Marine Geology, 113 (1993) I 11-125
Elsevier Science Publishers B.V., Amsterdam
111
Factors controlling 13C contents of sedimentary organic
compounds: Principles and evidence
J.M. Hayes
Biogeoehemical Laboratories, Departments of Geological Sciences and of Chemistry, Geology Building, Indiana
University, Bloomington, IN 47405, USA
(Revision accepted May 5, 1993)
ABSTRACT
Hayes, J.M., 1993. Factors controlling 13C contents of sedimentary organic compounds: Principles and evidence. In:
R.J. Parkes, P. Westbroek and J.W. de Leeuw (Editors), Marine Sediments, Burial, Pore Water Chemistry, Microbiology
and Diagenesis. Mar. Geol., 113:111-125.
The carbon isotopic composition of any naturally synthesized organic compound depends on (l) the carbon source utilized,
(2) isotope effects associated with assimilation of carbon by the producing organism, (3) isotope effects associated with
metabolism and biosynthesis, and (4) cellular carbon budgets. These factors are reviewed and quantitative considerations
summarized, particularly with regard to active and passive modes of carbon assimilation by phytoplankton and the existence
of discernible regularities in isotope effects associated with lipid biosynthesis. It is concluded that n-alkyl lipids are in general
depleted in 13C by about 1.59(~relative to polyisoprenoid lipids produced by the same organism. The effects of biological
reworking on isotopic compositions of organic carbon are examined and it is suggested that enrichment of 13C in sedimentary
organic carbon may result from loss of CH4 from zooplanktonic gut communities. Isotopic methods for estimation of ancient
COz levels are considered and a hyperbolic form favored for the relationship between concentrations of dissolved CO2 and
isotopic fractionation.
Introduction
Each molecule recovered from an ancient sediment carries information about the organism that
produced it. Through chemotaxonomic associations, the structure can indicate the identity of the
producer. The isotopic composition of the molecule can indicate the isotopic composition of the
parent organism and that, in turn, can reveal the
carbon source utilized by the producer and thus
its position within the ancient ecosystem. Because
these factors are in turn dependent on environmental conditions, the distribution of 13C among natural products is a sensitive paleoenvironmental
indicator and can provide a great deal of information about ancient biogeochemical processes.
The principles are straightforward, but details
0025-3227/93/$06.00
of analysis and interpretation can become intricate.
Given the variety of controlling factors, some well
organized strategy is required for the interpretation
of isotopic data. To disentangle the multiple signals
present in the molecular-isotopic record, it has
proven useful to consider processes sequentially,
focussing first on the products of photosynthetic
organisms and determining their isotopic compositions. Isotopic compositions of respiring heterotrophs and of fermentative organisms can then be
understood in terms of secondary isotopic fractionations. Quantitative treatment of fractionations
associated with equilibrium isotope effects and
with branch points in networks of largely irreversible reactions is straightforward, but m a n y persons
long interested in sedimentary phenomena are
unfamiliar with such considerations. This paper
aims to present a useful introduction.
© 1993 - - Elsevier Science Publishers B.V. All rights reserved.
112
J.M. HAYES
Biogeochemical principles
Isotopic fractionation
Consideration of isotopic compositions must be
based on fractionation rather than isotopic composition. It is the difference between two isotopic
compositions which is meaningful, not either f
value on its own. We will begin, therefore, by
introducing terms for isotopic fractionation and
considering the isotopic composition of marine
dissolved inorganic carbon (DIC), a prominent reference point for many considerations of fractionation. It is best to express kinetically and
thermodynamically controlled fractionations in
terms of a ratio of isotope ratios. This is accomplished-indirectly, to be sure--by use of the e
notation:
/~A/B~- (O~A/B- -
1) 103
etA/B--=RA/R B = (1000 + fA)/(1000 + fa)
(1)
(2)
where R-13C/12C. As implied by the form of Eq.
1, the units for e are parts per thousand or %0, just
as for f. For small values of e and especially when
f values are near zero, differences between AA/B
and eA/Bare small:
AA/B= fA -- fB ~ A / B
(3)
For example, for fA = --27 and fib = --31, AA/B is
4.0 and eA/B is 4.1%o, a difference that is probably
small in comparison to related uncertainties. In
contrast, for fA = 0 and fib = --28, AA/B is 28.0 and
eA/B is 28.8%0. This difference is also not very large,
but close comparisons of fractionations associated
with carbon fixation, for which these values would
be typical, are becoming more common. Confusion
will be minimized if e is uniformly employed in
such cases.
Inorganic carbon
H 2 C O 3 ~ H + + HCO3
(5)
HCO3 ~ H ÷ + CO 2 -
(6)
Fully deprotonated carbonate can combine with
any of a number of divalent cations to form
insoluble minerals, calcite and aragonite being the
most common:
Ca 2 + + C O 2 - ~ CaCO3(s )
(7)
An isotope effect is associated with each of these
equilibria. The isotopic compositions--6 values-of dissolved CO2 and carbonate minerals are,
therefore, not equal. A characteristic of such equilibrium isotope effects is that the isotopic difference
between reactant and product depends only on
temperature and not on the distribution of material
between product and reactant. Therefore, although
the relative abundances of the species indicated in
Eqs. 4-7 will be strongly dependent on pH, the
isotopic differences between those species will
depend only on temperature. Paleobiogeochemists
are interested in the substances at the beginning
and end of the sequence of carbonate equilibria:
CO2(aq) is the substrate for the carbon-fixing
enzyme, rubisco, and CaCO 3 is the commonly
preserved mineral that provides information about
overall abundances of 13C in ancient oceans.
The isotopic differences between these species
are summarized most conveniently in terms of
equilibrium isotope effects relative to bicarbonate.
