CYTOCHEMICAL STUDIES OF PLANETARY MICROORGANISMS
PROPOSED CONTINUATION

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
GRANT NGR-05-020-004

For The Period

December 1, 1977 to November 30, 1978

Principal Investigators:

Quel Cac . an

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if) mfiy / ;
Jott Le See f

 

 

 

Préfessor Joshua cies Elliott C. Levinthal, Adjunct Professor
Department of Genetics if Department of Genetics
“Stanford University School of Medicine Stanford University School of Medicine
Stanford, California 94305 Stanford, California 94305
Telephone: (415) 497-5801 Telephone: (415) 497-5813

Co-Investigator:

ye ft fell

Dr. Dennis H. Smith

Department of Genetics and Chemistry
Stanford University School of Medicine
Stanford, California 94305

Telephone: (415) 497-5289

AUG 8 1977

rc
PROPOSAL

This proposal is a request for the continuation of Grant
NGR-05-020-004 to the extent of $110,000 for the period December 1,
1977 through November 30, 1978.

In addition to outlining the proposed activities for the twelve
month period beginning December 1, 1977, we have also briefly
reviewed some of the accomplishments of the previous twelve month

period.
TABLE OF CONTENTS

GENERAL INTRODUCTION iii
RESEARCH PLANS 1977-78
A. Microbiological and Genetic Studies 1
B. Chemistry of Polymeric Carbon 8
APPENDIX I PROGRESS REPORT 24

BUDGET 27

ii
GENERAL INTRODUCTION

As has been the case for some time, our research effort is divided
into a) microbiological, and b) analytical-chemical studies. These are
discussed in two main partitions of this renewal application and progress
report. On this occasion, all of the methodologically oriented efforts of
past years have been ‘spun off' into programs supported from other sources;
our current proposals --truncated by progressive inflation at level-dollar
Support -- converge on the theme of the origin of life and fundamental
issues in the evolution of primitive microorganisms.

The overall program of work summarized here far exceeds what could
be accomplished within the actual budget requested from NASA. We have
the advantage, however, of considerable momentum from previous experience
and equipment. We also anticipate an advantageous overlap with support
that we are planning to request from ERDA and from the NIEHS with respect
to health- and energy-related aspects of the biology and chemistry of
polymeric carbon. (Only the most preliminary contacts have been made with
those agencies at this time.) The funds requested will enable us to sustain
the key professional staff during this transition, and to continue to
focus on those issues that are of specific interest to the NASA program office.
If other support cannot be obtained, we will be constrained to an
opportunistic pursuit of only the most promising research gambles that

present themselves during our explorations.

iii
RESEARCH PLANS 1977-78.

 

A. Microbiological and Genetic Studies

For several years, the main focus of microbiological research in this
laboratory has been on the construction of simple model systems for the genetic
diversification of simple genetic codes. This work entailed the use of
‘recombinant DNA' technology to construct bacteria that had simple monotonous
synthetic sequences, whose further evolution could then be studied in detail. The
technical task has proven to be far more difficult than was originally envisaged;
but on the way we have made a number of important advances in a) methods of DNA
segmentation and implantation, b) understanding of the conditions for expression
of genes exchanged among widely diverse bacterial species, and c) the prevalence
of mecnanisms for promiscuous mixing of genes among bacteria believed to be quite
separate in an evolutionary sense -- e.g. Bacillus and Staphyloccus. In a sense
tnis last finding may unexpectedly prove to be the most important of our
contributions to the theme of the origin and evolution of life (Green, 1964).

In the light of the unexpected ferocity and hostility of contention about
the puolic health hazards of work with recombinant DNA, -- although we have
endeavored to stay as far as possible from situations with credible pathogenic
potential -- we have decided to phase out our work under NASA support that
directly involves recombinant DNA; and this will no longer be part of our
continuation proposal. However, we will continue efforts along the lines of c)
above, namely to seek still broader understanding of the extent to which genetic
informaticn is shared with a common gene pool among microbes. Julian Davies, for
example, nas long speculated that antibiotic-producing streptomycetes are the

primary reservoir of plasmids that recur in a wide range of pathogenic bacteria,
and by conferring resistance to the antibiotics result in serious clinical
problems. However, direct support for this degree of panmixis has yet to be
achieved experimentally. In the course of our observations of the Staphylococcus
x Bacillus exchanges mentioned above, we believe we may have some hints how to
overcome these experimental barriers, and will apply these ideas to building
general "plasmid-traps" to capture the movement of plasmids among a variety of
species in their natural soil habitats. Tne insight sought here would of course
be of the greatest importance for a) the theory of evolution of microbes (if not
eukaryotes as well); b) practical problems of tne sources of antibiotic-
resistance; and ¢c) common-sense tempering of regulatory controls that have become
a serious hindrance to research in this field.

