Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1
DOI 10.1186/s40645-015-0032-y

REVIEW

Open Access

Biochemical and physiological bases for the useof carbon and nitrogen isotopes in environmental
and ecological studies
Naohiko Ohkouchi1*, Nanako O Ogawa1, Yoshito Chikaraishi1, Hiroyuki Tanaka2 and Eitaro Wada1,3
Abstract
We review the biochemical and physiological bases of the use of carbon and nitrogen isotopic compositions as an
approach for environmental and ecological studies. Biochemical processes commonly observed in the biosphere,
including the decarboxylation and deamination of amino acids, are the key factors in this isotopic approach. The
principles drawn from the isotopic distributions disentangle the complex dynamics of the biosphere and allow theinteractions between the geosphere and biosphere to be analyzed in detail. We also summarize two recently
examined topics with new datasets: the isotopic compositions of individual biosynthetic products (chlorophylls andamino acids) and those of animal organs for further pursuing the basis of the methodology. As a tool forinvestigating complex systems, compound-specific isotopic analysis compensates the intrinsic disadvantages of bulk
isotopic signatures. Chlorophylls provide information about the particular processes of various photoautotrophs,
whereas amino acids provide a precise measure of the trophic positions of heterotrophs. The isotopic distributionsof carbon and nitrogen in a single organism as well as in the whole biosphere are strongly regulated, so that theirmajor components such as amino acids are coordinated appropriately rather than controlled separately.
Keywords: Carbon isotopic composition; Nitrogen isotopic composition; Ecosystem; Food web; Chlorophyll; Trophic
position; Amino acid; Animal organ

Review
Introduction

The biological processes that significantly affect the Earth's
surface environment generally lack rigorous formulations
because they are intrinsically elastic. However, using thechemical signatures induced by molecular motion (vibrational, translational, and rotational), we can at least document them rigorously and accurately. In recent decades,
the natural abundances of stable carbon and nitrogen isotopes (represented as 13C/12C and 15N/14N ratios, respectively) have been widely used to probe biogeochemical andecological processes. Thanks in part to such isotopic evidence, we have come to understand our living planet moreclearly. Carbon and nitrogen are essential elemental constituents of organisms and form various chemical species* Correspondence: nohkouchi@jamstec.go.jp
1
Japan Agency for Marine-Earth Science and Technology, 2-15
Natsushima-cho, Yokosuka 237-0061, Japan
Full list of author information is available at the end of the article

on Earth. The isotopic compositions of these elements
provide process-related and source information.
Historically, this field started as early as the 1930s, whena custom-made mass spectrometer was constructed tomeasure subtle differences in the isotopic ratios of naturalcarbon (Nier and Gulbransen 1939). Although the carbon
and nitrogen isotopic compositions of diverse types of organic matter were measured and discussed by the pioneersof isotope geochemistry (e.g., Rankama 1948; Wickman
1952; Craig 1953; Hoering 1955; Parwel et al. 1957), theisotopic fractionation associated with various biochemical
reactions was only determined precisely after some time(e.g., Park and Epstein 1960; DeNiro and Epstein 1977;
Hoering and Ford 1960; Wada 1980). As our knowledgeof the distributions of carbon and nitrogen isotopes inorganic matter and the factors controlling them has
accumulated, the scope of isotopic measurements has
greatly expanded.
An important application of the analysis of carbon andnitrogen isotopic compositions is in ecological studies.

© 2015 Ohkouchi et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.

Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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In 1967, Y. Miyake and E. Wada first reported that marine animals incorporate dietary 15N in preference to 14N(Miyake and Wada 1967). Later, several seminal papers
provided robust data on the 15N-enrichment that occurs
during heterotrophic processes, based on diet-controlledlaboratory culture experiments (e.g., DeNiro and Epstein
1981; Minagawa and Wada 1984). This 15N-enrichment
is a reflection of biochemical processes that accompany
significant isotopic fractionation. The carbon isotopic
composition of an organism mainly reflects its dietary
signature (DeNiro and Epstein 1978), and coordinatedisotopic measurements of organisms provide a uniqueapproach to describing the dietary habits of animals, amacroscale ecological phenomenon. Such macroscale
functions in the biosphere strongly influence the Earth's
surface environment via the population dynamics of ecosystems (Begon et al. 2006). In this review, we summarize
our current knowledge on the theoretical backgroundunderlying this approach. To achieve this, we consider theprocesses sequentially, first describing the isotopic relationships between substrates and autotrophs. We alsopresent original datasets to address two related topics,
compound-specific and organ-level isotopic compositions,which are helpful in achieving this goal.
Isotopes and isotopic fractionation

In nature, organic matter contains substantial amountsof stable isotopes of the ‘light elements’, includinghydrogen, carbon, and nitrogen. For example, a humanbody weighing 50 kg contains over 200 g of the heavier
isotopes of these elements (Wada et al. 1995). Small variations in the isotopic compositions present in natureare generally described by comparing the ratios of theisotopes in the sample material with those of standard(or reference) materials. These are expressed in the conventional δ notation defined below:δ ð‰Þ ≡ 103 Rsample =Rstandard – 1ð1Þ

where the term R denotes the 13C/12C ratio for carbon or
the 15N/14N ratio for nitrogen. In general, Vienna Pee Deebelemnite (VPDB; 13C/12C ≃ 0.011179; Coplen et al. 2002)
and atmospheric nitrogen (AIR; 15N/14N ≃ 0.003677; Junk
and Svec 1958) are used as the standards for carbon andnitrogen, respectively. Because authentic standards suchas NBS-19 are not largely available, the reference materials
routinely used are prepared in each laboratory or community (e.g., Tayasu et al. 2011). When a sample contains less13
C or 15N than the standard, the δ value is negative. A δvalue of −20‰ indicates that the isotopic ratio of the sample is 2% lower than that of the standard (in the case ofcarbon, 13C/12C ≃ 0.01096).
All physicochemical processes cause isotopic fractionation, which can be observed by measuring the isotopic

compositions of both the reactants and products. The
magnitude of isotopic fractionation is described as eitherthe ‘isotopic fractionation factor’ denoted by α, or the‘isotopic fractionation’ denoted by ε, defined with thefollowing equations:
αr=p ≡ Rr =Rp