For dissolved CO2, Mook et al. (1974) have shown
that:
Ed/b
=
[(fd -j'- 1000)/(f b + 1000) - 1] 103
(8)
= 24.12- 9866/T
where the subscripts d and b are shorthand for
dissolved CO2 and bicarbonate and T is the absolute temperature. Serious confusion has existed
regarding equilibria between bicarbonate and carbonate minerals because isotope effects relating to
calcite and aragonite differ and this was not adequately recognized. Recently, however, Morse and
MacKenzie (1990) have shown that, for calcite:
The DIC from which marine biomass is synthesized is comprised of multiple chemical species
linked by a series of equilibria:
/~rn/b= [(frn "{- 1 0 0 0 ) / ( f
CO2(aq) + H 2 0 ~ H2CO3
where the subscript m is shorthand for mineral
(4)
b
"~ 1000) -- 1] 103
(9)
= -- 14.07 + 7050/T
t3C
CONTENTS
OF
SEDIMENTARY
ORGANIC
113
COMPOUNDS
calcite. As shown in Fig. 1, the resulting carbon
isotopic difference between calcite and dissolved
CO2 is large and quite strongly temperature
dependent.
Two additional factors complicate the picture.
First, it cannot be assumed that equilibrium was
attained between calcite and dissolved CO2. Where
the preserved carbonate is a product of biomineralization, the 13C content of CO 2- at the site of
precipitation may have been affected by metabolic
processes. For foraminifera, for example, such
"vital effects" can be as large as a few permil.
Pertinent data have been reviewed recently by
Spero et al. (1991). An exemplary case has recently
been considered by Jasper et al. (1993), who drew
a distinction between dim, the isotopic composition
of calcite in equilibrium with dissolved CO2, and
dif, the isotopic composition of a preserved foraminiferal calcite. For Neogloboquadrina dutertrei,
a planktonic foram abundant in sediments from
the central equatorial Pacific, they showed that
available data (Fairbanks et al., 1982) indicated:
Am/f ~ dim -- dif = -- 0.709"00
(10)
and improved the accuracy of their reconstruction
of did by applying this correction. Second, even if
12
. . . .
i
. . . .
i
. . . .
i
. . . .
11
o
9
Calcite vs.
Dissolved CO2
8
. . . .
I
I
10
i
,
,
i
20
. . . .
i
30
. . . .
40
Temperature, °C
Fig. I./~m/d,the equilibrium isotopic fractionation factor relating
mineral calcite and dissolved CO2, as a function of temperature.
Calculated from values of 8d/b, the fractionation factor relating
dissolved CO2 and bicarbonate (Mook et al., 1974) and 8mr.,
the fractionation factor relating crystalline calcite and bicarbonate (Morse and MacKenzie, 1990).
equilibration did occur, the site of that process
(i.e., in bottom waters or sedimentary pore waters
rather than in the photic zone) may be significant.
Thinking again in terms of foraminifera, if the
goal is reconstruction of the isotopic composition
of dissolved CO2 available to algae in open marine
environments, reference to planktonic rather than
benthic foraminiferal calcites is clearly preferred.
In a study of the Greenhorn Formation, Hayes
et al. (1989) found that micritic carbonates were
comprised largely of pelleted but otherwise uncorroded coccoliths and that stratigraphic variations
in their 6 values were smoother than those of
sparry cements or benthic bivalves. To estimate
changes in (as distinct from accurate values of)
photic-zone 6d, they therefore used the micritic
phase, since it was at least certain that it reflected
photic-zone DIC. Where carbonates that formed
from sedimentary pore waters are involved, diagenetically induced shifts in dim can be severe and
great care must be taken; Irwin et al. (1977) have
provided a very useful review of relevant
phenomena.
Organic carbon
Production of biomass can be viewed as occurring in two distinct steps: assimilation of carbon
and biosynthesis of cellular components. An overview is provided in Fig. 2. The 13C content of each
biomolecule depends on:
(1) The 13C content of the carbon source.
(2) Isotope effects associated with the assimilation of carbon.
(3) Isotope effects associated with metabolism
and biosynthesis.
(4) Carbon budgets at each branch point.
For an autotroph, the carbon source will be environmental carbon dioxide or bicarbonate. Factors
affecting their 6 values have been considered in the
preceding section. For a heterotroph, the isotopic
composition of the carbon source will be determined by feeding patterns and by isotopic fractionations occurring upstream in the foodweb. Isotope
effects associated with assimilation o f carbon are
of great importance in autotrophic organisms, but
assimilation isotope effects per se are significant
for heterotrophy only where very small molecules
114
J.M. HAYES
Unused Reactant
~$ource
Excreted
C
Cu 8u-
i
I
Supply of
Reactant
~;~
II
1
I
~R"
CR
I
I
~op =, Cp
Kinetic Isotope
Effect
~p,Sp ~, Product
i
I
I
/
Stream
Reaction Site
Fig. 3. A depiction of the flows and isotopic compositions of
carbon at a branch point.
C
Fig. 2. An overview of a cellular carbon budget and of pathways
leading to biosynthetic products. Kinetic isotope effects are
denoted by e and are arbitrarily numbered to indicate their
independence. Isotopic compositions are denoted by similarly
numbered 6 terms. The circled numbers indicate branch points.
(probably three carbons, maximum) are absorbed
and processed individually, as in methanotrophs.
In heterotrophs that ingest food particles, assimilation "effects," if present at all, derive from selective
absorption of specific compounds or compound
classes (including, in metazoans, substances that
may be produced by gut flora). In all organisms,
both autotrophic and heterotrophic, the biosynthesis of cellular components is based on cellular
pools of common intermediates; lipids, for example, always deriving from acetate. Isotope effects
associated with metabolic reactions that produce
or consume such intermediates affect the ~3C
contents of the biosynthetic products. Finally, at
each point within the cellular reaction network,
distribution of carbon among products will affect
isotopic compositions. A detailed example follows.
standing fractionation and is represented by the
equation:
¢7~RC~R= ~)UI~U+ ~pl~p
where the ~b terms represent flows of carbon
(moles/s) and the 6 terms represent isotopic compositions, the subscripts R, U, and P indicating
respectively reactant, unused reactant (which flows
to other reaction sites at which it is used to make
other products), and product. Because a kinetic
isotope effect is associated with the reaction
(CR--,Cp), the product is depleted in 13C relative
to the reactant available at the reaction site. Due
to that preferential withdrawal of 13C-depleted
reactant, the pool of CR at the reaction site is
isotopically enriched relative to the external supply.
Using 6f to represent the 6 value of the instantaneously available reactant, we note f* > 6R and
where e indicates the magnitude of the kinetic
isotope effect:
e - (12k/lak-
Fractionation in reaction n e t w o r k s
Isotopic compositions of organic compounds
are shaped within cellular reaction networks.
