Geochimica et Cosmochimica Acta, Vol. 68, No. 8, pp.

1691–1700, 2004
Copyright © 2004 Elsevier Ltd
Pergamon Printed in the USA. All rights reserved
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doi:10.1016/j.gca.2003.10.023

Isotopic order, biogeochemical processes, and earth history

Goldschmidt Lecture, Davos, Switzerland, August 2002
JOHN M. HAYES*
Department of Geology and Geophysics, Woods Hole Oceanographic Institution Woods Hole, MA 02543, USA

Abstract—The impetus to interpret carbon isotopic signals comes from an understanding of isotopic
fractionations imposed by living organisms. That understanding rests in turn on studies of enzymatic isotope
effects, on fruitful concepts of isotopic order, and on studies of the distribution of 13C both between and within
biosynthetic products. In sum, these studies have shown that the isotopic compositions of biological products
are governed by reaction kinetics and by pathways of carbon flow.
Isotopic compositions of individual compounds can indicate specific processes or environments. Examples
include biomarkers which record the isotopic compositions of primary products in aquatic communities, which
indicate that certain bacteria have used methane as a carbon source, and which show that some portions of
marine photic zones have been anaerobic. In such studies, the combination of structural and isotopic lines of
evidence reveals relationships between compounds and leads to process-related thinking. These are large
steps. Reconstruction of the sources and histories of molecular fossils redeems much of the early promise of
organic geochemistry by resolving and clarifying paleoenviron-mental signals. In turn, contemplation of this
new information is driving geochemists to study microbial ecology and evolution, oceanography, and
sedimentology. Copyright © 2004 Elsevier Ltd

1. INTRODUCTION specific organisms, and particular sources of signals (e.g., a

My aim is to trace the transformation of organic geochem-
istry into biogeochemistry. It has been an interesting revolution
with many leaders. I am honored to appear as a representative.
Forty years ago, organic geochemists thought mainly about
petroleum and its sources. In that milieu, Philip Abelson,
Melvin Calvin, and Geoffrey Eglinton were revolutionaries.
Abelson called his research “paleobiochemistry” (Abelson,
1954) and then “biogeochemistry” (Abelson and Hoering,
1960). Eglinton and Calvin (1967) took organisms and bio-
chemistry as the starting points for their interpretations of
sedimentary molecules. The impact of this new thinking was
great and the future looked very promising. The development
of the field was discussed using terms from information theory
(Shannon, 1948; Hamming, 1986). Sedimentary organic mol-
ecules were viewed as channels linking modern observers to
ancient events. The great diversity of molecular structures
meant that the number of channels, and thus the flow of
information, was practically unlimited. The implied system is
summarized in Figure 1, which is both conceptual and caution-
ary. As it illustrates the hoped-for flow of information, it also
reminds us that the molecular channels are coupled to remark-
ably diverse sources (in the information-theoretical sense) and
encoders. They are also affected by an important source of
noise.
The tools of modern organic chemical analysis were brought
to bear and data were immediately plentiful. But data can be
turned into information only by accurate decoding. For organic
geochemists, the essence of decoding is to know not only how
the atoms within the molecule are arranged now, but also to
know about their history. Decoding establishes relationships
between sedimentary molecules, initial biosynthetic products,
* Author to whom correspondence should be addressed
(jhayes@whoi.edu).
1691
paleoclimatic variation).
There are multiple approaches to decoding. By resolving the
structures of sedimentary molecules in great detail, many work-
ers have been able to constrain their histories. Stratigraphic
variations also provide useful evidence: molecules whose abun-
dances vary together might have a common source. The present
discussion will focus on isotopic variations.
An isotopic variation does not constitute an interpretable
signal unless the mechanism controlling it is known. For inor-
ganic processes, these are usually equilibrium isotope effects
(Chacko et al., 2001). For organic molecules, the mechanisms
prominently include kinetic as well as equilibrium isotope
effects (Hayes, 2001). Large and diverse isotopic variations are
common. The questions are what causes them and whether the
mechanisms are consistent enough that observed fractionations
can be interpreted reliably.
2. INTRAMOLECULAR PATTERNS OF ISOTOPIC ORDER
A key contribution was made by Galimov (1973), who
developed accurate techniques for calculating equilibrium dis-
tributions of isotopes within organic molecules. Because vibra-
tional environments differ significantly between carbon posi-
tions, these intramolecular distributions are not expected to be
uniform. In fact, Galimov’s results indicated an astonishing
world of isotopic order. Figure 2 provides an arresting example.
