Natural Abundance of the Stable Isotopes
of Carbon in Biological Systems
Bruce N. Smith
The author is with the Department of Botany,
University of Texas, Austin, Texas 78712.
226
1947). Consequently, to break a
12C_12C bond should re~uire less energy than to part a I C-12C bond.
Factors influencing the chemical behavior of a system such as temperature,
pressure, chemical composition, activity
coefficients of solutions, etc., will determine the isotopic fractionation for an
equilibrium reaction.
Methods
Since the techniques employed will
be new to many readers, let me briefly
outline the usual procedure. Organic
materials are converted to C02 at
800-900 C in an excess of oxygen (Fig.
I A), following the procedure outlined
by Craig (I953). During combustion,
the generated gases are continuously
recycled via a Toepler pump to ensure
total conversion to C02. Products are
collected in a liquid nitrogen-cooled
trap; upon warming to dry-ice temperature the carbon dioxide, free of any
water or gas contaminant, can be collected in a sample tube cooled by liquid
nitrogen. Respiratory CO 2 is collected
according to the method of Park and
Epstein (1960). As shown in Figure IB,
living tissue is placed in a sample
chamber flushed with air which has been
scrubbed free of C02. At a flow rate of
about 150 ml/min. the gas from the
sample chamber is passed through a
series of three traps. The first trap is
maintained at atmospheric pressure (as
is the sample chamber) and is bathed in
a solvent at dry-ice temperature. The
second and third traps are immersed in
liquid nitrogen and maintained at about
Yz an atmosphere of pressure (ca. 38 mm
of Hg). The dry ice trap greatly reduces
the amount of water reaching the
nitrogen traps. Most of the C02 is
collected in the first N2 trap, none at all
is found beyond the second nitrogen
trap. The low temperature traps must be
held at reduced pressure to prevent
liquification of oxygen. If 02 liquifies,
subsequent distillation of the oxygen
may pull along with it some of the C02
which could in turn lead to isotopic
fractionation.
A.
SAMPLE
FURNACE
TOEPLER
PUMP
B.
SAMPLE CHAMBER
I----®-.,
C02 SAMPLE
Fig. 1. A. Apparatus for combustion of organic matter and coUection of combustion products.
B. Apparatus for coUection of respired C02.
BioScience Vol. 22 No.4
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About 99% of all carbon is the 12C
isotope while I % is 13C. The other
isotopes of carbon are, by comparison,
extremely rare. For instance, only about
one atom in eve~y 10 12 carbon atoms is
14C. The precise ratio of the isotopes
will vary depending on the material
a n a I y zed. Limestones, atmospheric
C02, marine algae, and land plants each
possess characteristic 13C/12C ratios,
differing slightly from one another. The
lowest ratio so far observed is for
carbon from ancient blue-green algal
mats (Kaplan and Nissenbaum, 1966)
and the highest for carbonate carbon
from meteorites (Clayton, 1963). Fractionation of plant carbon is brought
about primarily by carbon dioxide assimilation in photosynthesis and is due
to preferential utilization of 12C and
exclusion of 13C. Curiously enough, it
has been found recently (Tregunna et
al., 1970) that higher plants which fix
carbon dioxide via the Calvin cycle
pathway differ in 13C/ 12C ratios from
plants which fix carbon dioxide via the
C4-dicarboxylic acid pathway. Thus, it
is now possible to determine whether a
given sample of sucrose was synthesized
in sugarcane or in sugar beet.
While it is the aim of this paper to
discuss a few recent studies on naturally-occurring 13C/ 12C ratios of biological materials, it is pertinent to note that
the subject has been reviewed from a
geochemical viewpoint (Bowen, 1966;
Degens, 1969; Kroepelin, 1966; McMullen and Thode, 1963; Schwarcz, 1969).
The various isotopic species of an
element differ slightly in chemical
properties from one another. Just as the
chemical properties of different compounds determine their formation under
various conditions, so the chemical
properties of different isotopic species
of the compounds will determine how
the isotopic abundances distribute
themselves in nature. Thus substitution
of a heavy for a light isotope lowers the
vibrational frequencies and the zero
point energy of a chemical bond (Urey,
Ii
bicarbonate
atmospheric CO 2
algae
lichens
•
gymnosperms
angiosperms
+1
-6
-14
OIl
C%
-11
-30
Fig. 2. Ii 13c values of plants.