Controlling factors are of two types: isotope
effects, specifically kinetic isotope effects except
where exchange with bicarbonate is involved, and
"branching ratios," the divisions of carbon flow
that occur wherever a single reactant leads to
multiple products. A schematic view is shown in
Fig. 3, which can be taken to represent a point
within a sequence of enzymatically catalyzed reactions. Mass balance provides the key to under-
(1 1)
1)10a
(13)
the k terms being rate constants for the indicated
isotopic species. Substitutions then transform the
mass-balance equation into useful expressions of
fractionation. Specifically, the fractional yield of
product, f, is given by q~p/~bR; by difference, the
fraction of incoming R that is unused is 1 - f ; and,
since no process acts to change the isotopic composition of the unused reactant, au =*. Isotopic
compositions of materials leaving the reaction site
are then given by:
au = aR +fe and ap = &R- (1 - f ) e
(14a, 14b)
13C C O N T E N T S
OF SEDIMENTARY
ORGANIC
115
COMPOUNDS
These relationships are represented graphically in
Fig. 4.
Emergent 03 Plants
e.~- o,~,~1
....
"1
Assimilation & Fixation Biosynthesis
~.~c,~. --.-. . . . . .
/
C5
Cycle
co~co~--+Lco.,,l
Fractionation associated with carbon fixation
Isotopic fractionations accompanying the assimilation of carbon by autotrophic organisms represent a special case. Not only does fractionation
depend on mass balance, but the value of ~ itself
is variable and dependent on environmental
factors. This occurs because assimilation comprises
at least two processes, mass transport and carbon
fixation, each with its own characteristic isotope
effect, and the overall isotope effect depends on
interactions between those processes. In some
organisms it is necessary to consider isotope effects
(plural) associated with fixation of carbon. This is
indicated in Fig. 5, in which a distinction is drawn
between organisms employing the Calvin Cycle
and possessing, therefore, a single pathway
through which essentially all fixed carbon flows,
and other groups of organisms. Green photosynthetic bacteria (the Chlorobiaceae) fix carbon
mainly by reversing the tricarboxylic acid cycle
(Sirevag et al., 1977). Together with nonphotosynthetic bacteria that can grow autotrophically while
fixing carbon and constructing biomass by use of
the "acetyl-CoA pathway" (i.e., acetogenic and
methanogenic bacteria, Wood et al., 1986), they
form a subset of organisms in which carbon is
.
.
.
.
i
.
I
,
.
.
.
~R + e
o
~
tiR
~R " E
,
0.0
,
,
,
0.5
=
,
,
1.0
f (= q)p/(l)R)
Fig. 4. The relationships between isotopic compositions and
fractional yield at a branch point.
I
• I
"~"ubisco
/
IL,"~;t~;:---;
'~nlrl2Uln'Tr~
~'
intermodiate~..l'l'~, ~
II ....... -.--~
II
"~
Calvin
|
06
C4 and CAM Plants
. PEPC
_.t' 11l
I
~
I H.CO~'C4
I
C5
Cs )~,.,...~,.b,,~o}'
in c~.g~M
t
_
Cycl.ea,.Cn(H20)nl-)./o
asbi°synthesiSabove
|
3
/
Subaqueous Plants and Chemoautrotrophs
H C O 3 m I # ~ I ~ H C O 3 / / f ~ C ~ l v i . n " C _ ( H ~ O ) - - ~ T O biosynthesis
A/
ii
i I 20
vyc=e
" = n /
as above
1l i1_ 1L
I
Acetogens, Methanogens, Chlorobiaceae
i
=1
.-~).
HC03/
41 ',~,
Multiple
Fixation
Sites
Products
.........
"~Y,Metabolic
.~¢
/~
',1 /t."~-~o,~-"'. nterr.od~ates,-~
CO - ' ~ l ' ~ c o "
~
Directly
.........
2-7- 2-.0..Fig. 5. Schematic views of the the differing pathways by which
carbon is supplied to biosynthetic reaction networks. In spite
of differences in assimilatory processes, sources of metabolic
intermediates are similar for all three systems in which carbohydrates are synthesized by the Calvin Cycle. Organisms in the
last groups noted, however, fix carbon at multiple reaction
sites. As a result, very large intramolecular isotopic contrasts
might appear where intermediates contain carbon from
multiple sites.
fixed at three or more different enzymatic sites.
The distribution of 13C between and within biosynthetic products from these organisms can directly
reflect isotope effects at and contributions from
individual fixation sites.
If mass transport occurs passively, with CO2
reaching the fixation site simply by diffusing in
response to concentration gradients, the isotope
effect associated with the carbon-fixing enzyme,
rubisco, tends to dominate. Relevant flows of
carbon and their isotopic compositions are shown
schematically in Fig. 6. All are cast in terms of
CO2 rather than bicarbonate because, even though
the latter substance is much more abundant, molecular CO 2 is both the actual substrate utilized by
rubisco (Cooper et al., 1969) and the form of
inorganic carbon that diffuses passively into and
out of cells. The isotope effect associated with
116
J.M.HAYES
/
Rearrangement yields an expression for the overall
isotope effect--the isotopic difference between
fixed carbon and the carbon source--denoted here
by ep (P for Photosynthesis or Primary
Production):
and ef is about 27%0 (Farquhar et al., 1982b), a
considerable range is expected.
Observed values of ep vary widely, sometimes
even beyond the expected range of 0.7 ~<ep~<27%0,
notably to ep< 0 (McCabe, 1985). Since all known
carbon kinetic isotope effects discriminate against
13C, the production of organic carbon that is
enriched in 13C relative to the CO2(aq) available
to the cell (i.e., ep < 0, an apparent preference for
13C) indicates that CO2(aq) was not the carbon
source and that some substance relatively enriched
in 13C must have been. As shown by Eq. 8,
HCO3 is a logical candidate, being enriched by
9%0 at 25°C. Thanks in part to such isotopic
evidence, it is now recognized that numerous algal
species actively transport or "pump" bicarbonate
into their cells (Badger, 1987). Carbonic anhydrase
subsequently catalyzes production of dissolved
CO2 that can serve as a substrate for carbon
fixation. As would be expected since such active
transport is energetically costly, the process is
inducible rather than ubiquitous and is commonly
employed when concentrations of CO2(aq) are too
low to sustain photosynthesis (Sharkey and Berry,
1985). Ideally, cells that had imported their internal
CO2 at significant metabolic cost would lose none
of it. In that case (i.e., ~bo=0), the carbon-flow
pathway (Fig. 7) would become a "one-way
street." Every bicarbonate pulled into the cell
would be committed to production of organic
carbon and the isotopic composition of the biomass would be determined by the isotopic characteristics of the pump. In fact, however, some CO2
leaks from the cell and the isotopic mass balance
associated with that leakage forms the principal
control on the isotopic composition of the biomass.