Each circle in the diagram at right represents a carbon position
in a molecule of chlorophyll, with each diameter proportional
to the abundance of 13C expected at isotopic equilibrium. Is
natural chlorophyll really like this? Even today, no analytical
technique can either confirm or refute the prediction. Moreover,
that issue is secondary. The shock is that an image this exotic
is required to describe the idea. Conventional depictions of
molecular structure (shown at left in the figure) have no isoto-
pic dimension.
Until then, conventional thinking about molecular structures
1692 J. M. Hayes
Fig. 1. Schematic view of the role of sedimentary organic molecules
as carriers of information. The elements portrayed here (“Message
source,” etc.) are those specified by information theory (Shannon,
1948). In this rigorous and formal system, the source of a signal is
distinct from the chemical or biological sources of molecules them-
selves.
had no isotopic dimension. But suddenly, such isotopic patterns
seemed likely to offer a means of constraining molecular his-
tories. The task would be to learn what isotopic order was built
into molecules during their biosynthesis and then to determine
whether and how those patterns were preserved in the geo-
sphere.
The impact of Galimov’s work was amplified because Abel-
son and Hoering (1961) had already demonstrated that measur-
able isotopic order existed within amino acids. They couldn’t
determine the abundance of 13C at each carbon position, but
they did show that the carboxyl groups were commonly en-
riched relative to the rest of the molecule. In both sign and
magnitude, the internal isotopic contrasts were for many amino
acids similar to those predicted by Galimov’s calculations. Was
this coincidental, or did it mean that thermodynamics, rather
than kinetics and biosynthetic pathways, actually controlled the
distribution of 13C within—and thus between— organic mole-
cules?
The clearest answer would come from examination of struc-
turally equivalent positions in a biosynthetic product. Lipids,
particularly the common fatty acids, provide the best example.
Biosynthetically, these abundant products derive from the po-
lymerization of acetate. As shown in Figure 3 all unbranched
Fig. 2. Two representations of the chemical structure of chlorophyll
a. On the left, the conventional structural formula. On the right, a
scheme that was invented by Galimov (1974) in order to describe
position-specific variations in the abundance of 13C. The isotopic
distribution indicated here is that expected at isotopic equilibrium.
Fig. 3. Factors affecting the distribution of 13C in n-alkyl chains. (a)
Biosynthetic sources of the C atoms in a typical n-alkanoic acid
(⫽ “fatty acid”). (b) The distribution of 13C expected if isotopic
equilibrium prevails throughout the biosynthetic process (Galimov,
1973). (c) The distribution expected if isotopic equilibrium prevails
during the biosynthesis of acetate but not during the polymerization of
acetate to produce the hydrocarbon chain. (d) The distribution expected
if kinetic factors govern the distribution of 13C in both acetate and the
final biosynthetic product.
carbon chains in nature contain two subsets of carbon atoms.
One is derived from the methyl position of the parent acetate,
the other from the carboxyl position. Since all but the termi-
nal-C positions are equivalent CH2 groups, equilibration calls
for isotopic homogeneity within the chain (Fig. 3b). Several
alternatives can be considered. The first (Fig. 3c) shows what
would be expected if the carboxyl position in the acetate
monomer were enriched in 13C and if that enrichment, rather
than equilibria established during biosynthesis of the final
product, controlled the distribution of isotopes.
The second alternative (Fig. 3d) is interesting because it is
expected if the distribution of 13C is controlled by kinetic
factors. DeNiro and Epstein (1977) showed that pyruvate de-
carboxylase, an enzyme similar to that which produces the
acetate biomonomer, has an isotope effect that discriminates
against 13C at the C-2 position in pyruvate. That isotope effect
would act like a dam. Carbon-13 would pile up behind it and be
diverted to alternate fates (Hayes, 2001). The C2 units produced
by pyruvate dehydrogenase would be depleted in 13C at the
carboxyl position. As shown in Figure 4, the magnitudes of the
enrichment and depletion would depend on the branching ratio.