ð2Þ



εr=p ≡ 103 Rr =Rp – 1 ¼ 103 αr=p – 1

ð3Þ

where the subscripts r and p designate the reactant andproduct of a reaction, respectively. The εr/p values for
enzymatic reactions are generally positive for both carbon and nitrogen.
There are two types of isotope effects: isotope (exchange) equilibrium and the kinetic isotope effect. Isotope equilibrium is observed in a closed, mixed system
in which a bidirectional reaction occurs and chemical
equilibrium is attained. A typical example of isotope equilibrium can be seen between gaseous CO2 and dissolved
bicarbonate (HCO3−):
13

CO2ðgasÞ þ H12 CO3 – ⇌12 CO2ðgasÞ þ H13 CO3 –ð4ÞIn this case, the isotope effect occurs during the formation and destruction of the bonds involving a carbon
atom. In this reaction, the equilibrium constant K isexpressed as follows:


 


K ¼ 12 CO2ðgasÞ  H13 CO3 Þ= 13 CO2ðgasÞ  H12 CO3 – Þ
ð5Þ¼ 13 C=12 C HCO3 − = 13 C=12 C CO

ð6Þ

¼ αHCO3 − =CO2ðgasÞ

ð7Þ

2ðgasÞ

In an exchange reaction containing only one exchangeable atom, the equilibrium constant K is equivalent to theisotopic fractionation factor α. At a water temperature of
25°C, the experimentally determined αHCO3 − =CO2ðgasÞ value
for the above reaction is 1.0079 (Mook et al. 1974). It indicates that at equilibrium, the carbon isotopic compositionof CO2 in the atmosphere is −7.9‰ when that of HCO−3
in the surface water is 0.0‰. Most of this fractionation occurs in the hydration stage (CO2(aq) + H2O ⇌ H2CO3)
and not during the exchange across the air-water interface(CO2(gas) ⇌ CO2(aq)) or in the dissociation of carbonic acid
(H2CO3 ⇌ HCO3− + H+).
Although the isotopes of a particular element have rather similar chemical behaviors, their specific thermodynamic parameters and rate constants in chemical andbiological reactions differ. The kinetic isotope effect isconsidered to best describe the isotopic fractionationthat occurs in the metabolic processes within a cell. The
kinetic isotope effect is observed in irreversible or unidirectional reactions, such as evaporation, diffusion, andOhkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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biologically mediated reactions. In such reactions, theisotopic compositions of the reactant (δr) and the cumulative product (δp) are conventionally described with thefollowing equations:δ r ¼ δ 0 –εr=p ln f

ð8Þ

δ p ¼ δ 0 þ f =ð1 –f Þεr=p ln fð9Þ

where f (0 ≤ f ≤ 1) is the fraction of unutilized substrateremaining and δ0 is the isotopic composition when f = 0.
It should be noted that these equations are approximate
forms of the Rayleigh distillation model, and the error
expands as f decreases. Figure 1 is a schematic representation of the isotopic compositions of the reactant(s) andproduct(s) in such a unidirectional reaction in a closed
system.
In general, the rate constant for the light molecule(the molecule containing the light isotope), which has
higher vibrational zero-point energy, is greater than thatfor the heavy molecule (the molecule containing theheavy isotope) (Bigeleisen 1949). Therefore, the productFigure 1 A schematic diagram showing the kinetic isotope
effect. The isotopic compositions of unconsumed reactant (blue,
solid line), instantaneous product (black, broken line), andcumulative product (red, solid line) are shown as a function of thefraction of unutilized substrate (f) for a unidirectional reaction.
Mathematical formulae for these reactants and products are also
shown. Subscripts r, ip, and p represent reactant, instantaneous
product, and accumulated product, respectively. Modified from
Mariotti et al. (1981).

of the reaction is depleted in heavy isotope (13C or 15N)
relative to the reactant (αr/p > 1 and εr/p > 0). The magnitude of the isotopic fractionation depends on various
factors, but in biochemical reactions, it is strongly related to the inherent characteristics of the enzyme catalyzing the reaction. The assimilation of CO2 and nitrateby autotrophs exemplifies reactions of this kind, which
will be discussed in the following “Carbon and nitrogenisotopic compositions of autotrophs: isotopic changes
during anabolism” section.
Carbon and nitrogen isotopic compositions ofautotrophs: isotopic changes during anabolism

Both the carbon and nitrogen that form autotrophic cells
are originally derived from inorganic substrates, such asenvironmental CO2 and nitrate, respectively (Figure 2).
In addition to CO2, methane (CH4), acetate (CH3COOH),
amino acids, and other small carbon compounds are also
used as carbon substrates by some specific autotrophs. Although we still do not know the quantitative importanceof these substrates or the ‘recycling’ of organic matter innature (Takano et al. 2010), here we assume that CO2 isthe overwhelmingly important substrate in both terrestrial
and aquatic environments. The situation is more complicated for nitrogen. Although nitrate (NO3−) and ammonium (NH4+) are often considered to be the substrates ofautotrophic processes, dinitrogen (N2), nitrite (NO2−),
urea (NH2CONH2), and some amino acids are also usedas substrates by some autotrophs. For simplicity, we will
mainly treat CO2 and nitrate as the carbon and nitrogen
substrates, respectively, in the discussion below.
When CO2 is assimilated by an aquatic alga, it diffuses
across the plasma membrane from the environment to
the intracellular CO2 pool, which is followed by carboxylation catalyzed by ribulose bisphosphate carboxylase/oxygenase (RubisCO). In contrast, nitrate is activelytransported across the membrane to the intracellular nitrate pool, where it is subjected to sequential enzymatic
reduction to nitrite and ammonium used in the synthesisof amino acids (summarized by Ohkouchi and Takano2014). In both cases, isotopic fractionation occurs during
the transport of the substrate across the plasma membrane and the enzymatic fixation of the substrate in thecell. The isotopic fractionation associated with the assimilations of CO2 and nitrate by photoautotrophs has
been successfully explained with a two-step model(Figure 2a,b; Park and Epstein 1960; Wada and Hattori
1978; O'Leary 1981; Farquhar et al. 1982; Hayes 1991).
In this model, the diffusional process across the membrane is reversible, whereas enzymatic fixation is irreversible. The overall kinetic isotope fractionation εp isexpressed with several types of formulae (e.g., Wada andHattori 1978; Farquhar 1983; Hayes 1991; Chikaraishi2014).

Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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Figure 2 Schematic illustrations of the autotrophic assimilation of inorganic substrates. Diagrams (a) and (b) indicate assimilation schemes
for CO2 and nitrate, respectively. Diagram (c) is a scheme of the normal two-step model, and (d) is a more rigorous scheme based on MichaelisMenten kinetics (Wada and Hattori 1978). The terms c, k, and ε indicate the concentration, rate constant, and isotopic fractionation, respectively.