13C per mil =
(13C/12C) sample - (l3Cf 12C) standard
(13Cf 12 C) standard
x 1000
Thus, a sam~le with a Ii 13C per mil of
-10 has a 1 C/12C ratio less than the
standard by 10 ~er mil, or 1.0%. For
example, the 1 C/12C ratio of atmospheric C02 is smaller than that of
the PDB 1 standard by about 7 per mil.
Therefore, the Ii 13C per mil of this C02
is -7. The precision of measuring Ii 13C
with the mass spectrometer is ± 0.1 per
mil.
Distribu tion of
Carbon Isotopes Among Organisms
Great differences in 13C/12C ratios
exist between different species of plants
(Fig. 2). This variation was surveyed by
Rankama (1948) and more thoroughly
by Wickman (1952). Wickman found
that marine plants, freshwater aquatics,
and some desert plants had relatively
more 13C than most terrestrial species.
Craig (I953, 1954 a ) confirmed the
isotopic difference between marine and
terrestrial plants and suggested that this
difference was due to the isotopic
composition of the source carbon since
bicarbonate is utilized by marine organisms and atmospheric C02 is fixed
by terrestrial plants. Bicarbonate generally has more 13C than atmospheric
April 1972
_
C02' The presumed difference between
marine and terrestrial carbon has been
used to distinguish marine from nonmarine petroleum (Silverman, 1964;
Silverman and Epstein, 1958), as well as
to differentiate between freshwater and
marine sediments (Clayton and Degens,
1959; Eckelmann et aI., 1962). Indeed
detection of water pollution by sawmill
wastes has been claimed by use of
13Cf 12 C ratios (Parker, 1967).
Wickman's (1952) report that some
terrestrial plants were relatively rich in
13C was overlooked for several years
even though occasional analyses gave
Ii 13C values similar to those for marine
plants (Craig, 1953; Broecker and Olson, 1959; Emery et aI., 1967; Oana and
Deevey, 1960). Bender (1968) was the
first to realize that isotopic differences
between some tropical grasses and some
temperate grasses represent heritable
metabolic adaptations to xeric environments. Species of salt marsh plants
growing side by side often exhibit very
large differences in isotopic composition
(Smith and Epstein, 1970). Isotopic
differences exist not only between grass
species but also between species of
succulent xerophytes (Vogel and Lerman, 1969; Bender, 1971). As data on
more plants has become available it has
been possible to propose, in addition to
the distinction between marine algae
and higher plants, that terrestrial plants
themselves consist of two categories (see
Fig. 2) - those with high 13Cf12C
ratios and those with low 13C/12C ratios (Smith and Epstein, 1971).
Most higher plants (Fig. 2), including
all lower vascular plants and all gymnosperms except Welwitchia, have Ii 13C
values of -24 to -34 % (Smith and
Epstein, 1971). Algae and lichens (Park
and Epstein, 1960) have Ii 13C values
generally intermediate between the two
groups of higher plants (Fig. 2).
Festucoid grasses (Bender 1968, 1971),
including bamboo, are in this group as
are the palms. Those plants highest in
13C are aquatics, desert and salt marsh
plants, and Panicoid grasses. On the
average the Ii 13C values of dicots are
slightly more negative than those of
monocots (Smith and Epstein, 1971).
Plants with relatively large 13Cf 12C
ratios are plants with a metabolic
syndrome (Tregunna et aI., 1970) that
includes fixation of much of their
photosynthetic carbon via phosphoenol
pyruvate carboxylase (Hatch and Slack,
1970) rather than via ribulose
diphosphate carboxylase.
Isotopically, animals reflect the plant
basis of their diet (Smith and Epstein,
1970). There is no evidence for successive fractionations along the food chain:
inorganic carbon to plant to herbivore
to carnivore (Parker, 1964; Sondheimer
et al., 1966).