3. DIRECT MEASUREMENTS OF INTRAMOLECULAR
ISOTOPIC ORDER
As a first step in searching for such effects, we learned how
to drive the Schmidt decarboxylation
* O H ⫹ HN ™™™™3 RNH ⫹ C
*O ⫹ N
RC 2 3 2 2 2 (1)
H2SO4
absolutely to completion so that we could analyze carboxyl
groups (Vogler and Hayes, 1979, 1980). We also synthesized
carboxylic acids in which the isotopic composition of the
carboxyl group was known absolutely (Vogler et al., 1978;
 Isotopic order, biogeochemical processes, and earth history 1693

Fig. 4. Branching flow of carbon at pyruvate dehydrogenase and its

effect on 13C abundances (DeNiro and Epstein, 1977; Monson and
Hayes, 1982). The scheme on the left summarizes the reactants and
products. A carbon kinetic isotope effect is associated with the action
of pyruvate dehydrogenase. As a result, the carboxyl position in acetyl
coenzyme A is depleted in 13C relative to the carbonyl position in
pyruvate. The graph at right depicts the relationship between the Fig. 5. Measurement of the isotopic compositions at specific posi-
magnitude of that depletion and the branching ratio, expressed in terms tions within hydrocarbon chains (Monson and Hayes, 1982). (a) Chem-
of the fraction of carbon flowing to acetate. ical reactions used to convert positions to carboxyl groups. (b) Mea-
sured isotopic compositions. Additionally, combustion of the parent
acids yielded overall ␦ values. The average result, ⫺12.8‰, does not
differ significantly from the average of the internal methyl (⫺9.5) and
Vogler and Hayes, 1978). Specifically, conventional Grignard internal carboxyl (⫺16.0). The terminal carboxyl positions have been
syntheses (Eqns. 2 and 3) were started with a known excess of fractionated by an additional process (Monson and Hayes, 1980).
CO2,

xRMgX ⫹ CO2 3 xRCO2MgBr ⫹ 共1 ⫺ x兲 CO2 (2) bling these chains and, in the process, quantitatively producing
new carboxyl groups. As shown in Figure 5a, this allowed
multiple, spatially resolved analyses of a parent fatty acid and
xRCO2MgBr ™™™™3 xRCO2H (3) its cleavage products. Carbon positions 10 and 9, highlighted in
H⫹,H2O
the figure and structurally equivalent in the parent acid, derive
the unconsumed CO2 was recovered and analyzed isotopically, respectively from the methyl and carboxyl carbons of an ace-
a mass balance showed that all the starting CO2 was accounted tate group.
for, and the ␦ value of the carboxylic acid was calculated by The results (Fig. 5b) showed that these structurally equiva-
difference. The acids served as standards that demonstrated the lent positions had different isotopic compositions and, there-
adequacy of the optimized Schmidt decarboxylation (Table 1). fore, that isotopic equilibrium did not prevail. As expected on
To analyze carbon positions within hydrocarbon chains, the basis of the isotope effect identified by DeNiro and Epstein
Monson and Hayes (1982) developed methods for disassem- (1977), the carbon position derived from the carboxyl group of
acetate was isotopically depleted. These and all other results
(Monson and Hayes, 1982) were quantitatively consistent with
Table 1. Tests of Schmidt decarboxylation. control of isotopic compositions by kinetic isotope effects at
two sites of fractionation. The first of these was at pyruvate
␦13CPDB, ‰ dehydrogenase, where a 23‰ kinetic isotope effect accounted
Substrate Predicted for carboxyl Found for CO2 Difference for the alternating pattern of isotopic order within hydrocarbon
chains. The second was at fatty acid synthetase, were a kinetic
Acetic acid ⫺44.4 ⫾ 1.6 a
⫺46.1 ⫾ 0.1 ⫺1.7 isotope effect of 13‰ at the branch point between liberation of
Na salt ⫺44.7 ⫾ 0.1 ⫺0.3 a free acid vs. continued elongation accounted for varying
Hexanoic acid ⫺57.6b ⫺57.4 ⫾ 0.5 0.2
Na salt ⫺58.7 ⫾ 0.2 ⫺1.1
isotopic abundances of terminal carboxyl groups (Monson and
Me ester ⫺57.3 ⫾ 0.2 0.3 Hayes, 1980).
