone in
the laboratory, but be unable to survive in competition
with the natural microflora existing in James River
sediments. Eventual success in a project of this type
requires that more information be gathered regarding
indigenous organisms.
Thus, the glamour and attention surrounding recombinant DNA may soon subside
in favor of increased interest in naturally occurring organisms. This is
particularly likely in non-pharmaceuticai industries, although the search for
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drug-like substances in higher plants and animals promises to reorient the focus
in the drug sector as well.
Biotechnologies other than recombinant DNA have received less pubiic attention
but, nevertheless, are expected to contribute significantly to the commercial
success of the "biology business." Modern fermentation technologies will be
applied to relevant operations in all industrial sectors, but the extent of their use
will obviously depend on the successful generation of new and useful microorgan-
isms that can be grown on a large scale. Likewise, immobilized bioDrocesses,
such as on-stream bioreactors for waste stream detoxification, will be utilized
only to the extent that other biotechnologies generate worthwiie organisms or
enzymes for the purpose of attachment. Cell fusion techniques will undergo
further development as an alternative to recombinant DNA methods for
producing genetically altered organisms. But applications of this technology are
limited for the most part to the biomedical field (e.g., monoclonal antiboaies)
and to the agriculture industry (e.g., fusions of plant cell protoplasts to generate
hybrids).
53. Overall assessment of risk
As with public perception of the possible benefits resulting from applied
genetics, considerations of potential risks associated with these technologies has
focused on recombinant DNA procedures. As mentioned in Section 4.1.3, several
workshops have been held during the past four years to review and summarize
the status of risk assessment in the recombinant DNA field. In addition, the NIH
(through its Recombinant DNA Advisory Committee, the RAC) has orepared a
Risk Assessment Plan which will summarize and update annually information
relevant to recombinant DNA risk assessment. The most recent update was
published in the September 17, 1980, issue of the Federal Register. A principal
component of the plan is to analyze risk data pertaining to three general
categories of host-vector systems in common use: prokaryotic (e.g., E. coli
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KI2), lower eukaryotic (e.g., Saccharomyces cerevisioe), ana higher eukaryotic
(e.g., mammalian cells). The following excerpt summarizes the current under-
standing of the risks:
...despite intensive study by the RAC Subcommittee on
Risk Assessment and NIH staff, several conferences and
workshops to consider specific issues and several experi-
ments, no risks of recombinant DNA research have been
identified that are not inherent in the microbiological and
biochemical methodology used in such research. (45 FR
61874)
Thus, in the absence to date of any compelling evidence to the contrary, and
despite assiduous efforts to identify any potential hazards, scientists are now
convinced that the practice of recombinant DNA techniques poses no health risks
over and above those encountered in normal microbiological research.
Since the NIH committee charged with the task of monitoring the field of
recombinant DNA has reached the conclusion that there exist no untowara risks
in practicing this technology, what will become of the committee itself? Inaeed,
what is the future of government involvement generally :n this, area? Several
comments can be made:
• During the past year or so, the RAC has divested itself of
oversight responsibilities by delegating many of its func-
tions to the local Institutional Biosafety Committees
(IBCs). The IBCs are naturally reluctant to take on the
added workload in dealing with what are now deemed
innocuous safety issues.
• As are other government agencies, the RAC is beginning
to examine issues from a cost/benefit stancpoint. The
RAC recognizes that other areas of potential health
concern exist in the biomedical research fiela. These
more conventional hazards, which exceed the threat of
recombinant DNA as potential risks, include exposure to
pathogenic aerosols, X-rays, radionuclides, and toxic
chemicals. Any future role for the RAC (or some other
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RAC-like organization) should include consideration of
these safety issues as a priority.
• Biotechnologies other than recombinant DNA have so far
received little attention with regard to potential hazards.
For example, cell fusions involving various cells derived
from human tissues may become increasingly popular as a
method for obtaining human biologies for drug manu-
facture. Large-scale application of this procedure entails
the speculative risk that pathogenic viruses will be
induced and propagated. Relevant government agencies
should be advised to monitor the application of any
biotechnology within their purview.
