Plankton Benthos Res 7(3): 135–145, 2012
Plankton & Benthos
Research
© The Plankton Society of Japan
Ecological aspects of carbon and nitrogen isotope ratios
of cyanobacteria
EITARO WADA1,*, K AORI OHKI2, SHINYA YOSHIKAWA2, PATRICK L. PARKER3, CHASE VAN BAALEN3,
GENKI I. MATSUMOTO4, MAKI NOGUCHI AITA1 & TOSHIRO SAINO1
1
Environmental Biogeochemical Cycle Research Program, Research Institute for Global Change, Japan Agency for MarineEarth Science and Technology (JAMSTEC), 3173–25 Showa-machi, Kanazawa-ku, Yokohama, Kanagawa 236–0001, Japan
2
Department of Marine Bioscience, Fukui Prefectural University, 1–1 Gakuenncho, Obama, Fukui 917–0003, Japan
3
Marine Science Institute, The University of Texas, 750 Channel View Drive Port Aransas, TX 78373, U.S.A.
4
Department of Environmental Studies, School of Social Information Studies, Otsuma Women s University, 2–7–1 Karakida,
Tama, Tokyo 206–8540, Japan
Received 5 January 2012; Accepted 28 May 2012
Abstract: Variation in the δ13C of a cultured marine cyanobacterium (Agmenellum quadruplicatum, strain PR-6) is
described with an emphasis on the relationships between growth rate, pCO2, and δ13C. An average nitrogen isotope
fractionation of 1.0017 was obtained during N2 fixation by cultured marine cyanobacteria. This result suggests that
nitrogen supply via N2 fixation may be characterized by a low δ15N, down to ca. −2‰ in marine environments, where
the average δ15N is higher (at ca. 6‰) for major inorganic nitrogenous compounds such as nitrate. The natural abundances of nitrogen and carbon isotope ratios for cyanobacteria collected from the McMurdo Dry Valleys, Antarctica,
were characterized by extremely low δ15N and widely ranging δ13C. Summarizing these and other data obtained, an
isotopic map of cyanobacteria and other plankton in aquatic ecosystems was constructed and the ecological implications are discussed. Our results suggest that the δ13C of marine phytoplankton is positively correlated with sea surface temperature and can be a useful parameter for estimating in situ growth rates in the open ocean.
Key words: Antarctica, C and N isotope ratios, cyanobacteria, marine plankton, N2 fixation
Introduction
Stable isotope studies can be useful for understanding
nutrient cycling, as well as the physiological nature of microorganisms in aquatic ecosystems. In photosynthetic organisms, carbon isotope fractionation is generally recognized to occur during photosynthetic carbon fixation. In
his comprehensive review, O Leary (1981) demonstrated
that the carbon isotope effect in the C3 pathway is large
during the carboxylation step. However, the diffusion of
CO2 into leaves is partially rate-limiting and this serves to
reduce in vivo fractionation, suggesting a possible correlation between the carbon isotope effect and the growth
physiology of marine phytoplankton. The CO2 content of
supplied gas has a major effect on the δ13C of microalgae
(Mizutani & Wada 1982). δ13C in marine plankton is
thought to range from −9 to −30‰ (Wada & Hattori
* Corresponding author: Eitaro Wada; E-mail, eitaro1939@yahoo.co.jp
1991), depending on temperature, pH (which affects the
HCO3–CO2 isotope exchange equilibrium), and carbon
availability. Based on latitudinal, hemispheric, and polar
discrepancies in plankton δ13C, Rau et al. (1982) and Goericke & Fry (1994) emphasized that geographic differences
in kinetic isotope effects associated with plankton biosynthesis and metabolism were important factors causing latitudinal variation in plankton carbon isotope abundance.
In a continuous culture of Chlamydomonas reinhardtii,
Takahashi et al. (1991) found a linear relationship between
the growth rate constant and the carbon isotope fractionation factor under light- and nitrogen-limited conditions.
The same trend was also reported for nitrate-uptake processes in the marine diatom Phaeodactylum tricornutum
(Wada & Hattori 1978). Because information on the
growth physiology of phytoplankton and epibenthic algae
can likely be obtained by measuring their C and N stable
isotope ratios, Wada & Hattori (1991) created a δ15N–δ13C
map of marine phytoplankton.
136
E. Wada et al.
In the open ocean, the nitrogen isotope ratios of plankton are closely correlated with the chemical forms of inorganic nitrogen used for primary production. The level of
δ15N in Trichodesmium sp. is low compared to those of
plankton commonly found in other areas, supporting the
idea that nitrogen is supplied to this community via molecular nitrogen fixation (Minagawa & Wada 1986, Saino &
Hattori 1987, Carpenter et al. 1997). The aerobic nitrogen
fixation ability of Trichodesmium has been confirmed
using isolated strains (Ohki et al. 1986, 1988, Chen et al.