This shift of empnasis to natural habitats is coupled with an effort to get
a better understanding of the role of solid surfaces in microbial physiology,
ecology and genetics. Some years ago, in connection with studies that helped to
prove that penicillin acted as an inhibitor of cell-wall synthesis, I was
impressed by the role that solid-agar had in controlling the morpnozenesis of
spheroplasts or L-forms in E. coli. Knowing that many, perhaps most, of the
microbes resident in soils have never been isolated in pure culture, I wondered
whether much of laboratory microbiology -- using homogeneous liquid media -~ is
not an artefact, far removed from conditions of the natural habitat. In the
interval, many soil microbiologists have made similar observations (summarized in
Marshall's book), at least of the influence of interfaces on tne growth and
physiology of particular species. The most extreme speculation that I propose to
investizsats is whether there are not many species that have an absolute
requirezsnt for a solid substrate -- from which it would follow that conventional
methods have oeen actively selecting against the recognition of an important part

2
of our biosphere. An even more interesting proposition is that these organisms
may also include representatives of the simplest, most primitive categories that
could give further clues about the baffling missing links in the 'Seala Naturae ,
Some (feeble) support for such a speculation can be gathered from the fact that
Mycoplasmas, which have the smallest genome-sizes of any presently known
freeliving organisms, lack a rigid cell wall. Some of these have, to be sure,
been cultivated in suspension, but often only with difficulty; and analogous L-
phases of larger bacteria generally require an agar substrate. The implication is
that a rigid wall, and the ability to proliferate readily in suspension, are
later evolved attributes of the 'typical' bacteria compared to more primitive
forms. So far, the most primitive mycoplasmas still have the complete repertoire
of the genetic code, and therefore can give us little insignt into its primary
evolution (unless one takes this as evidence of special creation or of
panspermia!).

We must concede that we have made little progress since Oparin in bridging
the hiatus between the chemosyntnesis of nucleotides and their assembly into
meaningful, autocatalytic sequences -- in part for.lack cf observable
representatives of the intermediate stages.

While this would be the most rewarding outcome, one does not have to stake
one's faith in that speculation to justify the research plan. The strategy will
be to construct media and devices that offer large surface areas to organisms in
soil habitats, but which can be extracted for closer study-- e.g. glass films
either bare or with various mineral coatings. These will be manipulated so as to
encourage organisms that nave a preference for sessile growth on the surface, and
tnen sampied further for organisms that may have a near~ or total- dependence on
such surfaces. (One may not have to look far: witness algal and cyanophytic films

3
in aquaria; but I do not know of critical comparisons of their growth potential
in adnerent vs. suspended mode. It is known that the sheathed habit, associated
with attacnment, of Sphaerotilus natans, is suppressed by high nutrient
concentrations -~ there may well be many organisms tnat correspond to the
attached phase of Sphaerotilus, being unable to grow except as epibionts. (The
associaticn of bacteria with surfaces has also attracted renewed interest in
recent years in connection with dental microbiology, and with other aspects of
infectious disease.) |

The associations of blue-green algae with fissures and voids in the
immediate sub-surface of dry Antarctic soils has been pointed out, by EI.
Friedmann, to refute the asserted sterility of this terrestrial habitat, and to
point to some caveats in the design of life-detection strategies for planetary
exploration. Saprophytic bacteria that would exploit the primary production of
the algae would oe very difficult to ascertain by conventional methods if they
were interface-dependent. This gap in our appreciation of the soil habitat is
thus equally important for laying the groundwork for future planetary biological
science and engineering. Furthermore, there are many anecdotal observations of
the enhanced resistance to thermal sterilization of microbes in soil matrices;
but the phenomenon has not been systematically investigated with regard to
mechanism. Thus, our projected studies of interface biology bear on problems of
survival in hostile environments (terrestrial and extra-terrestrial) as well as
upon practical problems of disinfection, e.g. for planetary quarantine. The
methods of microscopic examination of soils that will oe developed for these
Studies snould also have some bearing on planning for instruments appropriate to
future planetary and return-sample missions from an exobiological perspective.

Finally, few students of the origin of life have FAILED to comment on the

4
probaole role of clay and other neterozeneous surfaces for the local
concentration of chemosynthetic precursors of life. tie thus note that, while the
central ideas of this researcn plan are widely entertained, they have only rarely
been the subject of concerted investigation: doubtless because of the
overwhelming convenience of working with transparent, homogenous media.

Besides the minerals commonly associated with soil, we will also give
particular attention to polymeric carbon as an interphase -~ the prevalence, and
adsorptive and catalytic properties of this material which justify this choice

are elaborated in part B of this proposal.
REFERENCES PART A.

’

(This is not a systematic reference list, but the following sources will help to
illustrate and enlarge upon the ideas just summarized.)

Courvalin, P., weisblun, B., and Davies, J., 1977. "“Aminoglycoside-modifying
enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic
resistance in #scherichia coli." Proce. Nat. Acad. Sci... US., 74, 999.

Ehrlicno, S.D., 1977. "Replication and expression of plasmids from

Staphylococcus aureus in Eacillus subtilis." Proc. Nat. Acad. Sci. , US., ZH,

 

1680. (Reprint enclosed, from this laboratory)

Lederberg, J. and St. Clair, J., 1958. “Protoplasts and L-type growth of E.
coli." J. Bact., 75, 143. |

Lederberg, J., 1952. “Preservation of bacterial cultures on silica gel."
(unpublished note). Cf. Hunt, G.A., Gourevitch, A., and Lein, J., 1958.
"Preservation of cultures by drying on porcelain beads.", J. Raet., 76, 453.