The equation often seen in the literature is related tothe substrate concentrations outside and inside the cell
(specified as ce and ci, respectively).
εp ¼ εt þ εf –εt ci =ce

ð10Þ

where εt is the isotopic fractionation associated with the
diffusion or active transport of the substrate across theplasma membrane, and εf is the net fractionation causedby the enzymatic reaction that fixes the substrate (carboxylation or nitrate reduction; see Figure 2c). The valuesof εt during CO2 assimilation were determined experimentally to be 2.9‰ to 4.4‰ for terrestrial plants (Craig
1957; Farquhar 1983) and 0.7‰ to 0.9‰ for aquaticplants (O'Leary 1984; Jähne et al. 1987). In contrast, thevalue of εf is far greater than that of εt, ranging from18‰ to 30‰, which was estimated with pure RubisCO
isolated from various plants and assayed under physiological conditions (summarized by Chikaraishi 2014).
The equation given above has been applied when reconstructing the CO2 concentrations in water or the atmosphere in the geological past (e.g., Pagani et al. 2005).
It must be remembered that εp is not only controlled
by ce but also by other factors. In particular, in low-CO2
environments such as the present atmosphere, thegrowth rates of autotrophs have been shown to strongly
control εp. The equation below was developed by

Takahashi et al. (1991) to explain the CO2 assimilation by
the freshwater green alga Chlamydomonas reinhardtii:
εp ¼ εf þ εt –εf k f =k t

ð11Þwhere kt and kf are the rate constants for the transport ofthe substrate across the membrane and from the intracellular pool to the organic matter, respectively (Figure 2c).
Because 0 ≤ kf/kt ≤ 1, the value of εp is expected to vary between εt and εf. Because εt is much smaller than εf, it isclear that the overall isotopic fractionation (εp) correlates
negatively with the growth rate (kf ), which is controlled byenvironmental factors such as light intensity and substrate
supply. When the growth rate increases, the mass transport tends to be a rate-limiting step. This relationship was
clearly observed in laboratory culture experiments of not
only C. reinhardtii (Figure 3a; Takahashi et al. 1991) but
also the cyanobacterium Agmenellum quandruplicatum(Wada et al. 2012).
In contrast, Wada and Hattori (1978) derived a more
rigorous formula based on Michaelis-Menten kinetics to
explain the nitrogen isotopic fractionation that occurs
during nitrate assimilation by the marine diatom Phaeodactylum tricornutum (Figure 2d):εp ¼ ε0 þ þ ε2 XY –ε0 – X
X ¼ 1 –k 2 ½ES =k 0 þ ½S 0 ; Y ¼ k 1 – =ðk 1 – þ k 2 Þð12ÞOhkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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Figure 3 Experimental results indicating the relationship between isotopic fractionation and growth rate (μ) of an alga. Variations in(a) the carbon isotopic fractionation associated with CO2 assimilation (εCO2 ) by the freshwater green alga Chlamydomonas reinhardtii (modified
from Takahashi et al. 1991) and (b) the nitrogen isotopic fractionation associated with nitrate assimilation (εNO3 ) by the marine diatom Phaeodactylum
tricornutum (modified from Wada and Hattori 1978).

where subscripts 0, 1, and 2 represent the sequential reactions from substrate transport across the membrane tothe formation of organic matter (Figure 2d), and the superscripts + and − represent the forward and backward
reactions, respectively. In this equation, the overall isotopic fractionation (εp) correlates negatively but notlinearly with the growth rate (k2) (Figure 3b).
In natural environments, the isotopic compositions ofphotoautotrophs occur across wide ranges: from −38‰
to −10‰ for carbon (Figure 4a) and from −9‰ to +10‰
for nitrogen (Figure 4b). The approximately 10‰ depletion of 13C in terrestrial plants relative to that in aquaticplants may be explained by the fast diffusion of CO2 in
air relative to that in water. These wide ranges are partially ascribed to fluctuations in the isotopic compositions of the substrates in the aquatic and terrestrial
environments. For example, in a modern cultivated field,
nitrogen is heavily supplied as chemically synthesized
fertilizers whose δ15N values generally range from −6‰
to +7‰ (Heaton 1986; Vitòria et al. 2004). Therefore,
fertilizer significantly overwrites the isotopic signature of
the nitrogen substrate in the soil. The processes and dynamics of substrate assimilation also affect the isotopic
compositions of biosynthetic products (Hayes 1991; Fogel and Cifuentes 1993; Raven 1997; Chikaraishi 2014;
Ohkouchi and Takano 2014). A well-known example isthe carbon isotopic compositions of C3 and C4 plants.
The former assimilate CO2 simply via RubisCO, whereas
the latter assimilate CO2 through phosphoenolpyruvatecarboxylase (PEPC) after CO2 is concentrated in the cell
as a form of phosphoenolpyruvate. Because most CO2transported into the C4 plant cell is used for the synthesisof organic matter, the carbon isotopic composition of C4
plants is relatively close to that of the substrate (atmospheric CO2) (Figure 4a). In all cases, the substrate assimilation processes are controlled by Rayleigh-distillation-

type kinetics, and the isotopic composition of the product is not constant but intrinsically variable.
During nitrate assimilation, the isotopic fractionationof the enzymatic reaction catalyzed by nitrate reductase
ranges from 2‰ to 10‰ (summarized by Ohkouchi and
Takano 2014). Because the nitrate concentration in the
pelagic ocean is generally up to several micromolar (approximately 3 orders of magnitude lower than the concentration of dissolved CO2) except at high latitudes,
nitrate is actively transported into the cell (Falkowski1975). This may cause little isotopic fractionation during
the transport of nitrate into the cell. Dissolved N2 isslightly enriched in 15N relative to that in the atmosphere (Figure 4b), reflecting the slight isotopic fractionation associated with the equilibration of N2 between
the gaseous and aqueous phases ( εN2ðaqÞ =N2ðgasÞ ≃ 0:7‰ at
25°C; Klots and Benson 1963). Except for regions whereN2-fixation and denitrification are dominant, the nitrogen isotopic composition of oceanic nitrate usually
ranges from 5‰ to 10‰. The nitrogen isotopic composition of nitrate from the surface photic zone is generally
greater than 5‰ the mean value for oceanic nitrate(Sigman et al. 2009) because of the biological uptake ofnitrate. When nitrogen is supplied via biological N2fixation, the bulk cells display a narrow δ15N range, predominantly between −2‰ and 0‰ (Figure 4b; Hoeringand Ford 1960; Minagawa and Wada 1986). The small
isotopic fractionation associated with this process suggests that the cleavage of the strong triple bond of N2
takes place under irreversible conditions (Wada et al.
2012) with high utilization (i.e., f ≃ 0; Figure 1) of the N2
incorporated into the cell. In aquatic environments, most
cyanobacteria and photosynthetic bacteria have an ability
to conduct N2-fixation, and in the terrestrial environment, some plants such as legumes contain symbiotic
bacteria that fix N2 in their root nodules. In contrast,Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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δ13C (‰)