1996, Ohki 2008, Finzi-Hart et al. 2009). However, nitrogen fixation sometimes occurs at low rates relative to the
total nitrogen budget (Ohki et al. 1991, Capone et al. 1997).
Unicellular diazotrophic cyanobacteria contribute significantly to nitrogen cycling in the ocean (Zehr et al. 2001),
although δ15N data for these unicellular species are not yet
available.
Cyanobacteria are distributed across a wide range of
physicochemical conditions, such as pH, redox potential,
and temperature, and play important roles as primary producers in a variety of natural ecosystems. In the McMurdo
Dry Valleys, Victoria Land, Antarctica, there are a variety
of saline lakes and ponds that host undescribed types of
cyanobacteria with the lowest known δ15N values in the
biosphere. In the saline lakes and ponds the cyanobacteria
use nitrate supplied via precipitation. Because of the very
low humidity in the McMurdo Dry Valleys, the precipitated nitrate is present as a powdered crystal of NaNO3,
which is the nitrogen source used for growth by the cyanobacteria in the saline lakes and ponds in this area. The
δ15N of sodium nitrate (NaNO3), which can be as low as
−20‰, and nitrogen isotope fractionation during nitrate
uptake are possible causes of these low δ15N values (Wada
et al. 1981, Matsumoto et al. 1987, Matsumoto et al. 1993).
Hage et al. (2007) isotopically characterized coastal pondderived organic matter in the McMurdo Dry Valleys. Recently, Hopkins et al. (2009) reported isotopic evidence for
the provenance and turnover of organic carbon by soil microorganisms in the McMurdo Dry Valleys.
In this study, we investigated the effects of cyanobacterial growth rate on carbon isotope fractionation in a marine cyanobacterium (Agmenellum quadruplicatum; PR-6).
Marine diazotrophs were also investigated to elucidate the
role of nitrogen isotope fractionation in molecular nitrogen
fixation by various kinds of marine cyanobacteria. Cyanobacteria collected from saline lakes and ponds in the McMurdo Dry Valleys, Antarctica, were also studied in detail,
with an emphasis on cyanobacterial growth physiology.
Finally, we summarized the characteristics of cyanobacteria on a δ15N vs. δ13C map and discuss some ecological implications of algal isotope ratios based on our data in combination with previously reported data (Minagawa & Wada
1986, Wada & Hattori 1991, Yoshioka et al. 1994, Aita et
al. 2011).
Materials and Methods
Cultures
Agmenellum quadruplicatum, strain PR-6 (Synechococus
sp. ATCC27264/PCC7002), was kept in the laboratory of
one of the authors (Van Baalen) in an axenic form. The organism was grown in modified ASP-2 medium (Van
Baalen, 1962) in test tubes (100 ml) or Erlenmeyer flasks
(1 L) with continuous bubbling of 1% CO2 (δ13C PDB=
−36.64‰) and illuminated with fluorescent lamps 10 cm
from the growth apparatus (50–60 μmol photons m−2 sec−1).
To control the growth rate, light intensity was altered using
black mesh cloth to cover the test tubes. Growth of the liquid cultures was followed turbidimetrically and the specific growth rate constant (μ) was estimated using the
method of Kratz and Myers (1955), where μ=0.301 corresponds to a generation time of 1.00 d.
Cells for isotopic studies were harvested at the late logarithmic phase or early stationary phase for test tube cultures. In large-scale cultures, cells were harvested at intervals and collected on Whatman type-C glass fiber filters
preheated to 400°C for 5 h. Filters with residues were
treated with 0.1 N HCl and stored in a refrigerator until
mass spectrometric analysis.
Diazotrophs
Trichodesmium spp. NIBB1067 was isolated and cultured in a synthetic medium as described in Ohki & Fujita
(1982) and Ohki et al. (1986). Five unicellular and one filamentous marine cyanobacteria isolated from the sea
around Singapore (Ohki et al. 2008) and Crocosphaera
watsonii WH8501 (Waterbury and Pippka 1989) provided
by Drs. Waterbury and Dyhman of Woods Hole Oceanographic Institution, USA, were maintained in a liquid modified Aquil medium without combined nitrogen (Ohki et al.