5.3 Recommendations to the EPA
The wide array of industrial uses of applied genetics can be grouped into two
categories with respect to environmental issues: (1) those applications in any
industrial sector that constitute an adverse impact on the environment; and (2)
bioprocesses designee to assist in the effort to control pollution and constitute a
net positive impact on the environment. With this general distinction in mind,
specific recommendations can be made:
• The commercial applied genetics industry is at a nascent
stage of development and, so far, no incidents of environ-
mental concern related to this industry have materialized.
Any environmental risks arising from industrial use of
applied genetics are speculative. At this time, there
exists no compelling reason for the EPA to establish
regulations in this area.
• Should environmental hazards emerge in the future, it is
probable that they ccn be handled within the existing
regulatory framework. During the past five years, the
Office of the General Counsel at the EPA has examined
the applicability of existing legislation, particularly
TSCA, to commercial recombinant DNA activities. A
consensus appears to have been reached that EPA has the
authority to regulate commercial uses of this technology,
including: (I) requirements for premanufacture review of
industrial processes based on recombinant DNA
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methodologies (Section 5 - of T5CA); (2) restriction or
prohibition of manufacture, processing, distribution, or
use of recombinant DNA if such is deemed hazardous
(Section 6); and (3) dealing with imminent hazards
involving recombinant DNA (Section 7). Moreover, the
discharge of recombinant DNA material into the
environment could be regulated under existing statutes
within the Clean Air and Water Acts. In summary, no
additional legislation would seem to be necessary in order
for the EPA to regulate commercial activities involving
recombinant DNA.
The EPA should continue to take an active role in pro-
moting applied research and development of biological
waste management processes and techniques. Emphasis
should be placed on the biology of relevant systems rather
than on process engineering and design. A particularly
troublesome problem requiring more research is m situ
decontamination of chemical wastes. A more tractcble
problem deserving EPA attention involves the use of on-
line bioreactors for treating industrial effluents at the
source.
The EPA should sponsor further investigation into the
generation, dispersal, and control of biological aerosols.
To the best of its abilities, the EPA should monitor
commercial and scientific developments in the field of
applied genetics with the aim of identifying both immi-
nent environmental hazards and areas where this techno-
logy might be applied to pollution control operations.
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BIBLIOGRAPHY
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Crea, R., Kraszewski, A., Hirose, T., and Itakura, K., 1978, "Chemical synthesis
of genes for Human insulin," Proc. Nat. Acad. Sci., USA, 75:5765-5769.
Gefter, M.L., Becker, A., and Hurwitz, J., f967, "The enzymatic repair of DNA,"
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Grobstein, C., 1979, "The recombinant DNA debate," Scient. Amer., 237:22-36.
Hishinuma, F., Tanaka, T., and Sakaguchi, K., 1978, "Isolation of extrachromo-
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Itakura, K., and Riggs, A.D., 1980, "Chemical DNA synthesis and recombinant
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Kennedy, J.F., 1979, "Facile methods for the immobilization of microbial cells
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Kennett, R.H., McKearn, T.J., and Bectheol, K.B. (eds.) 1980, Monoclonal
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Mulligan, R.C. and Berg, P., f980, "Expression of a bacterial gene in mammalian
cells," Science, 209:1422-1427.
Novick, R.P., 1980, "Plasmids," Scient. Amer., 243:102-127.
Office of Technological Assessment, 1980, Impacts of Applied Genetics, Washing-
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Peppier, H.J. and Perlman, D. (eds.), 1979, Microbial Technology, 2nd Ed.,
Academic Press, NY.
Perlman, D., 1974, "Prospects for the fermentation industries, 1974-1983," Chem-
tech, 4:210-216.
Richards, J. (ed.), 1978, Recombinant DNA: Science, Ethics, and Politics,
Academic Press, NY.
Reinert, J. and Bajaj, Y.P.S., 1977, Applied and fundamental aspects of plant
cell, tissue, and organ culture, Springer-Verlag, NY.
Schaffner, W., 1980, "Direct transfer of cloned genes from bacteria to mammali-
an cells," Proc. Nat. Acad. Sci., USA, 77:2163-2167.
Sebek, D.K., and Laskin, A.I., (eds.), 1979, Genetics of Industrial Microorganisms,
Am. Soc. Microbiol., Washington, D.C.
Skinner, K.J., 1975, "Enzymes technology," Chem. Eng. News, 53:22-4f.
Svoboda, A., 1978, "Fusion of yeast protoplast induced by polyethylene glycol," J.
Gen. Microbiol., 109:169-175.