A number of plant families (Fig. 3)
including Gramineae and Chenopodiaceae have species in each of the two
categories (low and high 13C). Even
227
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Once collected, the gaseous C02,
whether from combustion or respiration, is analyzed on an isotope ratio
mass· spectrometer. With the best available instruments in which ion currents
are collected and measured singly,
changes in the isotopic ratio of the light
elements of 0.1 % are just detectable.
However, by collecting the two ion
beams for the isotopes in question at
the same time and measuring the isotope ratio directly, a much higher
precision may be obtained. Basic details
of the commonly used mass spectrometer in stable isotope work have been
described by Nier (1947). Improvements of the design (McKinney et aI.,
1950) have greatly increased the precision of the Nier instrument. The
13Cf 12C ratio in any given sample is
compared with a standard. The standard
used is C02 from the fossil carbonate
skeleton of Belemnitella americana
(PDB 1)' The function defining the
values reported is
228
BioScience Vol. 22 No.4
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tained more 12C. Of further significance
was the observation that carboxyl
groups of amino acids were generally
Gymnospermae
000
o
enriched in 13C by as much as 20 per
mil relative to the remainder of the
molecule.
These internal isotopic flucChenopodiaceae
00 0 cfl5>qf1 oc9o 0
00 c9 I?~
6'oOXl
tuations in the amino acids were not
reflected in the 13C content of the total
protein which was the same as the
Leguminosae
o
associated carbohydrate fraction. Winters l has recently confirmed that differences in isotopic ratios do exist for
Compositoe
o 0
o
o
different amino acids. On the other
hand, Parker (1964) found that different fatty acids of a given organism
Gramjneoe
00
have identical isotopic compositions as
would be expected if they arose through
the
same synthetic pathway. Animal
Potamogetonoceae
o
00
o
lipids (Smith and Epstein, 1970; Sondheimer et aI., 1966) are remarkably
like ingested plant lipids. Isotopic data
.f-2--+--------:J&·-------_1-14------_+22-------t_3-0-----~-3. also exist for different chemical fractions from phytoplankton (Degens et
Fig. 3. 613C values of representative plant families. 0 plants relatively high in 13C; 0 plants aI., 1968b), and potato tubers (J acobson et aI., 1970a). Comparison of the
relatively low in 13C.
major plant constituents in cotton and
sorghum has recently been made by
within one of the categories variation of isotopically. For example, Table 1 com- Whelan et al. (1970). All fractions
10 to 12 per mil occurs (Fig. 3). The pares isotopic values for various frac- (lipids, carotenoids, proteins, amino
physiological or ecological basis for the tions of potato tuber tissue. Park and acids, organic acids, and sugars) of
variations within the categories is not Epstein (1961) found a sizeable dif- sorghum differ substantially from all
well understood. A number of genera ference in 6 13C values between plant fractions of cotton. In both plants,
have species in each of the two cate- lipid and whole plant carbon for a lipidS and carotenoids are enriched by
gories (Tregunna et aI., 1970). In one number of plants. Small differences about 5 % in 12C with respect to the
instance two subspecies of A triplex were also noted between sugars, cel- total carbon of the whole leaf.
canescens differed by more than 5 % ulose, and lignin fractions from a par(Smith and Epstein, 1971). Bjorkman et ticular plant. Abelson and Hoering
Postulated Mechanism for
al. (1969) have shown by hybrid forma- (1961) noted similar isotopic differenCarbon Fractionation
tion between species of A triplex that ces between lipid and total carbon for a
A model has been proposed by Park
the prevalence of C4-photosynthesis is number of cultivated unicellular algae.
and Epstein (1960) to account for
genetically determined. It is presumed
TABLE I.
isotopic variation in plants. The four
that the processes which result in
different carbon isotopic ratios are also Carbon isotope abundance for chemical major fractionation sites are indicated in
Figure 4 by numbers. Step 1 represents
under genetic control.
fractions of potato tuber
the boundary effect associated with
uptake of C02 by the plant cytoplasm.