a)
-40

-30

-20

-10

0

5

10

CO 2 (atmosphere) 1
CO 2 (seawater, total) 2
Terrestrial C3 plants 3
Terrestrial C4 plants 3
Terrestrial CAM plants 3
Freshwater algae 3
Oceanic algae 3,4

δ15N (‰)

b)
-10

-5

0

N2 (atmosphere) 5
N2 (seawater) 60m

NH4+ (seawater) 6

500m
50 m

NO3- (seawater) 6,7
NO3- (soil) 8,9
Terrestrial C3 plants 8,10
Terrestrial C4 plants 4,11
Marine algae 6,9,12
N2 -fixing cyanobacteria
13,14

Figure 4 Compilation of a) carbon and b) nitrogen isotopic compositions of inorganic substrates and autotrophs from natural environments.
All the samples shown here were collected from Japan or the northwest Pacific Ocean, except the atmospheric CO2 and N2. Note that most terrestrial
plants were collected from cultivated areas, so their nitrogen isotopic compositions are more or less affected by chemical fertilizers. Sources: 1Cunz(2011), 2Kroopnick (1985), GEOSECS Station 224, 3Chikaraishi and Naraoka (2003), 4Wada et al. (1993), 5Mariotti (1983), 6Miyake and Wada (1967),
7
Minagawa et al. (2001), 8Wada et al. (1975), 9Wada et al. (1986), 10Chikaraishi et al. (2005), 11Chikaraishi et al. (2010), 12Chikaraishi et al. (2009), 13Minagawa
and Wada (1986), 14Wada et al. (2012).

when nitrogen is abundantly supplied to the soil via precipitation as nitrate, the isotopic signatures of plants are
generally consistent with that of the nitrate.
Figure 5 summarizes the δ13C and δ15N values for autotrophs from various environments, many of whichhave been investigated by one of the authors (EW) over
40 years of his career. In natural environments, the isotopic compositions of autotrophs range widely, depending upon the substrate, the assimilation process, and thesubstrate utilization. To our knowledge, an algal felt
from a penguin rookery in Antarctica displayed thehighest autotrophic δ15N value (approximately +31‰),
which was attributed to the evaporation of ammonia
from the soil, whereas the lowest value (−49‰) was observed in algae collected from a pond in the Dry Valley

area in Antarctica (Wada et al. 1981). Such 15N-depletion is probably attributable to the large isotopic fractionation during assimilation with a slow growth rate
and the elevated nitrate concentration in the pond. In
methane seeps, some archaea and bacteria assimilate
methane, causing strong 13C-depletion in the cellularcomponents (approximately −130‰; e.g., Hinrichs et al.
1999). The biochemical processes related to the 13C-depletion in these microorganisms are not well understood.
In aquatic environments, it is technically difficult torigorously separate autotrophs (i.e., algae and cyanobacteria) from heterotrophs and detritus. Consequently, thecarbon and nitrogen isotopic compositions of particulateorganic matter (POM) have long been substituted for
those of photoautotrophs in surface water. However,Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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30
Algal mat in penguin rookery 1(Antarctica)

Algae in Lake Biwa 3

Algae in oligotrophic ocean 4

Algae in Oyashio region 6

10Cyanobacteria red tide 4

Algae in Lake Baikal 5

Terrestrial C4 plants 4

δ15N (‰)

Terrestrial in C3 plants 40

Seagrass meadow 4(Texas)Cyanobacteria 4,7(East China Sea)
Algae in eutrophic ocean 4

Mountain forest 4
Ice algae 4-10
Algae from dry valley pond1,2(Antarctica)

-50

-40

-30

-20

-10

0

δ13C (‰)
Figure 5 Summary of the δ13C and δ15N values for autotrophs from various environments. Superscripts indicate the sources: 1Wada et al.
(1981), 2Wada et al. (2012), 3Yamada et al. (1998), 4Wada and Hattori (1991), 5Yoshii et al. (1999), 6Aita et al. (2011), 7Minagawa and Wada (1986)”.

these records must be more or less overprinted by theisotopic signatures of the detritus from other organisms.
Therefore, the isotopic compositions of chlorophylls areuseful for more accurately characterizing the isotopic
signatures of primary photosynthates. Chlorophylls areabundantly produced only by photoautotrophs and contain four nitrogen atoms. The biochemical processes ofchlorophyll synthesis in the photoautotrophic cell havebeen studied intensively (e.g., Beale 1995). This strong
research background is an advantage when interpreting
the isotopic signatures of chlorophylls (Katase and Wada
1990; Ohkouchi et al. 2006, 2008), although the number
of applications of this technique is still limited.
This approach is particularly useful for understanding
complex ecosystems. Lake Kaiike is a meromictic lake
located on the coast of Kamikoshiki Island, southwest
Japan. Because of its locality, seawater leaks into the deeper part of the lake through a gravel bar. In contrast, thesurface water of the lake is covered by freshwater flowing from the watershed. Therefore, the lake is permanently stratified with a strong density gradient, generating
an anaerobic water body below the chemocline at a
depth of around 5 m. Figure 6 illustrates both the carbon and nitrogen isotopic compositions of three types ofchloropigments isolated from the water column of thelake, chlorophyll a, bacteriochlorophyll a, and bacteriochlorophylls e (Nakajima et al. 2004). In this lake, thesechloropigments were derived from cyanobacteria, purple
sulfur bacteria, and brown-colored green sulfur bacteria,
respectively. The three types of chloropigments haveunique isotopic signatures, attributable to the distinctive
ecologies and physiologies of these photoautotrophs. At
the redox boundary of the water column, bacteriochlorophylls e are nearly 10‰ depleted in 13C relative to bacteriochlorophyll a, even though both the green and purple
sulfur bacteria assimilate CO2 (Figure 6a; Ohkouchi et al.
2005). This large variation in 13C is ascribed to the different assimilation pathways used by these photoautotrophs
in synthesizing these pigments: purple sulfur bacteria fix
CO2 via the C3 carboxylation pathway catalyzed by
RubisCO, whereas green sulfur bacteria use the reverse
tricarboxylic acid cycle catalyzed by α-ketoglutarate synthase. The latter process fractionates 13C far less than theformer process (Sirevåg et al. 1977; Quandt et al. 1977).
Bacteriochlorophylls e are far more depleted in 15N relative to either chlorophyll a or bacteriochlorophyll a(Figure 6b). This suggests that the green sulfur bacteria either fix N2 or grow very slowly by assimilating ammonium. In the aquatic environment where various processes
occur simultaneously, compound-specific isotope analysisOhkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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Figure 6 Carbon and nitrogen isotopic compositions of three types of chloropigments from Lake Kaiike, Japan. Carbon (a) and nitrogen(b) isotopic compositions of chloropigments (i.e., chlorophyll a, bacteriochlorophyll a, and bacteriochlorophylls e) from the water column ofmeromictic Lake Kaiike, Japan. The water column of the lake is strongly stratified with a chemocline at a depth of around 5 m. Below thechemocline, oxygen is depleted but hydrogen sulfide is abundant. The isotopic compositions of bacteriochlorophylls e are the mean values for
those of bacteriochlorophylls e1, e2, and e3. Redrawn from Ohkouchi et al. (2005).