Vasil, I.K., Ahuja, M.R., and Vasil, V., !979, "Plant tissue cultures in genetics and
plant breeding," Adv. Genet., 20:127-215.
Wade, N., 1980, "UCLA gene therapy racked by friendly fire," Science, 210:509-
5H.
Wetzel, R., 1980, "Applications of recombinant DNA technology," Amer. Scient.,
68:664-675.
Wingard, L.B., Katchalski-Katzir, E., and Goldstein, L. (eds.), 1979, Enzyme
technology, Academic Press, NY.
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1!. PHARMACEUTICAL
Bell, G.I., Swain, W.F., Pictect, R., Cordell, B., Goodman, H.M., and Rutter,
W.J., >913, "Nucleotide sequence of a cDNA clone encoding human prepro-
insulin," Nature, 282:525-527.
Bibb, M., Schottel, J.L., and Cohen, S.N., 1980, "A DNA cloning system for
interspecies gene transfer in antobiotic-producing Streptomyces," Nature,
284:526-531.
Bloom, B.R., 1980, "Interferons and the immune system," Nature, 284:593-595.
Cape, R.E., '979, "Microbial genetics and the pharmaceutical industry," Chem-
tech, 9:638-644.
Chemical Week, February 6, 1980, "Gene feat spurs interferon race."
Derynck, R., Remant, E., Saman, E., Stansses, P., De Clercq, E., Content, J., and
Fiers, W., 1980, "Expression of human fibroblast interferon gene in E. coli,"
Nature 287:193-197.
Fiddeo, J.C., Seeburg, P.H., Denoto, F.M., HalleweH, R.A., Baxter, J.D., and
Goodman, H.M., 1979, "Structure of genes for human growth hormone and
chorionic somatomammotropin," Proc. Nat. Acad. Sci., USA, 76:4294-4298.
Forbes Magazine, January 5, 1981, "Drugs."
Goeddel, D.V., et al., 1980, "Human leukocyte interferon produced by E. co>i is
biologically active," Nature, 287:4>I-4I6.
Goeddel, D.V., Heyneker, H.L. Hozumi, T., Arentzen, R., Itakura, K., Yansura,
D.G., Ross, M.J., Miozzari, G., Crea, R., and Seeburg, P.M., '979, "Direct
expression in E. coli of a DNA sequence coding for human growth
hormone," Nature, 281:544-548.
Goeddel, D.V., Kleid, D.G., BoMvar, F., Heyneker, H.L., Yansura, D.G., Crea, R.,
Hirose, T., Kraszewski, A., Itakura, K., and Riggs, A.D., 1979, "Expression
in E. coli of chemically synthesized genes for human insulins," Droc. Nat.
Acad. Sci., USA, 76:106-110.
Henriquez, P., Candia, A., Norambuena, R., Silva, M., and Zeme'man, R., 1979,
"Antibiotic properties of maine algae," Bot. Mar., 22, 451-454.
Kieslich, K., >980, "New examples of microbial transformations ir pharmaceuti-
cal chemistry," Bull. Soc. Chim. Fr., H2:9-I7.
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Lorz, H., and Potrykus, I., 1979, "Regeneration of plants from mesophy't
protoplasts of Atropa belladonna," Experientia, 35:313-3(4.
Marx, J.L., (980, "Interferon congress highlights," Science, 210:998.
MiHer, H.I., Guerigirian, J.L., Troendle, G., and Sobel, 5., 1980, "Aspects of the
drug regufatory process: recombinant DNA technofogy," Recomb. DNA
Techn. Butt., 3:72-74.
Miozzari, G., (980, "Strategies for obtaining expression of peptide hormones in E.
coti," Recomb. DNA Techn. Butt., 3:57-67.
Ross, M.J., '980, "Production of medicatly important polypeptides using recom-
binant DNA technology," Recomb. DNA Techn. Bult., 3:t-M.
Shine, J., Fettes, It, Lan, N.C.Y., Roberts, J.L., and Baxter, J.D., 1980,
"Expression of cfoned beta-endorphin gene sequences by E. coli," Nature,
285:456-461.
Shiner, G. (980, "Human growth hormone: potential for treatment are broad-
ened," Res. Resour. Report., 4:1-5.
Sun, M., (980, "Insulin wars: new advances may throw market into turbulence,"
Science, 210:1225-1228.