Gaudy, #&. and wolfe, R.S., 1961. "Factors affecting filamentous growth of
Spnaerotilus natans." Applied Microbiol., 9. 540.

wallace, D.C. and Morowitz, H.J., 1973. “Genome Size and Evolution,"
Coromosoma., 40, 121. (This article points out that Mycoplasma probably
represents a primitive genome size of about 509 megadaltons, with other bacterial
species tending to be integral multiples. Prof. Wallace is now a member of the
faculty of this department and will consult informally with us.)

Landman, O.E., Ryter, A.,and frenel, C., 1968. "Gelatin-induced reversion

of protoplasts of Bacillus subtilis to the bacillary form." J. Bact. 96, 2154.

 

Qu, Li-Tse and Alexander, M., 1974. “Effect of glass microbeads on growth,
activity anc morphological changes of Bacillus megateriun."” Arch. Microbiol.,

19i, 35.
Friedmann, E.I. and Ocampo, R., 1976. "“Endolithie blue-green algae in the
dry valleys: primary producers in the Antarctic desert ecosystem." Science, 193,
1247.

Marshall, K.C., “Interfaces in Microbial Ecology," Harvard, 1976.

Hattori, T., “ Microbial Life in the Soil," Dekker, 1973.
£. Cnemistry of Polymeric Carbon

1. Introduction

The methodological emphasis of our previous work is outlined in the progress
report (see Appendix 1). We believe that GC/MS techniques have now progressed to
the point where scientifically useful applications should be the main stress of
our effort, namely on the carbon chemistry of the solar system. We propose
specifie areas of study wnich bear directly on the origins of carbon compounds,
and their interactions and reactions in both ancient and modern environments.
These studies complement directly Part A of this proposal in that we will
investigate carbon substrates and adsorbed material as an environment for the

growth of microorganisms.
2. Sumnary of Proposed Studies

we propose to investigate certain aspects of the chemistry of polymeric
carbon. Polymeric carbon, defined in more detail in the subsequent section,
ineludes a variety of polymers, such as graphite and carbon black, characterized
in part by hign carbdon content, high structural order at the molecular level and
high surface area. The role played by polymeric carbon as a participant in the
evolution of carbon compounds over geologic time is poorly understood. Yet it is
the thermodynanically favored form of carbon under many conditions of temperature
and pressure in reducing atsiospheres, and is stable to decomposition under many
other conditions because of high activation energies toward decomposition.

{Tnere are several reasons for our interest in polymeric carbon:

@) Extraterrestrial Carbon. The carbon chemistry of the moon is quite

complex and not fully understood. A similar situation pertains to Mars, where
the carbon content is unknown but reactions of carbon compounds in the Viking
biology experiments produce unexpected, and unexplained results. An
understanding of behavior of low molecular weight carbon compounds and their
interactions with polymeric carbon under various conditions might facilitate an
understanding of some of the observations.

b) Prebiotic Carbon. There is considerable controversy over the presence of
polymeric carbon in prebiological times. No experiment of which we are aware
dealing with condensation of gases to yield precursors of biopolymers has
considered the effects of polymeric caroon, although the effects of clay on
condensation reactions have been studied. Similarly, complex models are proposed
for subsequent concentration and condensation of e.g., amino acids without
attention being paid to the extreme adsorptive power of polymeric carbon and its
catalytic activity. Again, however, other mineral surfaces have been proposed to
act in similar roles.

c@) Geo~ and Biochemical Significance of Carbon. Significant quantities of
the world's carbon are cycled through polymeric carbon, for example in natural or
man-made combustion products, and in compaction and diagenetic modification of
organic compounds into coal or related polymeric carbon deposits. Yet the
origins, distribution and fate of this material are not well known. For example,
we have no reliable information on the pathways by which charcoal generated by
forest fires reenters the global carbon cycle, although this must represent a
substantial percentage of the total annual photosynthate. The role of polymeric
caroon in mediating cnemical reactions in the biosphere and the ability of soil
organisms to utilize carbon for support and/or nutrition are not well understood.
Previous work in this laboratory, some years azo (Schneour, 1966) did not elicit
convincing evidence of microbial conversion of charcoal on a significant seale.

9
Our present speculation is that soil protozoa and other invertebrates must be
involved in ways yet to be experimentally examined.

We do not claim that polymeric carbon was necessarily an essential
ingredient in evolution of carbon compounds or in the subsequent origin of life.
But the combination of its properties, its intimate relationship to other formas
of organic carbon and the lack of attention which has been given to its role in
the above problems demands some investigation. We propose to begin some
preliminary studies, described in the following section.

Because our proposed studies deal with mixtures of organic compounds, GC/MS
will be the primary analytical method for characterization of mixtures and
identification of compounds. Thus, maintenance of our current capabilities in
GC/MS and computer techniques is necessary. In addition, we will have to carry
out some development work in both hardware and programs to support the new
studies.