Octanoic acid ⫺50.3b ⫺50.7 ⫾ 0.0 ⫺0.4 Particularly in retrospect, this dissection of controlling mech-
Na salt ⫺51.8 ⫾ 0.0 ⫺1.5 anisms has had multiple lines of significance. It provides direct,
Me ester ⫺50.6 ⫾ 0.2 ⫺0.3 quantitative support for the existence of intramolecular isotopic
Na salt ⫺58.2 ⫾ 1.0c ⫺59.1 ⫾ 0.1 ⫺0.9
Nonanoic acid ⫺25.0b ⫺25.2 ⫾ 0.2 ⫺0.2 order and for the concept that isotopic differences between
Na salt ⫺65.2 ⫾ 1.8c ⫺64.6 ⫾ 0.3 0.7 molecules are in fact the attenuated reflections of isotopic
differences within molecules. Together with the demonstration
a
Determined by pyrolysis of the sodium salt (Meinschein et al., that kinetic isotope effects and flows of carbon within reaction
1974).
b
Independently analyzed by the method of von Unruh and Hayes
networks govern isotopic fractionations, it provides a straight-
(1976). forward framework for the interpretation of molecular-isotopic
c
Determined by synthesis (Vogler and Hayes, 1978). signals.
1694
Fig. 6. Main processes by which phytoplankton can assimilate inor-
ganic carbon (Wolf-Gladrow and Riebesell, 1997; Laws et al., 1997;
Laws, 1998; Badger et al., 1998; Reinfelder et al., 2000; Morel et al.,
2002).
4. FRACTIONATION OF CARBON ISOTOPES DURING
PRODUCTION OF ORGANIC MATTER
The most sophisticated dissection of biochemical reaction
networks thus far has been that dealing with the assimilation of
carbon dioxide by photosynthetic or chemoautotrophic organ-
isms. Studies of land plants, summarized by Farquhar et al.
(1982), showed that isotopic fractionation was largely governed
by two processes which operate in series. The first, transport of
CO2 from outside the plant to the active site of a carbon-fixing
enzyme, has a small isotope effect. The second, fixation (the
bonding of carbon from CO2 to a larger organic molecule),
usually has a much larger isotope effect. The net fractionation
is either large or small, depending which of these processes
controls the overall rate. More recently, the research team at
Hawaii has beautifully elucidated the processes governing the
fractionation of carbon isotopes by algae (Laws et al., 1995,
2002). The relevant systems are summarized in Figure 6. The
prominent role of bicarbonate in some of these shows that
equilibrium isotope effects can play a role in determining
isotopic compositions of organic molecules. If there is a re-
versible reaction, if it has an isotope effect, and if there is time
for equilibration to occur, it will be a controlling factor. A
treatment of carbonate equilibria incorporating considerations
of mass transport as well as reaction kinetics has recently been
published by Zeebe and Wolf-Gladrow (2001).
For algae which obtain CO2 by passive diffusion, overall
fractionations observed to date can be summarized by this
expression (Popp et al., 1998):
␮ 共V/S兲
␧P ⫽ ␧f ⫺ ␤ (4)
ce
where ␧P is the overall fractionation, ␧f is the isotope effect
associated with carbon fixation, ␤ is a constant, ␮ is the specific
growth rate, V and S are the volume and surface area of the
algal cells, and ce is the concentration of dissolved CO2 exter-
nal to the algal cell. In the modern ocean, the most important
controlling factor seems to be ␮. Its power is seen in Figure 7,
which pertains specifically to the alkenone-producing hapto-
phytes, Emiliana huxleyii and Gephyrocapsa oceanica, thus
eliminating size and shape as factors. On the left, the data have
been plotted simply in terms of ␧P and ce. If concentrations of
dissolved CO2 were the dominant controlling factor, the results
J. M. Hayes
would be expected to fall along a straight line with an intercept
near 25‰ on the ␧P axis. On the right, the data have been
transformed and a strong correlation observed. The transfor-
mation is one in which the variable, b, serves as a proxy for
growth rate. For each point, b is estimated from (25 ⫺ ␧P)ce.
That is, given ␧f ⬇ 25‰, b is equal to the product ␤␮(V/S) in
Eqn. 4. Since ␤ and (V/S) are practically constant for popula-
tions of E. huxleyii and G. oceanica, variations in b must reflect
variations in ␮, the specific growth rate. The observed corre-
lation with concentrations of soluble reactive phosphate need
not indicate that phosphate itself is the growth-limiting nutrient.
Instead, the specific growth rate may be controlled by some
trace micronutrient that covaries with phosphate (Morel et al.,
1991).