For the system shown schematically in Fig. 7, a
mass balance analogous to Eq. 15 takes the form:
'~'P = (~ © -- (~f = 'St Jr ( ci/ ee)( F'f - ~t)
(~i((~e -- ~d/b -- ~a) = ~/~o(t~i -- ~t) + ~bf(t~i - ~f )
So
~i, Se. % ] . .
C02 ~ 90,~ "~ {
Ce
~k~
c~,
r~
CO 2
Ci
~'~" q
Rublsco
~ Cn(H20)n
.I
boundary
Fig. 6. The two-stepmodel for fixationof carbon by autotrophs
that obtain CO2 by passive diffusion(Farquhar et al., 1982).
The wavy line represents the boundary between a C-fixingcell
and the environment. Isotopic compositions of external CO2,
CO2 at the fixation site, and fixed carbon are denoted by 6c,
6i, and 6f, respectively. Flows of carbon (e.g., moles/s) are
denoted by ~band isotope effects by e. Isotopic compositions
of carbon travellingthe indicatedpathwaysare givenby 6c- et,
6 i - ef, and 6~-et. Concentrations of C02 outside and inside
the cell are specifiedby ce and cl, respectively.
mass transport is denoted by et, so the 6 value of
carbon diffusing into the cell is given by 6~-et. In
turn, the isotope effect associated with the fixation
reaction is ef, so the 6 value of fixed carbon is
given by 6i-ef. The mass balance for CO2 inside
the cell can then be written as:
(#i(~e --/~t) = ~bo(t~i -- ~t) "+" t#f(~i -- Fff )
(15)
If the fluxes of carbon into and out of the cell are
proportional to the concentrations, cc and ci, then
~bo/~bi= ci/ce and ~bf/q9i = 1 - ci/ce. Moreover, 6i can
be eliminated by the substitution (~i = (~f "~-/~fSO that
Eq. 15 becomes:
6¢ -- F.t = ( Ci/ C¢)( (~f -~- F.f -- ~t) + ( 1 -- Ci/ C¢)(~ f
(16)
(17)
This derivation, which follows an approach introduced by Farquhar et al. (1982a), has liberally
employed the approximation A ~e, but it can be
shown that the final relationship is exact.
According to Eq. 17, values of ep ought to vary
between et and ef depending on the magnitude of
the ratio ci/c,. Since et is very small (0.7%0 for
diffusion of CO2 in water at 25°C, O'Leary, 1984)
(18)
where ea is the isotope effect associated with the
active transport of bicarbonate. Defining L = ~bo/~bi
(i.e., L = fraction of imported bicarbonate that
leaks from the cell) and making substitutions analogous to those employed above leads to:
•p = 6 e -- (~f = ed/b + e a +
L(ef - et)
(19)
In such systems, ep--+ed/b+ea as L--,0 and, because
13C CONTENTS OF SEDIMENTARY ORGANIC COMPOUNDS
117
Fractionation associated with biosynthetic
pathways
HCO~
• i, Be- tab" %
Active
Transport
~oA-
C O 2 q:
Leakage
Bo
3
Rubisco
> Cn(H20)n
cell
boundary
Fig. 7. Employinga notational schemelike that used in Fig. 6,
this sketchrepresentsflowsand isotopiccompositionsof carbon
in an organismsthat activelyassimilatesinorganiccarbon from
its environment. For purposes of illustration it has been been
assumed that the "pumped" form of carbon is bicarbonate,
but there are also organisms in which CO2 is the actively
transported form (Badger, 1987). The equilibriumisotopeeffect
relating dissolvedC O 2 and bicarbonate is denoted by ed/band
dissolved CO2 and bicarbonate in the internal and external
pools of inorganiccarbon are assumed to be in equilibrium.
~d/b< 0 and ea, though positive, is likely to be small,
ep can assume negative values, with algal biomass
being enriched in 13C relative to dissolved CO2
(but not relative to bicarbonate).
It is not yet known to what extent the range of
biomass 6 values encountered in nature reflects
variations in Ci/C e (Eq. 17), the occurrence of
bicarbonate pumping and associated variations in
L (Eq. 19), or other factors. The latter include
variations in ef, there now being considerable
evidence that several variants of rubisco exist,
particularly in cyanobacteria (Raven and
Johnston, 1991; Guy et al., 1993), and phenomena
not contemplated by the simplified models presented above. These include possible variations in
fie in stagnant diffusion layers around rapidly
growing cells (pertinent to Eq. 17) and the existence
of processes and carbon flows in addition to those
considered in the derivations of Eqs. 17 and 19.
One generalization has, however, already proven
durable: whether due to a decline in Ci/C e o r the
onset of active transport, low concentrations of
CO 2 are commonly associated with low values of
ep. This raises the hope that a quantitative relationship might be found and a "paleobarometer" for
CO2 developed. Further consideration of that topic
appears in the concluding section of this paper.
Products of carbon fixation flow to additional
processes that synthesize the compounds needed
for construction and maintenance of biomass.
These include complex carbohydrates, proteins,
and lipids. With the prominent exception of porphyrins, nearly all sedimentary biomarkers derive
from lipids. Both for that reason and because
diverse information is now available regarding
isotopic fractionations associated with the biosynthesis of lipids, we will focus on that class of
compounds.
It has long been known (e.g., Abelson and
Hoering, 1961) that "lipids are light" (i.e., they
contain less of the heavy isotope, 13C, than do
other products of biosynthesis). For prokaryotic
organisms, the reaction network within which that
isotopic depletion must occur is shown in Fig. 8.