U.S. Environmental Protection Agency, 1976, pharmaceuticat Industry Hazardous
Waste Generation, Treatment, and Disposal, SW-508, Washington, D.C.
Va'enzuela, P., Gray, P., Quiroga, M., Zatdivar, J., Goodman, H.M., and Rutter,
W.J., 1979, "Nuc'eotide sequence of the gene coding for the major protein
of hepatitis B virus surface antigen," Nature, 280:8(5-819.
Woodruff, H.B., (980, "Natural products from microorganisms," Science,
208:1225-1229.
III. CHEMICAL
Buchanan, R.A., Cud, I.M., Otey, F.H., and Russet', C.R., 1978, "Hydrocarbon and
rubber producing crops: evaluation of U.S. species," Econ. Botany, 32:13'-
145.
Suchta,.K., >974, "Biotechnicat oroduction of organic acids,'' Chem. Zeit., 98:532-
538.
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Chemicaf Week, June 4, 1980, "Enzymes are a sweet way to do business."
Chemical Week, October 8, (980, "Biotechnology: research that coutd remake
industries."
Chemical Week, March 4, 198), "What applied genetics might do in chemicals."
Johnson, J.D. and Hinman, C.W., 1980, "Oil and rubber from arid land plants,"
Science, 208:460.
Khafagy, S.M., Metwatly, A.M., Eil-Ghazooly, M.G., and El-Naggar, 5.F., 1979,
"Sesquiterpene lactones from Varthemia candicans," Planta Med., 37:75-78.
Markwell, A.J., 1978, "Some chemical processes involving microorganisms,"
Chemsa, 4:44-45.
Miwa, T.K., (979, "Chemicals bloom in the desert," Chemical Week, 124:31-33.
Nyiri, L., >97', "Preparation of enzymes by fermentation," Intern. Chem. Eng., H:
447-458.
Pape, M., (976, "The competition between microbial and chemical processes for
the manufacture of basic chemicals and intermediates," Sem. on Microb.
Energy Conversion, United Nations Inst, for Training and Research, Oct-
ober, 1976.
Sanderson, J.E., Wise, D.L., and Augenstein, P.C., r979, "Organic chemicals end
liquid fuels from algal biomass," Biotechnol. Qioeng. Symp., 8:'3!-(5l.
Schwartz, R.D., Williams, A.L., and Hutchinson, D.3., 1980, "Microbial
production of 4,4-dihydroxy biphenyi: hydroxyfation by fungi," Appl.
Environ. Microbiol., 39:702-708.
Tilax, 3.D.,1978, "Prospect of manufacture of industrial chemicals from ceflulo-
sic raw materials," Symp. Proc. Byconversion CeKutosic Substances, New
Dethi, February, 1977.
Wang, D.I.C., Cooney, C.L., Demain, A.L., Gomez, R.F., and Sinskey, A.J., 1978,
"Degradation of cettutosic biomass and its subsequent utilization for
production of chemicaf feedstocks," MIT Program Rep. No. COO/4>98-6.
Yoshiharu, I., Ichino, C. and Tamis, I., 1978, "Production and utilization of amino
acids," Angew. Chem., I7:f76-I83.
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IV. ENERGY
Benemann, J.R., and Hallenbeck, P.C., 1978, "Recent developments in hydrogen
production by microalgae," Symo. on Energy from Biomass and Wastes,
Inst. Gas Techno)., Chicago. IL.
Chin K.K., and Gohr, T.N., J978, "Bioconversion of solar energy: methane
production through water hyacinth," from Symp. on Energy from Biomass
and Wastes, Inst, of Gas Technot, Washington, D.C., p. 215.
Clausen, E.C., Sitton, O.C., and Gaddy, J.L., 1979, "Biological production of
methane from energy crops," Biotechnol. Bioeng., 21:1209-1219.
Da Silva, E.J., 1980, "Biogas: fuel of the future?" Ambio, 9:2.
Dunlop, D.D., 1976, "Microbial oil recovery," from Sen. on Microb. Energy
Conversion, United Nations Inst, for Training and Research, October, 1976.
Gerson, D.F. and Zajic, J.E., 1979, "Bitumen extraction from Athabasca tar sands
with microbial surfactants," Petroleum Abstract, 19(32), No. 266,277.