Over long periods of geologic time, where the globally
averaged fractionation between dissolved inorganic carbon and
algal biomass has varied widely, we have a choice between
believing that all cells grew more slowly, or that the globally
averaged ratio of surface area to volume was higher, or that
concentrations of CO2 were higher. On those scales of time and
averaging, the CO2-related interpretations, in which the in-
creased depletion of 13C in marine organic matter prior to the
Miocene is attributed mainly to higher concentrations of CO2,
may still be largely correct (Arthur et al., 1985; Hayes et al.,
1999).
5. ISOTOPIC SIGNALS OF BIOGEOCHEMICAL
PROCESSES
Consideration of molecular-isotopic signals leads immedi-
ately to a focus on biogeochemical processes. These include not
only production but also a wide variety of aerobic and anaer-
obic secondary processes in the water column and sediments.
All of these interact to control the molecular and isotopic
composition of total organic carbon (TOC) in sediments. It’s
easy to focus on that end product. Throughout the Phanerozoic,
isotopic mass balances allow estimation of the fraction of
carbon that’s been buried in organic form (Holser et al., 1988).
For example, from the isotopic compositions of carbonate
carbon and organic carbon deposited 30 million years ago, we
Fig. 7. Two plots summarizing the same observations of the frac-
tionation of 13C by alkenone-producing algae (Bidigare et al., 1997;
Laws et al., 2001). On the left, measured values of ␧P are plotted as a
function of 1/ce, where ce is the concentration of dissolved CO2
external to the algal cells. On the right, the same data have been
transformed so that b, a proxy for growth rate (defined in the figure and
explained in the text) is plotted as a function of nutrient concentrations.
 Isotopic order, biogeochemical processes, and earth history
can estimate that 23% of the carbon being sequestered in
sediments was organic and that the rest was accounted for by
carbonates. In such calculations, the surface carbon cycle is a
black box that puts out carbonates and organic materials. Pro-
cesses within the box are not considered. Determining why
23% of the buried carbon was organic, or understanding dif-
ferences between sedimentary basins 30 million years ago,
requires consideration of processes.
Molecular-isotopic techniques make some key details acces-
sible. We can, for example, estimate the isotopic difference
between primary products and the TOC that was finally buried.
In our first investigations of this kind, we used porphyrins—
degradation products of chlorophyll—rather than algal lipids as
the isotopic proxy for primary products (Hayes et al., 1989).
Our aim was to partition the isotopic fractionation between
carbonates and organic carbon into two components. The first,
measured by the difference between carbonates and porphyrins,
would reflect fractionations associated with primary production
and with the formation of carbonate minerals. The second,
measured by the difference between porphyrins and TOC,
would reflect the sum of fractionations associated with second-
ary processes. The approach is summarized schematically in
Figure 8.
The section investigated was from the Western Interior Sea-
way in North America. It includes the boundary between the
Cenomanian and Turonian ages and its well-known carbon-
isotopic excursion (Arthur et al., 1988). Depth profiles for
carbonates, porphyrins, and TOC are shown in Figure 9. As in
many sedimentary sequences, the 4.1‰ range of variations for
the organic materials significantly exceeds the 2.3‰ range in
carbonates.
The differing ranges indicate that the isotopic composition of
the organic material is indeed a separate signal that encodes
information beyond that carried by the isotopic composition of
carbonates. Values of ␦carbonate vary as a result of changes in
the globally averaged inputs and outputs to the carbon cycle.
They can also be affected by diagenetic processes (Veizer,
Fig. 8. Schematic representation of the partitioning of the isotopic
difference between carbonate minerals and total organic carbon into
two components (Hayes et al., 1989). The first, denoted by ␧1, includes
␧P and the relatively constant offsets between carbonate minerals and
dissolved CO2 and between biomass and the carbon in porphyrin ring
systems (the heteroaromatic portion of a chlorophyll molecule). The
second, ⌬2, is the sum of fractionations associated with food webs,
diagenesis, and other secondary processes.
1695
Fig. 9. Isotopic profiles, Greenhorn Limestone (Hayes et al., 1989).
The upward triangles and solid line refer to the rising portion of the
isotopic excursion as seen in carbonates. The downward triangles and
dotted line refer to the falling portion.