l3C/ 12C Ratios of Chemical Fractions
This includes stomatal resistance to
At a first approximation all parts of a
diffusion, collision frequency of the gas
-25.8
plant have similar isotopic ratios. Closer potato tuber tissue
molecule with the mesophyll cell sur-23.8
scrutiny however indicates that small sugars
face, and uptake of the CO 2 by the
-25.5
isotopic differences between plant or- starch
cytoplasm. After passing through the
-26.6
gans do exist (Emery et al., 1967; protein
membrane, the dissolved C02 is partiLowdon, 1969; Parker, 1964; Smith and organic acids
tioned into enzyme-catalyzed conver-26.9
Epstein, 1970). Little change in amino acids
sion to starch (Step 2) and into removal
-27.1
13C/12C ratios was noted with age of lipid (chloroformof some of the dissolved C02 through
wood in growth rings of Sequoia trunks
the vascular system resulting in excremethanol extract)
-34.6
(Craig, 1954b), except for a slight
tion through the roots. Park and Epstein
increase in 13C concentration during They also demonstrated that the various (1960) found that conversion of "disthe first 150 years of the life of the tree. amino acids in a protein hydrolyzate solved C02" into carbohydrate via
Similar results have been noted (Jansen, from a given organism will cover a range ribulose diphosphate carboxylase (Step
1962) from the Kauri tree of New of about 16 per mil. Most enriched in 2) resulted in a fractionation of about
13C were serine, threonine, glycine, and 17 per mil. Whelan (1971) has recently
Zealand.
Different chemical fractions of an aspartic acid, whereas leucine, iso- found this fractionation to be even
organism also differ from one another leucine, and aromatic amino acids con- larger than reported by Park and
Respiration Studies
Generally, respired C02 has the same
13Cj 12C ratio as the total organic
Leaf
or
cell surface
atmospheric
CO,
CD!
!
physical
absorption
l
CD
"dissolved coi' elzymatic. starch
fiution
I
I
10
I
translocated
aid IIcreted
from rools.
10
I
lipid
Fig. 4. Model for fractionation of carbon
isotopes in photosynthesis (after Park and
Epstein, 1960).
lK. Winters, personal communication
April 1972
carbon of the organism (Baertschi,
1953). Isotopic fractionation during
degradation of a given substrate was
estimated by culturing an obligate
heterotroph, the fungus Fusarium
roseum, aerobically on an inorganic salts
medium with a sole carbon source of
either glucose or palmitic acid (Jacobson et ai., 1970a). The Ii l3C-values of
respired C02 were within 1.9 % of the
Ii -values of the corresponding substrates.
Thus it did seem feasible to detect
changes in respiratory substrate from
changes in 13C/12C ratios of respired
C02' Analysis of respiratory C02 from
potato tuber slices (Jacobson et ai.,
1970a) led to the conclusion that
respiration that develops 24 hours after
slicing represents starch degradation.
Respiration of aged potato tuber slices
is resistant to concentrations of cyanide
which inhibit the respiration of fresh
slices. The addition of cyanide indu~es a
shift from starch to lipid as the respiratory substrate. The shift can be
detected by changes in the 13C/12C
ratios of respired CO 2 (Jacobson et ai.,
1970b). Changes in respiratory substrate
during germination of peanut and sunflower seedlings have also been detected
by this method (Smith, 1971). Changes
in substrate for microorganisms have
been detected in a similar manner under
both aerobic (Kaplan and Rittenberg,
1964) and ana~robic conditions (Krouse
and Sasaki, 1968).
Animal respiration can certainly be
studied in the same way. Carbon
dioxide respired by man and dogs
differed by only 1.5 % (Duchesne and
Van de Vorst, 1968); birds show a
similar small difference from mammals,
while C02 respired by frogs had much
less 13C than did mammals or birds
(Duchesne et ai., 1968a). Phylogenetic
differences between groups of vertebrates are probably of less importance
than diet and metabolism in explaining
the isotopic difference in respired C02
from diverse animals. Duchesne et al.
(1968b) have indicated that this may be
so since changes in amounts of insulin
injected into chickens are reflected in
13Cj 12C changes in respired C02'
In related studies, relatively low
ratios of 13C/ 12C in crystals of calcium
oxalate from rhubarb (Hoeks, 1969)
and calcium carbonate from hackberry
seeds (Smith et al., 1971) were interpreted as due to inclusion of respiratory
C02 in the deposited crystals. Bluegreen algal mats from certain sediments
were shown to be, unlike living algal
mat s, relatively enriched in 13C
(Behrens and Frishman, 1971). This was
interpreted by the authors to mean that
anaerobic bacterial decomposition of the
mats, durin~ the sedimentation process,
released 1 C preferentially in the
respired CO! leaving the sediments
enriched in 1 C.