3. Proposed Research Studies in the Chenistry of Polymeric Carbon

Polymeric carbon is defined as those natural and synthetic materials which
consist structurally of fused polycyclic ring systeis composed predominantly of
carbon. Tnese materials range in homogeneity from graphite to coal and soot.
(For present purposes we ignore the crystalline form of diamond.)

The major forms and methods of preparation of carbon are as follows:

a) Graphite. Graphite consists of large two dimensional sheets of fused
aromatic rings. Tne layers are held together by weak van der waal's forces which
give the material its characteristic properties ~ lubricating ability and
softness. it is found naturally in a variety of places including deep Earth

deposits and meteorites. Tne majority of the graphite in commercial use is

10
prepared synthetically by high temperature conversion from less pure carbon black
or coke (Holliday et al., 1973).

b) Carbon Black. These materials, which are prepared by partial combustion
of various petroleum feedstocks, consist of fairly pure carbon (90-100 per cent
C) and structurally are composed of microcrystalline graphite-~like particles.
Carbon blacks have high adsorptive properties and have impurities which depend on
the feedstock, pyrolysis method and post-treatment with oxygen, sulfur, nitrogen
and occasional inorganic elements (Schubert, et al., 1959).

c@) Activated Carbon. These materials are prepared by controlled pyrolysis of
various organic substrates including sugar, bone, and coconut. They are
activated by partial oxidation to yield materials of optimum adsorptive
properties. This preparation leaves this carbon form very porous. Depending on
the feedstock activated carbon is more or less pure carbon (Hassler, 1974).

G) Coal and Petroleum, These biogenic materials vary greatly in their carton
content and their degree of polymerization. They are formed over geological time
by deposition and modification of biological materials followed by alteration by
heat and pressure (Eglinton and Murphy, 1969).

e) Other Carbons. Any incomplete combustion process will leave a residue of
carbon of variable form. Major processes of this type inelude soots from
petrochemical energy production and uncontrolled fires.

There are several methods of natural production of polymeric carbon which
may nave been important to the early carbon chemistry of the Earth. Among the
most important are:

a) Pyrovenie. Due to interest in the preparation of carbon black this area
has received considerable attention (Cullis, 1976; Abrahamson, 1977). The

generally accepted view is that acetylene is formed as an intermediate by

11
cracking of larger molecules or dehydrogenation of methane. Soot formation
(polymeric carbon) then occurs as a stepwise process of polymerization of
acetylene and partial dehydrogenation.

Db) Cosmological, Below 930 dezrees C carbon monoxide is unstable with
respect to disproportionation to carbon and C02. However, this reaction is very
Slow and néeds catalysis to achieve reasonable rates. Catalysts for carbon
monoxide decomposition include palladium, iron oxides and graphite itself. The
mechanistic aspects of this formation of carbon are no doubt quite complex and
have not been studied.

c) Thermal. Once complex molecules have been formed, wnether abiogenically
or piogenically, thermal processes serve to modify them further. Such reactions
have been important in more recent geological periods in the formation of
petroleum and coal and in present-day production of activated carbon.
Carbonaceous materials formed py pyrogenic or cosmolozical reactions are also
subject to thermal alteration, however, and such processes were presumably active
in early Earth history.

4. Specific Projects
@. Cnemical Characterization of Polymeric Carbon.

we propose to develop chemical methods for detection and characterization of
polymeric carbon. betection is important for those problems such as analysis of
lunar or chondritic material (and presumably martian soil also) where the levels
of caroon are relatively low and no direct methods for measurement of polymeric
carbon exist. Characterization of the structure of polymeric carbon is important
because of the widely differing physical and chemical properties of the various
Structural forms.

i. Detection of Polymeric Carbon. The analysis of low levels of graphitic

le
carbon has traditionally been accomplished using difference methods. For example
carbon black in rubber is analyzed by dissolving away the organic matrix with hot
nitric acid and weighing the residue (Sehubert et al., 1969). Graphitic carbon
in lunar soil was estimated after removal of volatiles, materials rendered
volatile upon acid treatment and materials rendered volatile below 750 degrees C.
Tne residual carbon was assumed to be graphitic and could be oxidized to CO2 at
1050 C (Cnang et al., 1970).

Our method for the analysis of carbon is based on unpublished work from our
laboratory. We have found that activated carbon upon oxidation with chromic acid
or potassiua permanganate gives rise to oxalic acid and a series of benzene
polycarboxylic acids (BPCA's). Similar results had previously been described
upon oxidation of graphite with sulfuric acid (Mantell, 1968) and of coal
gasification residues with oxygen in a basie solution at elevated temperatures
(Eisenberg and Solomon, 1977).

These acids are conveniently identified and quantitated after conversion to
their methyl- or trimethylsilylester derivatives. The method will be calibrated
using known amounts of carbon. We will study what, if any, interference is caused
by the presence of clays or other inorganic materials. This will lead us to a
method for measuring the polymeric carbon content of soils.

There is evidence in the literature that different forms of polymeric carbon
will react differently to the oxidizing conditions of our experiment. Chromic
acid has been reported (Oberlin and Mering, 1964) to oxidize graphite to COo2
wnile carbon black is relatively resistant to such treatment. Also, Eisenberg
and Solomcn (1977) have noted variations in the pattern of BCPA's formed upon
oxidation of different coal~-gasification residues.