is a useful tool for disentangling multiple coexisting signals. However, it must be kept in mind that isotopic discrimination occurs during chlorophyll biosynthesis, which
produces substantial isotopic differences between thechlorophylls and the whole algal cells (chlorophylls are approximately 0.5‰ depleted in 13C and approximately 5‰
depleted in 15N relative to the cell; Hayes et al. 1989; Sachs
et al. 1999; Ohkouchi et al. 2006). Nevertheless, the interpretation of the isotopic records of complex ecosystems
can advance by separating processes, which is made possible by compound-specific isotope analyses.
Food chain analysis with bulk isotopic compositions:isotopic changes during catabolism

Once produced by autotrophs, organic matter is utilized
by heterotrophs as an energy source. The heterotrophic
processes generally involve breaking down organic matter to smaller compounds. In these catabolic processes,
12
C and 14N tend to be preferentially metabolized, sothat the body tissues of higher animals including
muscle are generally enriched in 13C and 15N relative
to their diets. These isotopic discriminations provide
a useful approach for estimating prey-predator
relationships.
During the last several decades, the analysis of grazing
food chains has been a topic greatly advanced by the
analysis of stable carbon and nitrogen isotopes (e.g.,Peterson and Fry 1987; Wada and Yoshioka 1995;
Tayasu et al. 1997; Fry 2006). As theoretically expected,
diet-controlled experiments have clearly demonstrated
that a predator is markedly enriched in both 13C and 15N
relative to its prey (DeNiro and Epstein 1978, 1981;
Minagawa and Wada 1984). The principles are generalized
in the following equations:δ 13 CTP¼n ¼13 Δðn– 1Þ þ δ 13 CTP¼1

ð13Þ

δ 15 NTP¼n ¼15 Δðn– 1Þ þ δ 15 NTP¼1

ð14Þ

where TP represents the trophic position, and TP = 1, 2,
and 3 correspond to the trophic positions of plants, i.e.,
autotrophs, herbivores, and primary carnivores, respectively. The terms 13Δ and 15Δ are the magnitudes of thetrophic enrichment for 13C and 15N, respectively. A numberof studies have demonstrated that 13Δ generally ranges
from 0‰ to 1.5‰ with a mean value of 0.8‰, whereas 15Δranges from 0.5‰ and 5.5‰ with a mean value of 3.4‰
(McConnaughey and McRoy 1979; Fry and Sherr 1984;
Rau et al. 1983; Post 2002; Vander Zanden and Rasmussen
2001). Empirically, the 13Δ value tends to be smaller in lakes
and coastal oceans than in the pelagic ocean (Fry and Sherr
1984; Post 2002; Wada et al. 1987, 1993).
The diversity in the isotopic compositions of autotrophs is integrated and averaged by herbivorous andcarnivorous processes. Most heterotrophs forage overwide areas and depths within varying environments. For
example, zooplankton such as the copepod is known tomigrate by day and night in the photic zone to feed. This
migration of heterotrophs must homogenize the isotopic
signatures in time and space.
Why and how do predators become enriched in heavy
isotopes? Because amino acids in the form of proteins
are the major analytes investigated in food web studies,
they are logically the key compounds with which to answer this question. Among the processes that catabolizeamino acids, enzymatic decarboxylation and deamination potentially induce the isotopic fractionation of carbon and nitrogen, respectively (O'Leary 1977; Abell andO'Leary 1988). These enzymatic reactions are multistep

Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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Figure 7 Chemical schemes of the biologically mediated (a) decarboxylation and (b) deamination of amino acids.

processes (Figure 7). In both enzymatic decarboxylationand deamination, the amino acid is first bound to the
enzyme pyridoxal phosphate (PLP, a derivative of vitamin B6) to form amino acid-PLP (Figure 7). Simply put,
after the substrate combines with the enzyme, CO2 or
NH3 drops off. The first step (formation of the aminoacid-enzyme complex) is reversible, whereas the second
step is irreversible. Therefore, the magnitude of the isotopic fractionations associated with enzymatic decarboxylation and deamination is dynamically controlled.
The carbon isotopic fractionation that occurs during thein vitro decarboxylation of glutamic acid varies between
1.014 and 1.022 (at 37°C, 3.6 ≤ pH ≤ 5.5; O'Leary 1977). The
small apparent carbon isotopic fractionation associatedwith decarboxylation in vivo (0.8‰ on average) suggeststhat the first reversible step (e.g., the supply rate of aminoacids) may be a major limiting factor in this process.
The in vivo isotopic fractionation associated with the deamination of amino acids has been investigated more extensively than the isotopic fractionation associated with theirdecarboxylation (e.g., Chikaraishi et al. 2007). During thesemetabolic processes, most amino acids display strong (2.1‰
to 8.0‰) 15N-enrichment. In contrast, phenylalanine andmethionine display little 15N-enrichment (Macko and Estep
1984; Chikaraishi et al. 2007). We have ascribed this difference in 15N-enrichment between the two groups of aminoacids to differences in their metabolic routes (Ohkouchi and
Takano 2014). In the first step of metabolism, most aminoacids are deaminated to form the corresponding keto acids.
However, neither phenylalanine nor methionine is deaminated; instead, phenylalanine forms tyrosine by hydrationand methionine forms S-adenosylmethionine by reactingwith ATP. This evidence and theoretical considerations confirm that deamination (cleavage of the
α-carbon-nitrogen bond) is responsible for the15
N-enrichment in bulk organisms along trophic steps.
The variation in 15Δ may also be partly ascribed to differences in the amino acid compositions of organisms.
Lake Baikal, located in southern Siberia, is one of theaquatic environments in which this method has been
most successfully applied (Yoshii et al. 1999; Wada
2009; Ogawa 1999). The structure of its grazing foodOhkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