Environmental Effects
Craig (1953) suggested that environmental effects may account for some of
the isotopic fractionations observed in
organisms. Isotopic differences between
warm water (low latitude) and cold
water (high latitude) marine plankton
populations (Sackett et ai., 1965, 1966)
imply a connection between isotopic
ratios and ambient water temperature.
Plankton grown in the laboratory
exhibited a 7 per mil difference between
cultures maintained at 10 C and 30 C
(Degens et al., 1968a). However, if large
quantities of C02 were administered
(Deuser et ai., 1968) to the growing
plants (in the process lowering the pH
to about 6); no temperature effects
were noticeable - and the isotope
values at all temperature levels corresponded to those found in plankton
of cold-water environments. Thus the
effect of water temperature on isotopic
fractionations in these experiments may
be on carbon availability (solubility in
seawater, etc.) rather than on biological
processes. However, small changes in
Ii 13C values
in carbonate carbon
(Eichler and Ristedt) of Nautilus may
be due to changes in the ambient water
temperature.
Both atmospheric C02 (Keeling,
1958;
1961a)
and
marine
bicarbonate (Parker, 1964) undergo daily
changes not only in amount but also in
13C/12C ratios. These fluctuations are
thought to be caused by metabolic
activities of organisms in the ecosystems
(Keeling, 1961 b). The combustion of
fossil fuel tends to increase 12C concentration in the atmosphere (Friedman
and Irsa, 1967) which may be reflected
in plant ol3C-values (Smith and
Epstein, 1971).
Natural abundance ratios of the
isotopes of carbon can be used to study
aspects of organism-environment interaction since fractionations of the isotopes will occur at various points in the
carbon cycle. Knowledge of these fractionations may make possible further
insight into evolution, physiology, and
ecology of organisms. It is my hope that
enough workers will apply these techniques to biological problems to fully
test their potentiai.
229
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Epstein. Whelan has also found the
fractionation for phosphoenol pyruvate
carboxylase to be much smaller than for
ribulose diphosphate carboxylase.
Galimov (1966) found CO 2 in the soil
to be ligher than atmospheric CO 2 , but
heavier than organic carbon, thus confirming prediction from the model. The
translocation step determines how
rapidly CO 2 in the cytoplasm is removed from the plant system to avoid a
build-up of 13C in the cells. Further
fractionation may take place during
metabolic processes (Step 4, Fig. 4),
resulting in different Ii 13C values for
different chemical fractions (see Table
1) within a plant.
The largest fractionation yet reported is an 80 % enrichment in 12C in the
conversion of methanol to methane by
anaerobic bacteria (Rosenfeld and Silverman, 1969). Utilization .of biological
methane as the starting carbon source
for metabolism may result in extreme
reduction of 13C content (Kaplan and
Nissenbaum, 1966). However, many
enzymatic processes show little or no
isotopic fractionation (Williams et aI.,
1959).
All four steps in the model (Fig. 4)
affect the final fractionation that is
associated with the fixation of CO 2 by
plants. The relative rates and efficiency
of these various steps determine the
isotopic composition of the final plant.
In principle this allows for plants to
have the range of Ii 13C values from 0 to
-38 % (Park and Epstein, 1960). Except
for blue-green algal mats which utilized
methane as the carbon source (Kaplan
and Nissenbaum, 1966), all plant values
in the literature (see Fig. 2) fall within
this range.
References
Animal
Parasitism
CLARK P. READ,
Rice University
Presenting the development of
animal parasitism as a relevant subject for the consideration of biologists, this book
relates the subject to the
mainstream of modern biology.
It examines a selected group
of animal parasites in detail
and traces their life cycles.
Emphasizing the advances of
modern experimental biology,
the author examines the broad
questions relating parasitism
to human welfare.
1972/192 pp'/illus./paper:
(013-037663-9)/cloth:
(013-037671-x)
For more information, write:
PRENTICE-HALL
Box 903, Englewood Cliffs, N.J_
07632
230
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