Gulf Oil Chemicals Co., 1979, "Biomass feedstocks of the future," °rocessirg,
25:38-39.
Hal), D.O., Reeves, S.G., Dennis, G., and Rao, K.K., 1978, "Biocatalytic hydrogen
production," from Conf. on Sun: Mankind's Future Source of Energy, New
Delhi, Vol. 2, p. 805.
Hashimoto, A.G., Chen, Y.R., and Prior, R.L., '979, "Methane and protein
production from animaf feedlot wastes," J. Soil and Water Conservation,
34:16.
Keenan, J.D., 1979, "Review of biomass to fuels," Proc. Biochem., 14:9-12.
Khan, A.W., (979, "Anaerobic degradation of cellulose by mixed culture," Can. J.
Microbiol, 23:I700-'705.
King, 5.R., 1979, "Gasohol: ethanol from plant matter as motor fuel," F.
Eberstadt & Co., Inc., NY.
Laskin, A.I., '979, "Microbial transformations of hydrocarbons," 174th Am.
Chem. Soc. Mtg., 24:848-850.
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Loehr, R.C., 1978, "Methane from human, animal, and agricultural wastes" in:
Renewable Energy Resources and Rural Applications in the Developing
World, Westview Press, Boulder, CO.
Lonsane, B.K., Singh, H.D., and 8aruah, J.N., 1976, "Use of microorganism and
microbial products in secondary recovery of petroleum from economically
unrecoverable oil reservoirs," J. Scient. Industr. Res., 35:316.
Morris, W. and Whiteley, M., 1978, "Liguid fuels from carbonates by a microbial
system," Am. Chem. Soc. Symp. Series, 90:120-132.
Pankhurst, E.S., 1980, "Biogas," Gas Eng. Mang., 20:3.
Pimente', L. and Catvin, M., >979, "Brazil's Diomass program is one of the most
extensive," Chem. Eng. News, 57:35.
Reed, T.B., 1975, "Biomass energy refiners for production of fuel and fertilizers,"
J. Appl. Polymer Sci., 28:1-9,
Schwab, C., 1979, "Energy from vegetation: legal issues in biomass energy
conversion," Solar Law Reporter, 1:784.
Seeley, J.Q., 1974, "Geomicrobiologicaf method of prospecting for petroleum,"
Oil Gas J., 72:142-144.
Sitton, O.C. and Gaddy, J.L., ('979, "Design and performance of an immobilized
cell reactor for ethanot production," from 72nd Ann. Mtg. Am. Inst. Chem.
Eng., San Francisco, CA, Abstract No. 41.
Smith, G.D., 1978, "Microbiological hydrogen production," Search, 78:209.
U.S. Environmental Protection Agency, 1979, "Process design manual; sludge
treatment and disposal," EPA 625/1-29-001, September, 1979.
Tornabene, T.G., 1977, "Microbial formation of hydrocarbons," in Proc. Symp. on
Microbial Energy Conversions, Goettingen, Germany, Pergamon Press, NY.
Yen, T.F., 1976, "Microbial oil shale extraction," from Seminar on Microbial
Energy Conversion, United Nations inst. for Training, and Research, Oc-
tober, 1976.
Yen, T.F., and Meyer, W.C., 1976, "Enhanced dissolution of oi1 shale by
bioleaching with Thiobacilli," Appf. Environ. Microbiol., 32:610-6f6.
Zajic, J.E., Kosaric, N., and Brosseau, J.D., 1978, "Microbial production of
hydrogen," Adv. Biochem. Eng., 9:57-109.
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V. MINING
Bruynesteyn, A. and Duncan, D.W., 1971, "Microbiological teaching of sulphide
concentrates," Canad. Metal Quart., 10:57-63.
Duncan, D.W., and Bruynesteyn, A., 1971, "Enhancing bacterial acticity in a
uranium mine," Canad. Min. Metal. Bull., 74:rl6-l20.
Duncan, D.W., Landesman, J., and Walden, C.C., (967, "Role of Thiobacillus
ferrooxidans in the oxidation of sulfide minerals," Can. J. Microbial., 13:
397-403.
Duncan, D.W., and Walden, C.C., 1972, "Microbiological leaching in the presence
of ferric iron," Develop. Indust. Microbiol., 13:66-75.