Branch points in the flow of carbon are evident
(1) downstream from pyruvate and (2) downstream
from acetyl-CoA. In an elegantly simple approach
to locating the point of fractionation, DeNiro and
Epstein (1977) grew heterotrophic bacteria on glucose (a C6 carbohydrate), on pyruvate, and on
acetate, thus providing carbon inputs at two points
above and one point below the branch point
between pyruvate and acetyl-CoA. The resulting
lipids were found to be depleted in 13C relative to
either glucose or pyruvate, the depletions being
roughly equal. Lipids from acetate-fed bacteria
were equal in isotopic composition to the carbon
source. These results indicated clearly that processes responsible for the depletion were at the
branch point downstream from pyruvate.
Mechanistically, it appeared that an isotope
effect associated with pyruvate~dehydrogenase,
the enzyme catalyzing the pyruvate acetyl-CoA
reaction, was involved, causing specifically a depletion in 13C at the carboxyl carbon of acetate and
leaving the methyl carbon unfractionated.
Although DeNiro and Epstein (1977) miscalculated the fractionations that would derive from the
isotope effect they proposed, Monson and Hayes
(1980, 1982a) subsequently showed that the qualitatively expected alternating pattern of 13C depletion was present in acetogenic lipids. From the
results of isotopic analyses of specific carbon posi-
118
J.M.HAYES
Carbon skeletons
of biomonomers
Metabolic Intermediates
2C02
2C02..¢,.~J~
Product of C fixation
in Calvin-cycle
autotrophs
;
~- C5
3C 2
CF
2°°2 2o,._1/
Pyruvate
-
Cm
m
3
~'-2-
isoprene
....
>. C 2
C m - Cc
AcetyI-CoA
H3C - C - CO2
o
f
C10,Cls,C2o...C4o
polyisoprenoids
Cc.~ / C c
products
Lipids
m c
H3C - C - SCoA
acetate
C14,C16,C18...C38.acetogenic
m
I
~--]-
ii
o
Product of C fixation
in reverse-TCA and
acetyI-CoA autotrophs
Products of catabolic degradation
of food in all heterotrophs
Fig. 8. Pathways of lipid biosynthesis in prokaryotic organisms. In heterotrophs, any of the indicated multi-carbon organic
compounds might derive from the food source. In Calvin-Cycleautotrophs the effectiveinternal source of organic carbon is a C6
carbohydrate. For organisms using the reverse-TeAor acetyl-CoAsystemsof carbon fixation,the methyland carboxylpositions of
acetyl-CoA will derive from two differentfixaton sites. The letters m and c denote positions in the C~ and C2 biomonomersthat
are derived from the methyland carboxy positions in acetyl-CoAand indicate the differentm/c ratios in the two lipid families.
tions within lipid carbon skeletons they were able
to develop a quantitative model for isotopic fractionations in lipid biosynthesis. For heterotrophs
growing on carbohydrates or for autotrophs producing carbohydrates by way of the Calvin Cycle,
they suggested:
6a~ = (6 m +
6°)/2
(20)
where the subscripts al, m, and c represent acetogenic lipids and the methyl and carboxyl positions
of acetyl-CoA. This expression states simply that
the ~ value of lipids containing equal numbers of
carbon atoms derived from the methyl and carboxyl positions of acetyl-CoA must be equal to
the average of the methyl and carboxyl ~ values.
DeNiro and Epstein (1977) proposed and, by
isotopic analysis of specific carbon positions,
Monson and Hayes (1982a) demonstrated, that
the methyl position in acetyl-CoA was not isotopically fractionated relative to carbohydrate, thus
6m = 6¢h (where ch designates carbohydrate
carbon). Finally, in precise analogy with the derivation of Eq. 14b, Monson and Hayes (1982a)
found
6c = 6c~ - (1 -f)epd~
(22)
requiring that the 6 value of the carboxyl position
would depend on the 6 value of the reactant carbon
(pyruvate derived without fractionation from carbohydrate, in this case); on the fraction, f, of that
reactant which flowed to acetyl-CoA; and on the
isotope effect, epdh, associated with reaction catalyzed by pyruvate dehydrogenase.
Further research has revealed significant complexities. Blair et al. (1985) showed that prior
results did not exclude the possibility that fractionation occurred downstream from acetate, but then
Melzer and Schmidt (1987) showed that kinetic
isotope effects associated with pyruvate dehydrogenase (not previously studied in isolation) were
precisely in accord with estimates of er~h derived
by Monson and Hayes (1982a). Significant gaps in
understanding remain and some of these limit the
precision with which results of isotopic analyses
of sedimentary materials can be interpreted:
(1) All of the preceding investigators assumed
119
13C CONTENTS OF SEDIMENTARY ORGANIC COMPOUNDS
that, in carbohydrates produced by the Calvin
Cycle, the abundance of laC was equal at all
carbon positions. But it has now been shown
(Rossmann et al., 1991) that this is not true for
maize (a C4 plant) or for sugar beets (Ca). In each
case, one of the two carbon positions in glucose
from which carbon flows without fractionation to
the methyl position of acetyl-CoA is depleted in
13C by 4.4%o relative to the molecular average. As
a result, one expects on average 6m= 6oh--2.2 and
a consequent additional depletion of lac in lipids
relative to initial carbohydrate. The depletion of
6m was not observed by Monson and Hayes
(1982a), who, for 6ch = --9.96_+0.05, found 6m=
- 9 . 5 _+1.0%o (uncertainties are standard errors of
mean values, 6m being based on analyses of two
different fatty acids). With allowance for uncertainties in the Rossmann et al. (1991) result, the
disagreement between the investigations is probably not statistically significant, but is worth keeping
in mind.
(2) As indicated in Fig. 9, pathways of carbon
flow are more complex in eukaryotic organisms,
mainly because C2 units are produced within the
mitochondrion but can be exported from it (as
required for lipid biosynthesis) only as part of
citrate, the C6 product resulting from the condensation of acetyl-CoA and a C4 species (see Fig. 9).
Bonds to the methyl position in acetate are made
and broken during the formation of citrate and its
subsequent cleavage outside the mitochondrion;
these processes provide additional opportunities
for isotopic fractionation. In a detailed study of
isotopic fractionation accompanying the biosynthesis of fatty acids in yeast, Monson and Hayes
(1982b) concluded that the pattern of enrichment
and depletion observed in prokaryotes is reversed
in eukaryotic products, with the carboxyl position
in the C2 biomonomer being enriched in zaC
relative to the methyl position. No subsequent
investigation has tested this result directly, but
observed isotopic differences between isoprenoidal
and n alkyl lipids (discussed below) indicate that
it is probably not general.