The only non-polymeric materials which might give rise to benzene

13
polycarboxylates in this experiment are small to medium size aromatics. The
presence of these materials can be checked by extraction and volatilization
techniques.

il. Analysis of Carbon Structure. The many forms of elemental carbon
(graphite, carbon black, activated carbon) are normally characterized in terms of
their methods of preparation, particle size, surface area and other gross
properties (Schubert et al., 1969). Tne types of molecular sub-structures
present and elemental analyses of various carbons are also well-known (Garten and
Weiss, 1957). We would like to extend the knowledge of carbon structure to
include estimations of the size distributions of the individual polymeric
molecules present in carbon structures. Two methods for approaching this problea
are:

1) Solubilization with phenanthrene - it is known that hot phenanthrene will
solubilize 90% of the material present in coal (Haredy and Fugassi, 1966). Once
in solution, the carbon molecules could be sized by gel permeation
cnromatozraphy.

2) Chemical modification - we have speculated that a reaction which
increases the polarity of the individual polymer molecules might increase their
repulsive interactions and allow for a more facile solubilization. Possibilities
include sulfonation, mild oxidation to phenols or a nitration/reduction scheme to
yield a polyammonium species. Indeed, Boehm, et al. (1962) investigated the
breakdown of graphite into individual layer molecules upon oxidation. He
prepared graphite oxide, a partially oxidized material which retains all of the
original carbon atoms. After dispersion as a dilute suspension in base, he
reduced the saterial to carbon with hydrazine to form what were believed to be
erystals of only a few layers of carbon each, Further characterization by X-Ray

14
analysis proved inconclusive in terms of determining the layer diameters within
the crystals.

we propose to repeat this experiment with graphite oxide but do the sizing
experiments on the oxide itself rather than the rereduced material. Presumably
the oxygen functionalities on the surface of the graphite oxide (phenols, ethers,
quinones) will disrupt the interlayer attractive forces and facilitate the
production of single layers. We will size these by liquid phase methods (gel-
permeation chromatography). The data so generated will not be easily correlated
to an absolute molecular weight due to the possibility of holes in the graphite
polymer (Hutcheon, 1970). However, this method should allow for characterization
of different forms of polymeric carbon and such data will be important in tracing
the genesis of unknown carbons found in soil and other complex media.
o. Gas/solid Carbon Chemistry

we hypothesize that polymeric carbon played (and continues to play) a
Significant role in the carbon chemistry of earth in particular and the solar
system in general. Thermodynamic calculations (Urey (1953); Dayhoff, et al.
(19604); Eck, et al. (1966)) reveal the stability of. “asphalt-like" mixtures under
conditions simulating solar nebulas and planetary atmospheres. Yet such studies
have not performed the more complex calculations needed to deal explicitly with
elemental carbon (Dayhoff, et al. (1964); Eck, et al. (1956)). Few laboratory
Simulations of condensation of model planetary atmospheres into simple organic
molecules examine carbon polymers which are formed, although "aromatic polymers"
have been noted in at least one case (Anders, et al,. 1973). No one to our
knowledge has studied the effects of polymeric carbon on such condensations
althoush cther solid supports have been included in some studies (Harada and Fox,
1964). Seuss has discussed the participation of solid forms of carbon in early

15
earth history (Seuss, 1975). Evidence from carbonaceous chondrites and the moon
(which might be considered modern representatives of early earth history)
(Eglinton et, al., 1972) indicates a very complex carbon chemistry involving in
part polymeric carbon. The known adsorptive and catalytic properties of
polymeric carbon suggest that, if present, it can participate in a variety of
chemical reactions involving carbon compounds.

We suspect that solid, polymeric forms of carbon also play a role in the
carbon chemistry of Mars. Interactions of carbon present on the Martian surface
with gases or added nutrients (Viking Pyrolytic Release and Labelled Release
experiments, respectively) might contribute to the observed results, which to
this date have no widely accepted explanation,

We do not propose to attempt to simulate a variety of planetary atmospheres
or soils in order to study the potential role of polymeric carbon. Important
laboratory experiments must be performed first to promote our understanding of
the physical and chemical nature of polymeric carbon and its interactions with
molecules in the gas phase. ‘Tne experiments discussed below are aimed toward
laying the foundation for more complex studies in the future.

We propose to study the interactions of well~characterized polymeric carbons
with low molecular weignt gases under a variety of conditions. We will obtain
representative samples of polymeric carbon of various degrees of purity,
including graphite, activated carbon and channel black. We will select samples
based in part on now well they nave been characterized by the manufacturer in
terms of physical and chemical properties. We will characterize the materials
further using scanning electron microscopy and by utilizing the proposed chemical
procedures for characterizing polymeric carbon (see above, Section IJ.B.1).

We plan at least the following two specific experiments. The first

16
experiment is to study the interaction of CO and CO2 mixtures with polymeric
carbon under moderate temperatures and pressures, with and without heavy metal
catalysts. We will utilize 14C labelled gases or carbon substrates to determined
adsorbed and chemically bound or reacted species. We will use GC and GC/MS to
determine gas mixtures and identities of nigher molecular weignt materials if any
are formed. The results of this experiment may have relevance to observations
(by the Viking landers) of Martian soil chemistry.