Gates, J.E. and Pham, K.D., 1979, "An indirect fluorescent antibody staining
technique for determining population levels of Thiobacillus ferrooxidans in
acid mine drainage waters," Microb. Econ., 8:121-128.
McGoran, C.J.M., Duncan, D.W., and Walden, C.C., 1969, "Growth of Thiobacillus
ferrooxidans on various substrates," Can. J. Microbiol., 15:135-138.
Murr, L.E., Torma, A.E., and Brierly, J.A. (eds.), 1978, Metallurgical Applications
of Bacterial Leaching and Related Microbiological Phenomena, Academic
Press, NY.
Razzeli, W.E., and Trussel, P.C., >963, "Isolation and properties of an iron-
oxidizing T]TiobacH]us," I. Bacterial., 85:595-603.
Sakaguchi, H. and Silver, M., 1976, "Microbiological leaching of a chalcopyrite
concentrate by Thiobacillus ferrooxidans," Biotechnol. Bioeng., 18:1091-1101.
Torma, A.E., Wcfden, C.C., and Branion, R.M.R., 1970, "Microbiological leaching
of a zinc sulfide concentrate," Biotechnol. Bioeng., >2:501-517.
VI. POLLUTION CONTROL
Alexander, M., 1981, "Biodegradation of chemicals of environmental concern,"
Science, 2IHI32-I38.
Bellinick, C., Batistic, L., and Mayadon, J., >979, "Degradation of 2,'4-D in the
soil," Rev. Ecol. Biol. Sol. (France), 16:161-168.
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Brown, M.J. and Lesfer, J.N., 1979, "Metat removal in activated sludge: the role
of bacterial extracellular polymers," Water Res., 13:817-838.
Chakrabarty, A.M., Frietlo, D.A., and Bopp, L.N., >978, "Transposition of the
plasmid DNA segments specifying hydrocarbon degradation and their
expression in various microorganisms," Proc. Nat. Acad. Sci., USA, 15:3109-
3H2.
Chemical Week, July 23, 1980," Building 'superbugs' for the big cleanup."
Crawford, R.L., (977, "Novel methods for enumeration and identification of
microorganisms for potential use in biological delignification," from Symp.
on Biological Delignification, Weyerhaeuser, August, 1976, pp. 55-72.
Davis, A.J. and Yen, T.F., (976, "Feasibility studies of a biochemical cesuifuriza-
tion method," Am. Chem. Soc. Symp. 74:'37.
Deschamps, A.M., Mahoudeau, G., Conti, M., and Lebeault, J.M., 1980, "Bacteria
degrading tannic acid and related compounds," J. Ferment. Techno'., 5: 93-
97.
Detz, C.M. and Barvinchak, G., '979, "Microbial desulfurization of coal, "Mineral
Cong. J., 65:75-82.
Finnerty, W.R., 1980, "Microbiol desulfurization ana denitrogenation," !80th Am.
Chem. Soc. Mtg., Las Vegas, NV.
Grady, C.P.L., and Grady, J.K., 1979, "Industrial wastes: fermentation industry,"
J. Water Pollut. Contr. Fed., 81:1325.
Harbo'd, M.S., 1976, "How to control biological waste treatment processes."
Chem. Eng., 83:157-160.
Kowal, N.E. and Pahren, H.R., 1978, "Wastewater treatment: health effects
associated with wastewater treatment and disposal," J. Water Pollut.
Contr. Fed., 50:1193.
Lee, D.D., Scott, C.D., and Hancher, C.W., 1979, "Ffuidized bed bioreactor for
coal conversion effluents," J. Water Pollut. Contr. Fed., 51:974-984.
Lehtoma, K.L. and Niemela, S. 1975, "ImDroving microbial degradation of oil in
soil," Armbio, 4: (26.
McKenna, E.J. and Heath, R.D., 1976, "Biodegradation of polynuctear cromatic
hydrocarbon pollutants by soil and water microorganisms," Univ. of Illinois
Report No. UTLU-WRC-76-0H3.
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Munnecke, D.M., 1979, "Chemical physical, and biological methods for the
disposal and detoxification of pesticides," Residue Review, 70:1-26.
Nelson, R.F. and Siegrist, T.W., 1979, "Industrial wastes: chemicals and allied
products," J. Water Potlut. Contr. Fed., 51:1419.
Orndorff, S.A. and Colwell, R.R., 1980, "Microbial transformation of kepone,"
Appl. Environ. Microbiol., 39:398-406.