chain has been extensively studied with field observations
and is suggested to be composed of five major ecologicalgroups: phytoplankton, mesozooplankton, macrozooplanktonic amphipods, fish, and seals. Because the ecological diversity is low and the dietary options for eachspecies are consequently limited, the food chain in thispelagic lake is clearly demonstrated in the δ13C-δ15N diagram shown in Figure 8 (modified from Yoshii et al.
1999). The δ13C values for these organisms are similar to
those in pelagic phytoplankton (−27‰ to −25‰), suggesting that neither coastal phytoplankton nor terrestrialorganic matter is an important carbon source (or energy
source) for the pelagic food web. From the phytoplankton
at the base of the food chain to the seals as the top
predator, these organisms show a clear trend in stepwise
15
N-enrichment, beautifully demonstrating the simple
prey-predator relationships in this ecosystem.
Another case study introduced here was undertaken in
Lake Biwa, the watershed of which has a human population
density much higher than that of Lake Baikal. We havebeen particularly interested in this industrialized and urbanized watershed because it displays significant eutrophication
as a consequence of elevated nutrient loading (e.g., Tezuka
1992; Nakanishi and Sekino 1996). Figure 9 summarizes
the isotopic compositions of various organisms collectedfrom Lake Biwa in the 1990s (Yamada et al. 1998). The
δ15N–δ13C diagram not only provides information about
the grazing food chain, but also a diagnosis of the effects ofhuman activity on the watershed. The nitrogen supplied
from the surrounding mountains to the lake (approximately
0‰) is substantially depleted in 15N relative to the phytoplankton (Wada and Hattori 1991; Wada et al. 1998),

Figure 8 A δ15N-δ13C diagram for various organisms belonging to
the pelagic ecosystem in Lake Baikal. Modified from Yoshii et al. (1999).

Page 10 of 17

suggesting the presence of either a source(s) or a process(es) that causes 15N enrichment. A heavy loading of domestic sewage substantially disturbed the natural nitrogen cycle
in both the lake and the watershed. In particular, it induced
denitrification, leading to the enrichment of 15N in the nitrogen pool of the lake (Miyajima 1994; Ogawa et al. 2001;
A. Makabe and K. Koba, personal communication). A recent microbiological study found abundant denitrifying
methane-oxidizing bacteria in the surface layer of the sediment, supporting this view (Kojima et al. 2012). Isotopic
analyses of sediment cores and formalin-fixed fish archives
revealed that the denitrification was enhanced from theearly 1960s to the 1980s, the period of severe nutrient loading in the lake (Ogawa et al. 2001, 2013).
The accumulation of such case studies over decades has
provided great insight into the isotopic signatures of carbon
and nitrogen. Except for lagoons and sea-grass meadows,
where no or little carbon isotopic fractionation has been
observed (i.e., 13Δ ≃ 0; Fry and Sherr 1984; Wada et al.
1993), linear relationships have often been observed between 13C- and 15N-enrichment factors. In the cases of
Lake Baikal and Lake Biwa shown in Figures 8 and 9, respectively, the slopes of the plots in the δ13C-δ15N diagrams
were calculated to be ca. 2.1 and 1.5, respectively, using
analysis of covariance (Wada et al. 2013). In many ecosystems, the magnitudes of 13C- and 15N-enrichment at each
trophic step are cross-linked with the following equations(Aita et al. 2011; Wada et al. 2013):
15

Terrestrial:
Marine:15

Δ=13 Δ ¼ 1:61  0:41

Δ=13 Δ ¼ 1:24  0:24

ð15Þ
ð16Þ

Such empirical relationships may be explained from akinetic perspective. The 15Δ/13Δ ratio will be constant if
the limiting steps in the intrinsic metabolic processes
have adapted to the external substrate conditions,whereas the ratio will vary if the balance between theavailable nitrogen and carbon varies. The relationships
between 15Δ and 13Δ can also be interpreted from aphysiological perspective. Primarily, the equations shouldreflect the well-regulated dynamics between enzymatic decarboxylation and deamination, the key processes involved
in isotopic enrichment (i.e., 15Δ and 13Δ) and shared by
the whole biota. In the cell, deamination mainly occurs inamino acids, whereas decarboxylation occurs not only inamino acids but in various carboxylic acids including citricacid and oxalic acid, which are intermediates of the citricacid cycle, and in fatty acids, the components of membranes and energy storage materials. The differences in
the compound groups subjected to deamination and decarboxylation potentially produces the variations observedin the balance between the 13C and 15N distributions
among heterotrophs.

Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

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Figure 9 A δ15N-δ13C diagram for the ecosystem in Lake Biwa. Both pelagic and littoral food chains are shown. The estimated isotopic rangeof Lake Biwa before substantial eutrophication (ca. 1960s to 1980s) is shown as the area surrounded by a broken line. POM, particulate organic
matter. Colors indicate the food web groups estimated by Yamada et al. (1998): green, primary producers; black, primary consumers; red,secondary consumers; blue, tertiary consumers. Modified from Yamada et al. (1998) and Wada (2009).