The second experiment we will perform will be to study the formation of .
organic materials using mixtures of methane, ammonia, hydrogen, water with
polymeric carbon, with and witnout heavy metals, as a catalyst. An adaption of
the apparatus of Harada and Fox (Harada and Fox, 1964) will be used to carry. out
the reactions. GC/MS will be used subsequent to derivatization of products to
determine the amounts and types of materials formed. Conparisons will be made to
pudlished results from other methods of simulation of prebiotic synthesis
(Calvin, 1969).

Ge. Liguid/Solid Carbon Chemistry

Solid surfaces have assumed a major role in most accounts of prebiotic
enenistry as vehicles for adsorption and concentration of organic molecules
(Nissenbaum, 1976) and catalysis of synthetic and polymerization reactions
(Poncelet et al., 1975; Paecht~Horowitz, 1975). As the most likely candidates
for this activity, the wide-spread clays have received considerable attention
(for a review see Anderson and Banin, 1975.)

Although not important surface solids at present, presumably because they
are metabolically recycled, the graphitic carbons should have been abundant in
prebiotica tines and may have functioned for the adsorption (protection and
concentration) or transformation of hydrophobic molecules.

17
There is ample precedent for the catalytic activity of polymeric carbon.
For example, activated carbon has been shown to catalyze numerous liquid phase
reactions including: 1) racemization of 1,1'-binapnhthyl (Pincock et al., 1973),
2) Olefin isomerizations and polymerizations (Meier and Hill, 1974), 3) the
inversion of sucrose (Puri et al., 1972), 4) the hydrolysis of ethyl acetate
(Puri et al., 1972), 5) the reduction of Co (III) by iodide (Murcinik and Spero,
1974).

Activated carbon by itself will also catalyze the oxidation of various
substrates by molecular oxygen (Garten and Weiss, 1957). Especially prone to
carbon-catalyzed: oxidation are difunctional molecules such as amino acids and
malonic acids. The use of carbon as a support for metal catalysts (Hassler, 1974
boersman, 1974) and inorganic reagents (Kagan, 1976) is well known.

Paecht-Horowitz (1976) has snown that montmorillonite clays will catalyze
the polymerization of amino acids and derivatives in aqueous solution. The
derivatives used were amino acid adenylates in neutral solution. Such
derivatives show a natural tendency to polymerize under aqueous conditions, but
in the presence of clay both a higher degree of polymerization and a high degree
of order in the resulting polymers are obtained. |

Our proposed experiment is to repeat this polymerization reaction in the
presence of suspended polymeric carbon. Catalysis of this reaction by clays was
very pH dependent (Paecht-Horowitz, 1976), proceeding only at near neutral pH's
due to the competing requirements of protonated amino groups (for adsorption to
clay) and free amino groups (for polymerization). On carbon the neutral species
will be tne preferred adsorbate and this fact may lead to increased rates of
polymerization of adsorbed amino acid adenylates.

G. Scliz/Solid Carbon Chemistry

18
i. Experiments on Prebiotic Synthesis. One can speculate that materials
such as amino acids generated on carbon surfaces (for example, in gas/solid
reactions, Section II.8.2) or adsorbed from aqueous solution might be
sufficiently concentrated and criented so that the probability of thermal
polymerization is increased. In a paper on thermal polymerization of amino acids
(Fox and Harada, 1958) it was found that an excess of one or two amino acid
monomers was necessary to decrease the melting point of an amino acid mixture
sufficiently below decomposition temperature. Under these conditions, some
polymerization could occur without extreme decomposition. On carbon surfaces we
have a very different situation energetically. Crystal forces are unimportant
and monomers are free to interact (if the concentration is high enough) at lower
temperatures.

we propose to study tne thermal polymerization of various amino acid
mixtures after adsorption onto polymeric carbon. Analysis of product mixtures
for degree of polymerization and for the presence of any induced specificity or
preference for certain sequences will be noted.

adi. Carbon as an Environment for Microorganisms. In Part A of this proposal
we speculated about the influences of solid surfaces on microorganisms and the
potential role of such surfaces as environments for novel organisms. Research
planned for the first part of this proposal will address questions of the utility
of surfaces such as polymeric carbon for the growth and nourishment of organisms.
In this part we propose to examine another aspect of organisms and polymeric
carbon, namely, can the carbon itself be utilized by microorganisms as a carbon
source.

In

oO

xperiments conducted in these laboratories several years azo (Schneour,
1966) preliminary evidence was obtained for the microbiological breakdown of

19
elemental carbon. Using radiolabelled carbon, it was shown that a) soil from
several sources showed activity toward metabolism of elemental carbon, and b)
this activity was enhanced in soils taken from areas expected to have high
concentrations of polymeric carbon due to recent forest fires.

We propose to reopen this investigation, using techniques developed in
conjunction with gas/solid studies (Section II.H.2) to monitor radiolabelled
gases. We will use well-characterized carbons (see Section II.B.1) together with
different organisms including those commonly culturable from soils. If activity

is found, we will search for intermediate products of carbon oxidation.