1983). Under the guidance of Brian Popp, such influences were
stringently minimized in this work by analyzing only non
luminescent, micritic carbonates (Hayes et al., 1989). Values of
␦roc tend to follow those of ␦carbonate because the dissolved
inorganic carbon is the reactant from which the TOC has been
produced. They also include the effects of variations in ␧P, the
isotopic fractionation associated with primary production, and
⌬2, the isotopic shift associated with secondary processes,
namely all of those between primary products and diageneti-
cally stabilized TOC. These include fractionations imposed by
the food chain in the water column; by the benthic community;
by the sedimentary microbial community; and, depending on
the sediment’s history, by thermal processes.
It’s useful to think in terms of relationships between precur-
sors and products. Those associated with primary production
are summarized in Figure 10. In the upper graph, the deviations
from slope ⫽ 1 show that the isotopic composition of primary
products did not precisely follow that of dissolved inorganic
carbon. In the lower graph, the same data are recast in terms of
␧1. As values of ␦carbonate increased during the late Cenoma-
nian, values of ␧1 and thus of ␧P dropped and then returned to
(nearly) their starting value. Thanks to the Hawaiians, we know
that the variations must represent some combination of changes
in concentrations of CO2, rates of growth, and algal shape and
size. Of course all may have varied, with partially offsetting
effects. Broadly, however, the signal on the rising side of the
isotopic excursion represents a perturbation in which concen-
trations of CO2 were drawn down and then recovered, in which
rates of growth increased and then returned to their starting
levels, or in which the algal community temporarily was dom-
inated by fatter cells (larger V/S).
A second precursor-product linkage exists between primary
products (the biomass produced by photosynthetic organisms,
here represented isotopically by the porphyrins) and sedimen-
tary TOC. The resulting isotopic shift is symbolized by ⌬2 (Fig.
11). Remarkably, the slope relating reactants and products is
not one but instead 1.5. As porphyrins are enriched in 13C,
something is enriching the TOC even more strongly. This
corresponds to an increase in ⌬2. In this case, the perturbation
1696 J. M. Hayes

values of ⌬2. Increased heterotrophy could result from rising

levels of O2 in the atmosphere and surface waters. But the
isotopic excursion marks an oceanic anoxic event! With only
slightly more confidence than perplexity, we concluded that
“oxygenation of environments other than deep ocean basins
must have increased” (Hayes et al., 1989). Now, stratigraphic,
sedimentological, and geochemical studies by Sageman and
coworkers (Meyers et al., 2001, 2004) have elegantly con-
firmed this interpretation.
Other isotopically distinct biomarkers can serve as indicators
of significant paleoenvironmental conditions or processes. The
first example was provided by Summons and Powell (1986).
They initially recognized that the aromatic-hydrocarbon frac-

Fig. 10. Two views of primary processes, based on the data in Figure
9. Lines and symbols as in Figure 9. (a) Isotopic composition of
porphyrins, a proxy for primary biomass, plotted as a function of the
isotopic composition of micritic carbonates, a proxy for dissolved
inorganic carbon. The nonunit slope shows that the isotopic composi-
tion of the organic product does not simply follow that of the inorganic
precursor. (b) The fractionation between porphyrins and micritic car-
bonates plotted as a function of the isotopic composition of the latter.
Decreases in the fractionation can be attributed to the causes noted at
lower left in the graph. Increases in ␦carbonate indicate increases in the
fraction of carbon buried in organic form.

maximizes near the top of the isotopic excursion and returns to

its preexcursion value at the end of the event.
Most of the carbon loss on the pathway between primary
products and TOC is due to the activities of respiring hetero-
trophs in the marine food chain (Longhurst and Harrison,
1989). In modern systems, each trophic step produces a 13C
enrichment of 0.5–1.5‰ (Yoshii et al., 1999). The magnitude
probably depends on the composition of the input (e. g., the
relative abundances of lipids, carbohydrates, and proteins).
Cretaceous effects may have differed. In any case, increases in
the intensity of heterotrophic processes are expected to increase
Fig. 11. Two views of secondary processes, based on the data in
Figure 9. Lines and symbols as in Figure 9. (a) Isotopic composition of
TOC as a function of that of porphyrins, a proxy for primary biomass.
As in the relationship summarized in Figure 10, the isotopic composi-
tion of the product (TOC) again does not precisely follow that of its
precursor (primary biomass). (b) The same variations summarized in
terms of ⌬2, which is seen to rise and fall over the course of the isotopic
excursion.