Patrick, F.M. and Loutit, M., 1976, "Passage of metals in eff>uents through
bacteria to higher organisms," Water Res., (0:333.
Prensner, D.S., Muchmore, C.B., Gilmore, R.A., and Oazi, A.N., :976, "Waste-
water treatment by heated rotating biological discs," Biotechnol. Bioeng.,
18:1615.
Reese, E.T., 1977, "Degradation of polymeric carbohydrates by microbial en-
zymes," Recent Adv. Phytochem., H:3H-367.
Robichaux, T.J. and Myrick, H.N., 1972, "Chemical enhancement of the biodegra-
dation of crude oil pollutants," J. Petrol. Technol, 24:16-20.
Suzuki, T., 1977, "Metabolism of pentachforophenol by a soil microbe," J.
Environ. Sci. Health, 3(2:113-127,
Walker, J.D. and Colwell, R.R., 1976, "Enumeration of petroleum-degrading
microorganisms," Appl. Environ. Microbiol., 31:198-207,
Walker, J.D., and Colwell, R.R., 1976, "Oil, mercury, and bacterial interactions,"
Environ, Sci. Techno!., 10:r(45.
Watkinson, R.J., 1978, Developments in Biodegradation of Hydrocarbons. Applied
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Yamasaki, N. Yasul, T, and Matsuska, K., 1980, "Hydrotherma! decomposition of
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VII. RISK ASSESSMENT
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GLOSSARY
This list of definitions is intended to help the reader and should not be considered
all inclusive.
antigen
aerobe
anaerobe
bacteriophage
chloroplast
chromosome
clone
codon
coiicin
conjugation
crown gal I
DNA
any chemical substance, natural or man-made, that elicits
an immune response in animals
organism requiring oxygen
organism able to live in the absence of oxygen; some
anaerobes are "obligate"; i.e., they are killed in the
presence of oxygen
one of a subgroup of viruses that infect bacteria; consists
of a relatively small amount of DNA contained in a
protein coat
a cellular organelle in higher plants; site of photosynthesis
the basic macrostucture of heredity; organization of DNA
in cell nuclei containing large numbers of genes
a collection of cells each having an identical genetic
composition
a triplet of nucleotides on a DNA chain that specifies a
particular amino acid or otherwise controls protein syn-
thesis
a bacterial toxin, the coding for which is found on a
olasmid; some forms are toxic to humans
one-way transfer of DNA between bacteria in cell contact
plant tumor caused by infection with Aqrobacterium
tumefaciens; genes located in the Ti-plasmid of the
Agrobacterium are responsible for tumor induction
deoxyribonucleic acid; the molecular basis of genes; made
from the sequential arrangement of four nucleotide build-
ing blocks: adenine, cytosine, guanine, and thymine; nor-
mal configuration is in double-stranded helical form
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cDNA
en tomopathogen
enzyme
eukaryote
F factor
gene
gene library
genome
HEP A filter
host-vector system
intron
tac operon
lambda
iigase
lignocellulose
complementary DNA; laboratory-created DMA that is
complementary to mRNA extracted from a cell
insect pathogen, usually microbial in nature, such as a
bacterium, protozoan, or virus
organic catalyst of biochemical reactions in a eel!; com-
posed of protein
an organism composed of cells that are distinguished by
the presence of a nucleus and multiple chromosomes;
fungi, protozoa, and all differentiated multicellular forms
of life are eukaryotic
fertility factor; plasmid that specifies gender in bacteria
a defined length along a chromosome, made of DNA and
coding for a protein molecule
the result of a shotgun experiment in which each cloned
bacterial colony contains different segments of DNA
all the genes of cn organism or individual
high efficiency particulate air filter
in the recombinant DNA field, the particular organism
(host) into which the gene is cloned, and the vehicle
(vector)—usually a plasmid system—that carries the ger.e
into the host
an intervening sequence of DNA of unknown function
found only in eukaryotic genes; this sequence is not
expressed in the transcription to mRNA
an operon in E. coli that codes for three enzymes involved
in the metabolism of lactose
bacteriophage that infects E. colj; commonly used as a
vector in recombinant DNA research
an enzyme that catalyzes the linking of sequential bases
in single-stranded DNA
complex biopoiymer comprising the bulk of woody plants;
consists of polysaccharides and polymeric phenols
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lymphocyte
lysis
non-conjugable
nucleotide
nucleus
oi igonucleotide
operator
operon
opines
peptide
phage
plasmid
polymerase
polypeptide
prokaryote
a type of cell found in the blood, spleen, lymph nodes, etc.