20
REFERENCES PART B.
Abrahamson, J., 1977. Nature, 266, 323.
Anders, E., Hayatsu, R., and Studier, M.H., 1973. Science, 182, 781.
Anderson, D.M., and Banin, A., 1975. Origin of Life, 6, 23.
Boehn, H.P., Clauss, A., Fischer, G., and Hoffman, U., 1962. “Fifth
Conference of the American Carbdon Committee," Vol. I, p. 73.

Boersman, M.A.M., 1974. Catal. Rev. Sci. Enz., 10, 243.

 

Calvin, M., 1969. "Chemical Evolution," Oxford Univ. Press, London.

Carnart, R.E., Varkony, T.H., and Smith, D.H., 1977. in "Computer-Assisted
Structure Elucidation," D.H. Smith, Ed., American Chemical Society, Wash., D.C.,
in press. (See Appendix 1)

Chang, S., Smith, J.W., Kaplan, I., Lawless, J., Kvenvolden, K.A., and
Ponnamperuma, C., 1970. “Proceedings of the Apollo 11 Lunar Science Conference,
Vol. 2, p. 1857.

Cullis, C.F., 1976. "Petroleum—Derived Carbons," M.L. Deviney and T.M.
O'Grady, American Chemical Society Symposium Series 21, Washington, D.C., p. 348.
Dayhofr, H.0., Lippincott, E.R., and Eek, R.Vey 1964. <Seience>, Anal.

Cnem,, 48, 1368. 146, 1461.

Eck, R.V., Lippincott, E.R., Daynoff,M.0., and Pratt, ¥.T., 1966. Science,
153, 628.

Eglinton, G., Maxwell, J.R., and Pillinger, C.T., 1972. Scientific American,
227, 81.

Fox, S.W., and Harada, K., 1958. Science, 128, 1214.

Garten, V.A., and Weiss, D.E., 1957. Hev. Pure Appl. Cnem., 1, 69.

Green, K.V., 1964. "Carbon Monoxide," in Encyclopedia of Cnemical
Technolozy, 4, 424.

21
Harada, K., and Fox, S.W., 1964. in "The Origins of Prebiological Systems
and of Tneir Molecular Matrices," S.W. Fox, Ed., Academic Press, New York, N.Y.,
p. 190.

Hassler, J.W., 1974, "Purification with Activated Carbon," Chemical
Publishing Co., New York, N.Y..

Heredy, L.A., and Fugassi, P., 1966. "Coal Science," Adv. in Chem., 55,
HUB.

Horowitz, N.E., Hobby, G.L., and Hubbard, 1976. Sctence, 194, 1321.

Hubbard, J.S., 1976. Origins of Life, 7, 281.

Hutcheon, J.M., 1970. ""Modern Aspects of Graphite Technology," L.C.F.
Elackman, Ed., Academic Press, London, p. 2.

Kagan, H.B., 1976. Chemtecn, 1976, 510.

Nissenbaun, A. 1976., Origins of Life, 7, 413.

Meier, J.A., and Hill, L.W., 1974. J. Catalysis, 32, 80.

Murcinik, R.d., and Spiro, M., 1974. J. Chem. Soc. Dalton, 2486.

 

Oberlin, tM., and Mering, J., 1964. Caroon, 1, 471.

Paecht-Horowitz, M., 1976. Origins of Life, 7, 369.

Pincock, R.E., Johnson, W.M., Wilson, K.R., and Haywood~Farmer, J., 1973. J.
Am. Chem. Soc., 95, 6477.

Poncelet, G., Van Assche, A.T., and Fripiat, J.J., 1975. Qrigins of Life,
6, 401.

Puri, b.k., Kumari, S., Halra, K.C., 1972. J. Ind. Cnem. Soc., 49, 127.

Sehneour, E.A., 1966. Science, 151, 991.

Senuvert, &., Ford, F.P., and Lyon, F., 1969. "Analysis of Carbon Black,"
in Eneveicoedia of Industrial Chemical Analysis, 8, 179.
Seuss, M.E., 1975. Origins of Life, 6, 9.

22
Smith, D.H., Achenbach, M., Yeager, W.J., Anderson, P.J., Fitch, W.L., and
Rindfleisch, T.C., 1977. Amal. Chem., in press. (See Appendix 1)

Smith, D.H., and Carhart, R.E., 1977. in "Chemical Applications of High
Performance Mass Spectrometry," M.L. Gross, Ed., in press. (See Appendix 1)

Urey, H.C., 1953. Proc. Roy. Soc. (Lond.), A, 219, 281.

23
APPENDIX 1

PROGRESS REPORT

Part

 

A. Recent work has been summarized in tne introductory and in discussion of
the rationale and research plan for Part A. Additional details may be found

in the attached reprints.