 Isotopic order, biogeochemical processes, and earth history
Fig. 12. (a) Isorenieratane, an aromatic polyisoprenoid and (b) isore-
nieratene (Schaeflé et al., 1977), a carotenoid pigment unique to the
green photosynthetic bacteria, the Chlorobiaceae.
tions of several North American Paleozoic oils contained a
prominent series of compounds related to isorenieratane (Fig.
12a). In turn, it was apparently derived from isorenieratene
(Fig. 12b), for which the only known source was and is the
green photosynthetic bacteria, the Chlorobiaceae. These organ-
isms are obligate anaerobes. They fix carbon and produce
biomass by using energy from photons, but they rely on sulfide as
an electron donor and are killed by exposure to O2. Accordingly,
the depositional environments for the source rocks of these oils
must have had waters that were both anaerobic (and sulfidic) and
sunlit. In large bodies of water, this is a rare condition.
For confirmation, Summons and Powell (1986, 1987) iso-
lated individual compounds, converted their carbon to CO2,
and measured the abundance of 13C. The aromatic polyisopre-
noids were enriched in 13C relative to saturated hydrocarbons
in the same oils by as much as eight permil. That isotopic signal
firmly associates these compounds with the Chlorobiaceae,
which are unique in their reliance on the reversed tricarboxylic
acid cycle as a means of fixing carbon. Isotope effects within
that cycle are much smaller than those in other pathways of
carbon fixation (Hayes, 2001). Moreover, lipids can be en-
riched in 13C relative to biomass (van der Meer et al., 1998).
In subsequent work, the group led by Jaap Sinninghe Damsté
and Stefan Schouten at the Royal Netherlands Institute for Sea
Research has developed improved methods for the detection of
these important indicators of water-column anoxia (Koopmans
et al., 1996). They are notably abundant, for example, in North
Atlantic sediments from the Cretaceous anoxic events (Sin-
ninghe Damsté and Köster, 1998). The presence of products
from fastidious anaerobes in open-marine sediments is notable.
Extraordinary levels of stratification are required. These may
have been stabilized—at least well enough to allow transient
growth of the Chlorobiaceae and generation of the molecular-
isotopic signal— by the estimated sea-surface temperatures of
35 to 38°C (which derive from a new, molecular-organic tem-
perature indicator; Schouten et al., 2002).
Molecular-isotopic techniques have also been useful in stud-
ies of methane cycling. This is because, as a result of isotope
effects associated with its production by either biological or
thermal processes, methane is commonly depleted in 13C
(Whiticar, 1994). The offset is so large that, for practical
purposes, methane carbon is labeled almost as effectively as
many artificial tracers. Values of ␦ for lipids from meth-
anotrophs frequently drop below ⫺100‰ (Hinrichs et al.,
2000).
1697
The isotopic depletion in methanotrophs is doubly interest-
ing. It results in part from isotope effects associated with the
assimilation of methane (Reeburgh et al., 1997). As kinetic
effects, these are unsurprising: the product (biomass) is de-
pleted in the heavy isotope. If the fractionation derived instead
from equilibrium isotope effects, its sign would be reversed,
with the biomass being enriched relative to the methane (Gali-
mov, 1985). For the question of organic 13C control, kinetic or
equilibrium, the relationship between methane and meth-
anotrophs is, therefore, decisively informative: the control is
kinetic (if equilibrium effects prevailed, the methanotrophic
products would be enriched in 13C relative to the methane). For
the other relationship between a C1 carbon source and biomass,
namely that between CO2 and plants, the kinetic and equilib-
rium isotope effects both predict depletion, which is of course
observed. Indecisive though it is, proponents of equilibrium
and kinetic controls have both pointed to the “fit,” with out-
siders and students being frustrated by the confusion.
Isotopically depleted biomarkers signaling the recycling of
methane turned up in some of the first sedimentary extracts
examined using continuous-flow techniques (Freeman et al.,
1990). It was no great surprise; the samples came from the
Messel Shale, which formed from the mud of an Eocene
swamp. The molecular structures were characteristic of aerobic
methanotrophic bacteria, which are known to thrive in such
settings.