(3) Not only acetogenic but, as indicated in
Fig. 8, polyisoprenoid lipids are produced from
acetyl-CoA. In eukaryotic organisms, the latter
include abundant sterols as well as acyclic polyisoprenoids such as phytol. Formation of polyisoprenoids is less important in prokaryotes, but
acyclic compounds are required by photosynthetic
species and pentacyclic hopanoids are also widely
distributed. Although both acetogenic and polyisoprenoid lipids derive from acetate, their isotopic
compositions are, for several reasons, not expected
to be equal. First, as noted in Fig. 8, three of the
carbon positions in isoprene, the C5 biomonomer
from which polyisoprenoids are built, derive from
the methyl position of acetyl-CoA but only two
derive from the carboxyl position. As a result, 6i~,
Product of C fixation
/
i
/
C
= 2~
6
.
v3
I
II
II
I
I
f
2CO
_ 2
~~r-
~ r'"2
oilier
/
2C 4
- %
t,,
products. . . . .
- ~-
it_
\
/
H~cm C- 002
~
/
\
O
I
-~
T C A cycle
)
,-,r, ~ J
,
I
"~6~
j(
V - - - ~
Pyruvate
\t
.......
2CO2 ~= 2CO2 ~
It Mitochondrion
in autotrophs
I
"~_//
......
2C 4 4 " " I
~
2C 2 -- --> Iipids
.
m c
i"
ACatyI-COA, H3C - C - SCoA
./
Products of catabolic degradation
of food in heterotrophs
Fig. 9. Pathways of lipid biosyntbeis in eukaryotic organisms. Exotic mechanisms of carbon fixation are not found in ¢ukaryotes,
but complexity is introduced by the presence of subcellular organelles. Because no form of acetate is able to pass through the
mitochondrial wall, C 2 units produced within the mitochondrion by oxidative decarboxylation of pyruvate must be packaged as C 6
species (citrate) for export. The formation and subsequent lyation of the C 6 units introduces additional branch points and possibilities
for isotopic fractionation.
120
J.M. HAYES
the molecular-average 6 value for polyisoprenoid
lipids, will be given by:
6il :
(36m+ 26¢)/5
(23)
and will be equal to 6al only if the methyl and
carboxyl positions of acetyl-CoA have the same
a3C content, a coincidence that has never been
observed. Second, the production of both polyisoprenoid and acetogenic lipids represents a branch
point in the flow of C2 units. An isotope effect at
that branch point could cause isotopic compositions of methyl- and/or carboxyl-derived carbon
positions in polyisoprenoids to differ from those
of analogous positions in acetogenic lipids.
Biosynthetic effects in general
As emphasized in Fig. 2, the 13C content of any
cell is the result of a mass balance between inputs
and outputs. For a cell containing mce. moles of
carbon, we can write:
Sedimentarysystems
Primary and heterotrophic fraetionations
Ancient systems
One of the first applications of compoundspecific isotopic analyses to the study of a sedimentary
record
focussed
on
the
Cenomanian-Turonian boundary, specifically in
the Greenhorn Formation of the North American
Western Interior Seaway (Hayes et al., 1989). The
"specific compound" available for analysis was in
fact a compound class: the porphyrins. This fraction, thought at the time to derive almost entirely
from chlorophylls, was chosen for its significance
as an indicator of the isotopic composition of
primary materials. Knowledge of 6pot (where por
designates the porphyrin fraction) allowed estimation of 6p values:
~p = 6por +
mcell= min - mout
(24)
and
6eell = ( m i n f i n -- m o u t ¢ ~ o u t ) / ( m i n
- mout)
(25)
Values of 6i. and gout depend on isotope effects
associated with the assimilation and excretion of
carbon. Associated fractionations will have pancellular effects. Equally,
3¢~H= E(m,f,)/Xm,
(26)
where m~ and 6~ are respectively the molar amount
and 13C content of the /th biosynthetic product
and Y.indicates summation over a complete cellular
inventory. Finally, 6o,ii must also be equal to the
weighted average 6 value of the pool of metabolic
intermediates which serves as the carbon source
for biosynthesis. Conceptually,
6i = l i n t -- Ai = 3cell -- Ai
(27)
where flint is the average 6 value of the pool of
metabolic intermediates and A~ is the overall isotopic fractionation characteristic of the biosynthesis of the ith product. Isotope effects and branching
ratios on pathways linking the pool of metabolic
intermediates to biosynthetic product will affect
the isotopic compositions of individual molecules
but not 6c.,.
Apor
(28)
where 6p is the isotopic composition of Primary
Products (in an open marine setting, the average
6 value for the biomass of phytoplankton).
Comparison of 3p and hd then allows estimation
of Cenomanian and Turonian ~p values. Moreover,
comparison of 8p and 8TOc (TOC=sedimentary
total organic carbon) allows estimation of all
secondary isotopic fractionations (=Ar), i.e., the
isotopic shifts associated with reworking of organic
carbon prior to burial.
ep ~ 6 d -- 6p = 6 m -- era/d -- (0po r +
Apor)
Ar = 3p- 3TOC= 6por+ Apor- 6TOC
(29)
(30)
Values of ep provide information about the nature
of primary producer organisms and their environment, those of Ar about the intensity of reworking
and thus trophic structure. Notably, this approach
transforms purely chemical data into information
about ancient biogeochemical processes, and takes
as its starting point the primary production of
biomass.
As might be inferred from the preceding discussion of isotope effects associated with lipid biosynthesis, the value of Ar~~ is uncertain. Considering
literature reports and analyzing chlorophyllides
and biomass from extant organisms, Hayes et al.