of higher animals; one sub-class of lymphocyte manufac-
tures and secretes anitbodies
process of cell disintegration; cell bursting
refers to bacterial plasmids that cannot be transferred
between organisms
any of a class of compounds consisting of a purine or
pyrimidine base, bonded to a ribose or deoxyribose sugar
and to a phosphate group; the basic structural units of
RNA and DNA
the cell region containing chromosomes and enclosed :n a
definite membrane; found only in eukaryotic ceils
the sequential arrangement of more than one nucleotide
a region of DNA that controls the expression of adjacent
genes by interacting with a repressor protein
a gene unit consisting of one or more genes and the
controls for that unit; the lac operon, for excmple, is
made up of three genes, the operator, and the terminator
unusual amino acids synthesized by genes located on Ti-
plasmids; nopaline and octopine are examples
two or more amino acids joined together
shortened form of the word bacteriophage; bacterial virus
a small circle of double-stranded DNA that exists and
replicates autonomously in bacteria; often codes for resis-
tance to antibiotics; may be transferred between bacteria
during conjugation
enzyme that catalyzes the assembly of nucleotides into
RNA and of deoxynucleotides into DNA
a molecular chain of many amino acids joined together;
synonymous with protein
cellular organism distinguished by the lack of a defined
nucleus and by the presence of a single, naked chromo-
some; bacteria and blue-green algae are the only major
examples
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promoter
protein
protoplast
replication
repressor
restriction
endonuclease
R factor
ri bosom e
RNA
mRNA
shotgun cloning
sticky end
the region on a DNA strand that indicates the place to
start the transcription of the gene into RNA
a sequence of amino acids; the ultimate expression of a
gene; primary component of enzymes and many hormones
a bacterium or plant cell
been removed
from which the cell wall has
T-ONA
the process of making copies of DNA in a cell; depending
upon the plasmid, man copies may be replicated after
insertion of DNA into the host
a gene product that prevents the transcription of an
operon by binding to an operator region
one of a class of enzymes that cleave both strands of
DNA at sequence-specific sites; used extensively in
recombinant DNA experiments
resistance factor; refers to plasmids coding for resistance
to antibiotics
a large molecular array, composed of RNA and protein,
that is responsible for translating messenger RNA into
protein
ribonucleic acid, used to form complements to DNA in
gene expression
messenger RNA. formed in the ceil nucleus in the process
of gene expression; complementary to the base seauence
of the DNA of the gene
cloning procedure in which aH the chromosomes of a
donor organism are enzymatically fragmented and placed
into hosts for expression; results in a gene library
refers to double-stranded DNA that has been cleaved in
such a way by certain restriction endonuclease enzymes
that the end of one strand extends beyond the end of the
other; the end is called "sticky" because the bases are
exposed and can thus mate with complementary sticky
ends •
a region of the Ti-olasmid that contains genes required
for crown gall tumor induction and maintenance
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terminator
a region on a gene that codes for the termination of
transcription
Ti-plasmid
transcription
transduction
transformation
translation
transposons
vector
virus
a large plasmid found in Aqrobacterium tumefaciens;
induces crown gail tumors in plants infected with the
bacterium
the process of copying DNA into RNA; the
messenger RNA
result is
the transfer of genetic material from one ceil to another
by means of a viral vector (for bacteria, the vector is
bacteriophage)
the process of inserting into the host organism a vector
containing a gene that is to be cloned
the process of making a peptide from mRNA; performed
by ribosomes
short DNA segments containing one or c few genes thct
are readily translocated between ceils or to different
sites within the same ceil; responsible for antibiotic
resistance in bacteria; also found in some eukaryotic
organisms; transposons may serve as suitable vectors for
genetic engineering in various organisms
an agent consisting of a DNA molecule known to au-
tonomously replicate in a cell to which another DNA
segment may be attached experimentally to bring about
the replication of the attached segment
any of the submicroscopic infective agents composed of
RNA or DNA wrapped in a protein coat end capable of
growth and multiplication only in living cells
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