The challenge has been to find biomarkers characteristic of
the microorganisms catalyzing the anaerobic oxidation of
methane at the expense of sulfate. Depth profiles of the con-
centrations of methane and sulfate in sedimentary pore waters
made it perfectly clear that this process was occurring (Iversen
and Jørgensen, 1985). Success eventually came to Bian (1994),
who found isotopically depleted crocetane (Fig. 13) in samples
from transition-zone muds provided by Niels Iversen and Bo
Fig. 13. Depth profiles of porewater sulfate and methane, the rate of
oxidation of methane, and the 13C content of the isoprenoid hydrocar-
bon, crocetane in cores raised from the seafloor of the Kattegat (Bian
et al., 2001). Complementary depth profiles of sulfate, methane, and
methane oxidation were first observed by Iversen and Jørgensen
(1985). The role of crocetane as biomarker for methane-consuming
anaerobes was discovered independently by Bian (1994) and by Thiel
et al. (1999).
1698 J. M. Hayes
Fig. 14. A gas chromatogram of lipid biomarkers extracted from
sediments at gas seeps in the Santa Barbara Basin (Hinrichs et al.,
2000). The peaks marked in light gray represent products derived
exclusively from producers and consumers in the oceanic water col-
umn. Those marked in darker gray represent monoalkyl-(M) and dial-
kyl-(D) glycerol ethers produced by sulfate-reducing bacteria within
the methane-consuming consortia. The peaks marked with a heavy
black line are products of methanotrophic archaea.
Jørgensen. Crocetane and other products of anaerobic methan-
otrophy were discovered independently by Michaelis, Thiel,
and coworkers (Thiel et al., 1999). Ether-linked lipids identical
to those in some methanogens, but with isotopic compositions
indicating derivation from methanotrophs, were discovered by
Hinrichs, DeLong, and coworkers (Hinrichs et al., 1999).
Much additional information about lipids from methanotro-
phic communities has now accumulated. The Hamburg group
was the first to show that 13C-depleted products probably from
sulfate-reducing bacteria were associated with those from the
methane-consuming archaea (Thiel et al., 1999). A more spe-
cific connection to sulfate reducers was established by Pancost
et al. (2000). Additional results from Hinrichs et al. (2000),
which serve to summarize these findings, are shown in Figure
14. Important further reports have followed (Pancost et al.,
2001a, b; Thiel et al., 2001a, b).
At the time of its publication, the initial report from Hinrichs
et al. (2000) was unique in its combination of molecular-
isotopic and genomic lines of evidence. The genomic approach
was promptly extended by Boetius et al. (2000) at the Max
Planck Institute for Marine Microbiology, who used fluores-
cently labeled oligonucleotide probes to show that archaea
closely related to those found by Hinrichs et al. (1999) were
closely associated with bacteria closely related to known sul-
fate reducers. This provided support for key aspects of the
“consortium hypothesis” introduced by Hoehler et al. (1994).
Further multidisciplinary investigations have confirmed that the
aggregates observed by Boetius et al. (2000) are indeed meth-
anotrophic (Orphan et al., 2001b) and revealed considerable
diversity in anaerobic methanotrophic communities (Orphan et
al., 2001a, 2002).
These most recent studies illustrate the convergence of three
lines of inquiry: studies of lipid biomarkers (isotopic and struc-
tural), microbial physiology and ecology, and microbial
genomics. This synergy promises to revolutionize, first, our
understanding of terminal processes in the modern carbon cycle
and, ultimately, our understanding of the evolution of the
carbon cycle over the course of earth history. The young
scientists pursuing these quests are progressing rapidly. Some
are certain to be recognized as fitting representatives of V. M.
Goldschmidt’s scientific legacy. I do not claim to reach their
level, still less Goldschmidt’s. I have, however, helped to build
the interdisciplinary connections that now characterize biogeo-
chemistry. Soon to be surpassed, I nevertheless have confi-
dence in the value of that role.
Acknowledgments—My work has been supported by the programs in
Exobiology and Astrobiology of the National Aeronautics and Space
Administration and by the National Science Foundation. Most of the
work described was completed while I was a member of the faculties
in Chemistry and in Geological Sciences at Indiana University, Bloom-
ington. I am grateful to that institution and to my colleagues there,
particularly Stephen A. Studley, who worked with me on the develop-
ment of mass spectrometric techniques from 1972 until I moved to
Woods Hole in 1996. This published version of my lecture has bene-
fited from constructive comments provided by Jaap Sinninghe Damsté,
David Des Marais, and an anonymous reviewer.
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