13C CONTENTS OF SEDIMENTARY ORGANIC COMPOUNDS
(1989) selected Apor=-0.5%o (i.e., porphyrins
enriched in 13C relative to biomass). More recently,
Kennicutt et al. (1992) have reported Ach~= - 1.7%o
(range - 5.8 to 0.3%o, s = 1.7%o, n= 12), where chl
designates chlorophyll and thus indicates that the
carbon analyzed included not only the substituted
heteroaromatic ring which is the precursor of
sedimentary porphyrins but also the phytyl ester
group. Correction for the presence of that lipid
carbon pushes values of Apor beyond -5%0. The
authors did not discuss the disagreement between
their findings and those of previous workers (Hayes
et al., 1989, and references cited therein) nor did
they discuss mechanisms that would produce the
required enrichment of laC. The same paper
reports that amino acids, which share biosynthetic
precursors with the porphyrins, have on average
Aaa = 1.3%o. If Apor
5%0, indicated values of
6p in many sedimentary systems (Hayes et al.,
1987; Freeman et al., 1990; Boreham et al., 1989)
would fall below the ~ values of coexisting lipids
of probable phytoplanktonic origin (pristane, phytane, steranes) and enormous heterotrophic shifts
(A,) would be required to explain observed values
of 6a-oc. There are numerous elements of consistency which can be sought in isotopic data sets. If
they are absent, caution is in order.
Subsequent investigations of the Greenhorn
samples (Hayes et al., 1990), using isotope-ratiomonitoring gcms, have shown that acyclic polyisoprenoids are depleted in 13C relative to the porphyfin fractions by 3.8 to 5.50, averaging 4.5%0 (n =
20). The same isotopic difference is observed in
modern systems (e.g., Madigan et al., 1989) when
chlorophyllides and isoprenoids are compared. The
hypothesis that sedimentary porphyrins derive
mainly from chlorophyll as opposed to alternatives
such as cytochromes or hemes is supported by this
observation, but the clearest evidence on this point
has come from studies of 6 values of individual
porphyrins. Simultaneously and independently,
research groups at the Bureau of Mineral
Resources, Canberra, and the University of
Strasbourg showed that C32 etioporphyrins were
uniquely depleted in ~3C relative to all other
porphyrins, notably including C32 DPEP structures
clearly deriving from chlorophyllides (Boreham
et al., 1989; Ocampo et al., 1989). Because the C32
w e r e
-
121
etioporphyrins comprise only a few percent of
typical porphyrin mixtures, fractions like those
examined in the Greenhorn investigation can usually be taken as representative of chlorophyll. The
observed depletion of laC in the C32 etioporphyrins makes derivation from hemes or cytochromes
produced by heterotrophs feeding in aerobic portions of the paleoenvironment unlikely and suggests an origin from cytochromes produced
abundantly by sulfide-oxidizing bacteria (Prince
et al., 1988) which, as a result of their location at
the aerobic-anaerobic interface, may utilize a
carbon source depleted in 13C.
Heterotrophic shifts in modern environments
In the context of Fig. 2, the outgoing flow of
carbon in a heterotrophic organism is comprised
of respired CO2 and fecal carbon and C H 4 (a
product of gut bacteria). Carbon dioxide results
mainly from operation of the tricarboxylic acid
cycle, in which it is produced by decarboxylation
of organic acids. Because the same acids can also
flow to biosynthetic uses (chiefly the production
of amino acids), each decarboxylation is associated
with a branch point. If the decarboxylation reaction has a normal kinetic isotope effect, the respired
CO2 will be depleted in 13C and, by difference, the
organism enriched. DeNiro and Epstein (1978)
pioneered systematic investigations of this phenomenon and showed that it is quite general. For
many animals, biomass is enriched in 13C relative
to the food source by approximately 1%o.
Examination of isotopic shifts associated with heterotrophy has become a widely used tool for
investigation of predator-prey and grazing relationships within ecosystems. A particularly elegant
example is provided by Fry's (1988) investigation
of the food web in the water column at Georges
Bank in the North Atlantic.
Effects of heterotrophy on the organic carbon
being buried in sediments are not well known. As
indicated above, Hayes et al. (1989) interpreted
the difference between 6p and 6roc in terms of
heterotrophy, and the isotopic shift is in the direction expected, but the record of molecular structures found in marine sediments has been
interpreted very differently, in fact mainly in terms
of the preservation of primary products. These
122
views are not necessarily contradictory--it is possible that little bulk primary material would survive
but that a selection of algal biomarkers would
(Bradshaw et al., 1992). Investigations focussed on
the biomarkers could then suggest that primary
primary products were well preserved, but 6TOC
would in fact reflect secondary overprints. A recent
report titled "Stable carbon isotope ratios of plankton carbon and sinking organic matter from the
Atlantic sector of the Southern Ocean" (Fischer,
1991) is particularly interesting in this regard. It
was found that sinking organic particles (mostly
fecal material, though undoubtedly containing
phytoplanktonic biomarkers, Bradshaw et al.,
1992) were enriched in ~3C relative to plankton by
2-5%o. The fecal material does not represent heterotrophi biomass. Its enrichment in 13C is very
plausible related to the enrichment of a3C noted
in sedimentary deposits, many of which contain
abundant pelletal material. The enrichment may
be associated with heterotrophy, but cannot be due
to respiratory metabolism. Instead, we can speculate here that it reflects loss of 13C-depleted CH 4
from zooplanktonic gut communities. Shallow subsurface maxima are often found when the distribution of CH4 in marine water columns is examined
(Brooks et al., 1981), and at least one study has
appeared in which such maxima are specifically
associated with high concentrations of zooplankton (Traganza et al., 1979). It appears likely that
this mechanism, rather than accumulation of ~3C
along food chains, provides an explanation for the
enrichment of ~3C in some sedimentary organic
carbon.
Although the Southern Ocean is highly productive and thus of all modern sites one of the
most likely to support extensive heterotrophy, it is
also true that the observed shifts significantly
exceed those ascribed to heterotrophy in the
Greenhorn sediments (Hayes et al., 1989) and that
a strong correlation between paleontologically
assessed trophic complexity and Ar has been
observed in recent investigations of the Oxford
Clay (Kenig et al., 1993). Additional enrichment
of ~3C occurred at the sediment-water boundary
(Fischer, 1991). Effects of bacterial heterotrophy
may also have been underestimated. Results of a
recent investigation in which ~3C contents of bacte-
J.M. HAYES
rial nucleic acids were used to estimate 6 values
for bacterial biomass produced under natural conditions indicate isotopic shifts as large as 3%0
(Coffin et al., 1990).