Food chain analysis based on the nitrogen isotopic
compositions of amino acids

The analysis of the nitrogen isotopic compositions of individual amino acids has recently emerged as a tool infood web studies. The nitrogen in an organism is predominantly contained in proteins (i.e., mixtures of aminoacids), and as a logical consequence, individual aminoacids can provide more accurate information on preypredator relationships than the bulk isotopic method described above.
Careful observation both in the field and in the laboratory has shown that predators are enriched in 15N tovarious extents in most amino acids including alanine,
valine, and glutamate (a mixture of glutamine and glutamic acid). In contrast, phenylalanine and methionine
from predators are only slightly enriched in 15N relative
to their prey (McClelland and Montoya 2002; Chikaraishi
et al. 2007). The former group of amino acids is often referred to as ‘trophic amino acids’ and the latter as ‘source
amino acids.’ Enzymatic deamination (i.e., cleavage of thecovalent α-carbon-nitrogen bond) to form the corresponding keto acid is the dominant metabolic route forthe trophic amino acids. This biochemical reaction is responsible for fractionating the isotopes of nitrogen bonded

to the α-carbon (Macko et al. 1986; Chikaraishi et al.
2007). In contrast, phenylalanine and methionine, whichdisplay little 15N-enrichment, are metabolized to tyrosine and S-adenosylmethionine, respectively, during
which the α-carbon-nitrogen bond is not cleaved. If the15
N-enrichment factors for most amino acids were reasonably constant among various organisms, it would indicate a well-controlled common reaction network interms of amino acid metabolism over a wide range of organisms. Because both phenylalanine and methionine areindispensable (essential) amino acids, the nitrogen isotopic
compositions of these amino acids in any heterotroph are
inherited from autotrophs, the ultimate nitrogen source inthe food chain. Consequently, the nitrogen isotopic difference between the source and the trophic amino acids is a
first-order function of the trophic position of a given organism in the food web. The balanced nature of aminoacids is in strong contrast to the nature of the secondary
metabolites (e.g., most lipids, alkaloids, etc.), whose production and degradation are mainly controlled by thephysical environment rather than by the physiology of thecell.
Diet-controlled culture experiments in various organisms have shown that the differences in the nitrogenOhkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

isotopic compositions of glutamic acid and phenylalanine
are most useful for the accurate quantification of trophic
position (Chikaraishi et al. 2009). Trophic position isexpressed with an equation in the following form:½T P  ¼ δ 15 NGlu –δ 15 NPhe þ β =7:6 þ 1

ð17Þ

where β denotes the isotopic difference between glutamic acid and phenylalanine in autotrophs ([TP] = 1).
The analysis of a number of plants has shown that themean value of β is −3.4 for the aquatic plants with a 1σ
error of 0.9 (Figure 10a; Chikaraishi et al. 2009). In terrestrial plants, the value of β was estimated to be 8.4 ±
1.6 (Figure 10b; Chikaraishi et al. 2011; Steffan et al.
2013). The difference in the β values of aquatic and terrestrial plants may be attributable to the differences intheir metabolism of phenylalanine. In terrestrial plants, asignificant fraction of phenylalanine is metabolized forthe synthesis of lignin phenol, making it a branching pointin the metabolic pathway (Ohkouchi and Takano 2014).
The advantage of amino acid isotopes over bulk isotopes
for reconstructing food chains is twofold. First, the isotopic
composition of the autotroph ([TP] = 1) is not required toestimate the trophic position. Second, the error in the estimated trophic position can be minimized.
With this approach, we reexamined the pelagic ecosystem of Lake Baikal (Figure 11). The results confirmed
not only the simple food chain but also revealed new aspects of it. The estimated trophic position of the zooplankton Episula baikalensis collected in different years

Page 12 of 17

was distributed in a narrow range from 1.9 to 2.1, confirming it as an herbivore rather than an omnivore. The
seals were in the highest trophic position with an average value of 5.1. These results basically confirm those of
the bulk isotope data described in the previous “Foodchain analysis with bulk isotopic compositions: isotopic
changes during catabolism” section (Figure 8). However,
an important aspect is newly revealed. The trophic
length of this food web estimated with the amino acid
method is approximately 1 unit longer than that estimated with the bulk method (Yoshii et al. 1999). Assuming that the amino acid method provides the correct
information, the 15Δ value in Lake Baikal was recalculated to be approximately 2.5‰ rather than 3.3‰. The
mean trophic position of the endemic fish omul (Coregonus migratorius) was 3.6 rather than 3.0, suggesting thatit feeds not only on herbivorous zooplankton but also on
carnivores or omnivores. Sculpin, whose mean trophic
position was 4.3, may be the bridge between the omul
(and probably other fish species with similar trophic positions) and the seal. This view is consistent with thebulk isotope results, which showed approximately 3‰
enrichment in 15N for the adult sculpin relative to theomul (Figure 8). However, these conclusions are inconsistent with the microscopic observation of the sculpin's
stomach contents, which included few fish fragments(Miyasaka et al. 2006). A confident solution has yet tobe reached, but the ‘snapshot effect’ of the stomach contents might explain the inconsistency among the othertwo approaches.

Figure 10 A schematic diagram showing the relationship between trophic level and δ15N values of amino acids. Relationships between
trophic positions and nitrogen isotopic compositions of phenylalanine and glutamate in (a) aquatic and (b) terrestrial C3 and C4 plants. Verticalaxis indicates the relative δ15N values of the amino acids. The mathematical formula for these relationships are given in the text as equation 17.

Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

Page 13 of 17

et al. 2004; Choy and Richards 2009; McMahon et al.
2011; Larsen et al. 2013). This perspective includes not
only the sources of diets (e.g., terrestrial vs. aquatic, C3
plants vs. C4 plants), but also the dynamics of aminoacids in the cell and during diagenesis.
Isotopic variations in animal organs and thecompartmentalization effectFigure 11 A δ15NPhe-δ15NGlu diagram for various organisms
belonging to the pelagic ecosystem in Lake Baikal. The brokenlines indicate the isopleths of trophic positions 1 to 5 based on theformula described in Equation 17 and Figure 10a.