Part B. We have fulfilled the objectives of our previous research proposal. The

Our

emphasis of that proposal was on extending the analytical methodology of
GC/MS because we perceived that the proposed extensions would have a strong
impact on our chemical and clinical research, partly supported by other
grants. We realized at that time that the concepts were sufficiently general
that many other studies employing GC/MS would benefit if the concepts were
transformed into working systems. We proposed a) to develop methods for
quantitative comparison of mixtures analyzed by GC/MS; b) to utilize GC/MS
data acquired at both low resolving powers (GC/LRMS) and high resolving
powers (GC/HRMS) to determine structures of unknown compounds encountered in
complex mixtures; and ¢c}) to upgrade and improve our libraries of mass
spectral data.

efforts have culminated in a series of papers which discuss several aspects
of the use of computer techniques to assist in data analysis and
interpretation. The first paper (Smith, et al., 1977; Appendix 1) discuss
our approach to the quantitative comparison of GC/MS profiles of complex
mixtures. This approach embodies the plan described in our previous
proposal. GC/MS data are processed to locate points of component elution and
to obtain mass spectra free from background and contributions from

overlapping spectra (Dromey, et al., 1976) (See Fig. 1 of Appendix 1).

24
Two

Subsequently, spectra of added hydrocarbons are located and their elution
times utilized to determine relative retention indexes (RRI's). Then spectra
of internal standards are located and the concentration (based on peak areas)
of each component relative to the standard is determined. These data, the
collection of "clean" mass spectra, RRI's and relative concentrations,
constitute the GC/MS profile for a mixture. We have developed methods
(Smith, et al., 1977) for collecting these profiles into libraries to obtain
a historical record of past observations. We have also developed the
necessary programs for comparing new data to an historical library. The
results of the comparison quickly indicate which components are present in
abnormal amounts. The collection of programs available in. our laboratories
constitutes a powerful tool for analyzing and comparing complex mixtures of
any organic compounds which can be suitably derivatized for analysis by
GC/MS. As Appendix 1 indicates, we have used tne historical library
approach to validate our analytical procedures for isolation of organic
materials in human body fluids. More recently, we have been using the
approach to establish baselines on organic constituents of both urine and
asmiotic fluid.

additional papers (Smith and Carhart, 1977; Carnart, et al., 1977; Appendix
1) discuss approaches to interpretation of mass spectral and other chemical
data in terms of molecular structure. These studies, carried out together
with our collaborators in the SUMEX and DENDRAL projects, have used actual
unknowns discovered during the course of our GC/MS analyses of mixtures.
GC/LEMS and GC/HRNS experiments provided mass spectral data on unknowns.
Suoseguently, structural candidates for these unknowns were propoed based on

comouter-assisted analysis of the mass spectral data together with other

25
chemical information. Additional details may be found in Appendix 1. The
techniques discussed in these papers are also quite general, and like the

historical library approach, can be used in the study of diverse chemical

problems, including those outlined in subsequent sections.

We have made considerable progress in improving and extending our library of mass
spectral data. We have added new spectra to the library by running spectra
of standard compounds and processing the spectral data with the CLEANUP
program (Dromey, et al, 1976). We have improved the quality of existing
spectra using the nistorical library approach outlined above ("HISLIB",
Smith, et al., 1977). HISLIB averages spectra of the same compound observed
in several GC/HS runs. Thus, statistical variations in ion abundances are
reduced as additional spectra are averaged. The resulting spectrum is
frequently of much higher quality than spectra in existing libraries. We
have implemented a mechanism for adding averaged spectra to or replacing
spectra in our primary library. This provides a mechanism for gradual
improvement of our libraries with time. In addition, relative retention
indexes are included with the spectra now, enabling us to improve the

certainty with which subsequent spectra are matched to the primary library.

26
NASA SRT BUDGET
Period 12/1/77 - 11/30/78

Prepared 8/2/77

PERSONNEL
J. Lederberg Principal Investigator $ 579*
E. Levinthal Co-Principal Investigator 06 2,509
D. Smith Research Associate 25 6,295
H. Pettigrew Sr. REsearch Assistant 25 4,416
We. Fitch Postdoctoral Scholar 25 3,24]
T. Everhart Research Assistant 50 74235
N. Veizades Research Engineer 06 1,606
E. Steed Research Engineer 07 1,333
P. Evans Life Science Technician 25 3,400
H. Pham Laboratory Assistant 25 2,/88
W. Harlow Machinist 03 621
D. Pearson Electronics Technician 06 1,047
H. Bush Glassware Washer 25 2,403
M. Wyche Glassware Washer 50 6,800
M. Allan Secretary 20 2,785 $ 47,058
*This portion of Dr. Lederberg's salary will
be cost shared by the university. The
remaining effort will be without funding
from this source.
Staff Benefits 9/1/77 - 8/31/78 at 20%
9/1/78 - 8/31/78 at 20.8% $ 9,506
EXPENDABLE SUPPLIES
Laboratory Supplies $ 6,540
Books 156
Postage 96
Communications 1,092
Miscellaneous office supplies 684
Equipment Maintenance 5,184 $ 13,752
TOTAL DIRECT COSTS $ 70,316
INDIRECT COSTS at 58%:. $ 40,784
TOTAL BUDGET $111,100
LESS UNIVERSITY COST SHARING $ 1,100
TOTAL REQUESTED BUDGET $110,000

27