Relationships between carbon skeletons
When considering 13C contents of lipid carbon
skeletons isolated from ancient sediments, it is-or wouM be--often helpful to associate two or
more compounds with a single source or closely
related sources. When, for example, two steranes
or two internally methyl branched alkanes happen
to have nearly identical 6 values, the structural
similarity supports but does not prove the association. In contrast, when fundamentally different
structures are possibly to be ascribed to the same
source, uncertainties can multiply rapidly. The
most basic comparison is that between n-alkyl and
isoprenoid carbon skeletons. Should we expect the
former always to be depleted in 13C relative to the
latter, or vice versa?
Monson and Hayes (1982a) showed that, in
Escherichia coli, the carboxyl carbon of acetylCoA was depleted in 13C relative to the methyl.
Therefore, if no isotope effect intervenes in the
synthesis of the C5 biomonomer from acetyl-CoA,
we should expect that isoprenoids, with a 3/2
methyl/carboxyl ratio, would be enriched in 13C
relative to n-alkyl carbon skeletons, in which the
ratio is 1/1. But how much significance can be
ascribed to results of analyses of a single bacterial
species and, in particular, what differences might
occur in eukaryotes? A summary of relevant observations follows:
(1) Wong et al. (1975) found "organic acids"
enriched in ~3C relative to carotenoids in cultures
of the purple
photosynthetic
bacterium,
Chromatium vinosum, thus suggesting that n-alkyl
carbon skletons might be enriched in 13C relative
to isoprenoids. However,
(2) Galimov and Shirinskiy (1975) found fatty
acids to be depleted in ~3C by 1-2%o relative to
polyisoprenoids in every case examined: a
cyanobacterium, zooplankton, a vascular plant,
and a eukaryotic alga.
(3) Sharkey et al. (1991) examined fractionation
of carbon isotopes during the biosynthesis of iso-
~aC CONTENTSOF SEDIMENTARYORGANICCOMPOUNDS
prene, C5Hlo, by red oak tree leaves. They found
that isoprene and carotenoids were depleted in 13C
relative to total photosynthetically fixed carbon by
,,~3%o and that fatty acids were depleted by an
additional 2%o.
(4) Jones et al. (1991) found that 24-ethylcholestanol from lacustrine sediments (and ascribed by
them to a terrestrial-plant source) was enriched in
13C by 4%0 relative to n-alkyl plant waxes in the
same sediments (Rieley et al., 1991).
Remarkably, there is at present no thorough
and systematic investigation of n-alkyl vs. polyisoprenoidal 6 values in a suite of biological sources.
Presumably this will change. At present, points
(2)-(4) above favor the idea that, within a single
source, isoprenoid lipids are enriched in 13C relative to n-alkyl species.
CO 2 paleobarometry
The two-step (mass transport + fixation) model
of photosynthetic carbon fixation clearly suggests
that ep might be pCO2-dependent. In qualitative
terms, if CO2 is abundant, mass transport is
unlikely to be rate limiting and the much larger
isotope effect associated with rubisco should be
dominant. In semi-quantitative terms (Eq. 17), ep
should vary with ci/G in systems in which active
transport is not a factor. But ep tends to maximize
at high values of c~ and, because Ce appears in the
denominator of the variable term in Eq. 17, the
relationship may appear inverted. Questions can
also be raised about the proper functional form
for G vs. ep calibrations. McCabe (1985) used a
logarithmic form in the first systematic examination of G-eP relationships. P o p p e t al. (1989) and
Jasper and Hayes (1990) followed that example,
but Rau et al. (1989, 1991a,b) preferred a simpler
linear form. Freeman and Hayes (1992) considered
both logarithmic and linear forms and, finding a
marginally better correlation in former, continued
the practice begun by McCabe (1985).
When considering short-term variations in ep
observed during the course of the North Atlantic
Bloom Experiment, Rau et al. (1992) took up the
idea that G - c l (i.e., the concentration gradient)
must reflect CO2 demand or growth rate and that,
under some conditions, this might remain constant.
For such cases, modification of Eq. 17 leads to a
123
relationship in which ep is expected to increase
with ce. Specifically, if ci = ce - ),, where ~, represents
the hypothetically constant CO2 demand, then
rearrangement of Eq. 17 yields:
~p = ~f + ( # c ~ ) ( ~ , - ~f )
(3 l)
a form in which maximal values of ep and ca are
associated. A more elaborate consideration of this
"hyperbolic form" appears in a forthcoming paper
by Francois et al. (1993). At least for cases in
which construction of an ev- G relationship does
not represent raw empiricism (e.g., Freeman and
Hayes, 1992), this approach seems likely to yield
the best connection between mechanism and effect.
It is, however, clear that c, is not the only factor
controlling ep. Biochemical and biophysical considerations have recently been very thoroughly and
helpfully reviewed by Goericke et al. (1993), and
other investigators have interpreted variations of
ap observed in natural systems in terms of varying
levels of enzymatic activity and pathways of carbon
fixation (Descolas-Gros et al., 1990; Fontugne
et al., 1991), growth rate and cell size (Takahashi
et al., 1991; Fry and Wainright, 1992), and speciesrelated physiological differences (Falkowski, 1991).
Fogel et al. (1992) have called upon many of these
factors to explain observed variations in 6p in a
seasonal study of the complex Chesapeake estuary.
The question to be settled is whether all of these
additional factors obscure G-related variations in
ep or whether, in spite of them, usefully accurate
estimates of ancient ce levels can be provided. It
seems very likely that, if success is achieved, it will
be based to a great degree on the use of compoundspecific analyses not just to derive ep but also to
provide as much additional information as possible
about the paleoenvironment and the producer
community to which it was host.
Acknowledgements
I am grateful to Peter Westbroek and Jan de
Leeuw for the invitation to participate in this
Symposium, and to the many coworkers and collaborators whose work is discussed in the preceding
text. Core support for the program in isotopic
biogeochemical research at Indiana comes from
the
National
Aeronautics
and
Space
124
J.M. HAYES
Administration (NAGW-1940) and from the prog r a m in m a r i n e c h e m i s t r y at t h e N a t i o n a l S c i e n c e
Foundation (OCE-9216918,
J.M. Hayes).
to J.P.
Jasper
and
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