The amino acid method has been used to analyze not
only ecological samples but also samples from related
fields including anthropology, paleontology, and oceanography to investigate issues related to diet (e.g., Naitoet al. 2010; Kashiyama et al. 2010; Popp et al. 2007;
Miller et al. 2012; Ohkouchi et al. 2013). The nitrogen in
the amino group of amino acids forms the peptide backbone (–C–N–C–C–N–C–)n, so it is not readily exchangeable with ambient nitrogen (e.g., NH4+) unless it ishydrolyzed (Ohkouchi and Takano 2014). Consequently,
the nitrogen isotopic signatures of the α-amino groups ofamino acids are potentially preserved for a long time.
Bone collagen and proteins encrusted in carbonate shell
are the two major examples of situations in which thispreservation occurs. Analysis of the bone collagen ofancient humans (approximately 5,000 years BP) from several archaeological sites in Japan has clearly demonstrated
that this method is useful for reconstructing the diets of
our ancestors (Naito et al. 2010, 2013). However, it must
be kept in mind that the amino acid method provides
information only on the protein source and not on the
total diet. The carbon isotopic compositions of individual amino acids potentially offer a different perspective
on food chains and more insight into 13Δ values (e.g.,
Macko et al. 1987; Minagawa et al. 1992; McCarthy

Higher animals are compartmentalized systems in which
numbers of organs work together systematically to perform metabolic activities. Not surprisingly, individual organs and tissues from a single organism have distinctive
isotopic signatures (DeNiro and Epstein 1978, 1981;
Mizutani et al. 1991). In higher animals such as humans,
the isotopic composition of the hair or nails has provenuseful in dietary analyses e.g., (Hare et al. 1991; O'Connell
et al. 2001; Petzke et al. 2005). Here, we summarize theavailable information on the distinctive isotopic signaturesof individual organs and tissues from a single organism.
The isotopic compositions of the individual organs andtissues of the salmon, globefish, cormorant, Mongolian
sheep, rat, and three primates are illustrated in Figure 12
as deviations from those of muscle. In animals such as therat, salmon, globefish, and Japanese macaque, the brain isenriched in 15N and depleted in 13C relative to most otherorgans and tissues (DeNiro and Epstein 1981). In contrast,
the cormorant brain is substantially depleted in 15N(Mizutani et al. 1991). However, this dataset is still limited,and some organs appear to show isotopic signatures inconsistent with those of similar organisms. For example,
the liver of the salmon displays a similar δ15N value to thatof muscle, whereas the livers of the globefish and rainbow
trout are approximately 1‰ depleted in 15N relative totheir muscle (Pinneger and Polunin 1999). Isotopic variations of a few permil among organs may be explained by
the differences in the turnover rates of proteins. As explained in the last section, the carbon and nitrogen isotopic compositions vary quite markedly, even among the
20 essential amino acids. In an organism with [TP] = 4, forexample, glutamic acid is over 25‰ enriched in 15N relative to phenylalanine. The heterogeneous distribution ofamino acids with distinctive isotopic compositions may
partly explain the isotopic heterogeneity among organs.
In the guinea pig, the turnover times for carbon andnitrogen in the liver are several months because of theregular mitosis of hepatocytes, whereas the turnover ofcarbon and nitrogen is much slower in the cerebellum
than in the liver. The isotopic signatures of cerebellar
DNA acquired in utero are retained for at least the first
6 months of life (Strable et al. 2011). It has also been
demonstrated that the neuronal DNA in the humancerebellum turns over little after maturity (Slatkin et al.
1985). This slowness or absence of protein turnover inthe brain could explain why its isotopic signatures differOhkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

Page 14 of 17

Figure 12 Carbon and nitrogen isotopic compositions of individual organs from various animal samples. These are shown as differences
relative to the isotopic composition of muscle (i.e., Δ13C = δ13Ctissue − δ13Cmuscle, and Δ15N = δ15Ntissue − δ15Nmuscle). The letters next to the symbols areabbreviations of the organs or tissues. All institutional and national guidelines for the care and use of laboratory animals were followed.

from those of other organs with normal cellular turnover. If tissues are not metabolized, their isotopic signatures should be fixed at the point of production orequilibrated with the chemical substances around them.
It is still too early to draw a clear picture, but thecompartmentalization effect complicates the isotopic
signatures of higher animals. Such isotopic variations
potentially confound the interpretation of isotopic signatures, and caution is required when construing them, especially when the bulk isotopic method is applied tohigher animals.

Conclusions
Here, we have reviewed the rules for and the theoreticalbackground of the isotopic approach to environmental
and ecological problems and have described the principles regulating the distribution of isotopes in the biosphere, which is inhabited by millions of species.
Although we still have limited evidence, we stress that
this approach relies strongly on the biochemical andphysiological processes common to the whole biota.
Even during biochemical reactions, isotopic fractionation
originates solely from the physicochemical properties ofmolecules, the differences in molecular motion. Generalities can be drawn from comprehensive enzymatic processes with equivalent thermodynamic properties in the
animal community. Therefore, a deep understanding of
the isotopic fractionation that occurs through metabolic
pathways is crucial to extend our knowledge of thephysiological ecology of animals. Compound-specificisotopic analysis is an effective tool for this purpose.
In this review, we have focused on the grazing foodchain, but clarifying the detritus food chain, the otherside of the energy flow in ecosystems is essential for understanding the surface environment of this planet. Fragmentary evidence has suggested that compound-specificisotopic analysis is also effective in analyzing the detritus
food chain (Macko and Estep 1984; Kohzu et al. 1999;
Yamaguchi, 2012; Maki et al. 2014). If true, this approach will demonstrate our ‘isotopically ordered world’even more vividly.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NO and EW proposed the topic and conceived and designed the study.
NOO, YC, and HT carried out the experimental study. All authors have readand approved the final manuscript.
Acknowledgements
We thank our many colleagues and collaborators who have supported our
research for many years. They include the scientists at the Mitsubishi-Kasei
Institute of Life Sciences, students and colleagues at the Center for EcologicalResearch, Kyoto University, and the members of the Department of
Biogeochemistry, JAMSTEC. We are especially grateful to Y. Takano, I. Tayasu,
T. Nakano, T. Nagata, A. Kohzu, K. Koba, N. Ohte, F. Hyodo, N. Ishikawa,Y. Yamada, S. Steffan, Y. Naito, and C. Yoshikawa for their informal discussionsof this topic, Y. Kudo for supplying rat organs and for discussions, and
Y. Kabaya, H. Suga, and N. Nishizawa for supporting the laboratory work. We
also thank two anonymous reviewers for their comments. This work waspartly conducted as a cooperative research program of the Primate ResearchInstitute, Kyoto University, and the Japan Monkey Centre.

Ohkouchi et al. Progress in Earth and Planetary Science (2015) 2:1

Author details
1
Japan Agency for Marine-Earth Science and Technology, 2-15
Natsushima-cho, Yokosuka 237-0061, Japan. 2Primate Research Institute,
Kyoto University, Inuyama 484-8506, Japan. 3National Research Institute ofFisheries Science, 2-12-4 Fukuura Kanazawa-ku, Yokohama 236-8648, Japan.
Received: 15 October 2014 Accepted: 7 January 2015

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