1986). Two freshwater heterocystous cyanobacteria in sections IV and V (Nostoc sp. PCC7120 and Fischerella
sp1ATCC29114, respectively) were cultured in BG0 (Rippka & Herdman 1992), and used as references. Cultures
were kept under fluorescent lights (daylight type) at
10 μmol photons m−2 sec−1 (12 : 12 L : D) at 26°C (Ohki et
al. 2008). Several milligrams of cells were collected, centrifuged at 3,000×g for 4 min at 4°C, and lyophilized for
stable isotope measurements.
Cyanobacteria from the McMurdo Dry Valleys, Antarctica
Observations of the McMurdo Dry Valleys area during
the 1980–1981 field season were reported in detail by
Wada et al. (1984). Lake Vanda, several ponds in the Labyrinth, Lake Bonney, and other lakes were visited during
the season. Epibenthic cyanobacteria were collected from
unnamed saline ponds in the Labyrinth, near the terminus
of the Wright Upper Glacier, and from Lake Vanda and its
surroundings. Samples were also collected from the Taylor
137
C and N isotope ratios of cyanobacteria
Fig. 1. Locations of sampling sites in the McMurdo Dry Valleys of southern Victoria Land, Antarctica (revised from Matsumoto et al. 1990). Dotted areas are ice-free. South side of Lake Vanda (77°31′0″S, 161°40′0″E): V1, V2, V3, V4, V5, V6, V7, V8,
V9, V10, and V11. Surroundings of Lake Canopus (77°33′00.0″S, 161°31′00.0″E): C2 (dried cyanobacteria), C3 (wet cyanobacteria), C4 (wet cyanobacteria), C8 (wet cyanobacteria), and C9 (dried cyanobacteria). Don Juan Pond (77°33′55″S, 161°11′26″E):
D1 (dried cyanobacteria), D2 (dried cyanobacteria), and D3 (dried cyanobacteria). Unnamed ponds in the Labyrinth (77°33′S,
160°50′E): L11, L12, L13, L14, and L20. Lake Bonney (77°43′S, 162°22′E): B1 (cyanobacteria in the meltwater stream at the east
side of the lake), B2, and B3 (cyanobacteria in the meltwater stream from Hughes Glacier).
Valley (Fig. 1).
Samples were frozen and brought back to Japan. Preliminary results from these samples were reported by Wada et
al. (1981). Sterol distribution in the saline ponds can be
found in Matsumoto et al. (1982). The geochemical features of the McMurdo Dry Valley lakes and ponds were reviewed by Matsumoto (1993).
To identify cyanobacteria, small aliquots of freeze-dried
samples (5–50 mg) were taken, and DNA was extracted
using a FastDNA Spin kit (MP Biomedicals, Solen, OH,
USA). Most of the 16S rRNA gene fragments were amplified using cyanobacteria-specific primers as follows: 8F
(forward, Wilmotte et al. 1993) and PISTE-Cyano-R (reverse, CTCTGTGCCAAGGTATC, Harverkamp, unpublished data). The conditions for polymerase chain reaction
(PCR) were the same as those used in Ohki et al. (2008).
The PCR products were ligated into a pGEM-T vector
(Promega, WI, USA), and transformed into Escherichia
coli cells to construct clone libraries. Clones containing an
insert were selected and sequenced after the plasmid was
purified. Homology-based searches of the DNA Data Bank
of Japan (DDBJ) were performed using the BLAST program on the DDBJ website (http://www.ddbj.nig.ac.jp/
index-j.html).
Isotope analyses
The samples collected from Antarctica were analyzed
using an RMU-6R mass spectrometer (Hitachi) fitted with
a double collector for ratiometry. The cultured diazotrophs
were measured by Flash 2000, CoFloIV, Delta V (Thermo
Fisher Scientific) in SI Science Co., Ltd., Japan (Aita et al.
2011). The isotopic data are presented in terms of δ13C as
the per mil deviation relative to the PDB isotopic standard.
For δ15N, atmospheric nitrogen was used as a standard.
The precisions of δ13C and δ15N measurements were within
0.2‰.
d 13C or d 15 N (‰) = (Rsample / Rstandard －1) ×103
(1)
where R is 13C/12C or 15N/14N, respectively.
The δ13C values of PR-6 are reported relative to the CO2
used as the carbon source.
Carbon isotope discrimination (Δδ13C) is generally expressed as a difference in the δ13C value between source
and product (Takahashi et al. 1991):
∆d 13C =
δ13C (source)－δ13C (product)
1 +δ13C (source) /1000
(2)
Spontaneous discrimination factor at biomass Nt1 and Nt2
138
E. Wada et al.
correlation with growth rate; that is, the δ13C of the cells
decreased with decreasing growth rate. The other group,
shown within the dotted circle in Fig. 2, which had rather
low discrimination factors, were collected in the late logarithmic phase or early stationary phase. Under those conditions, cell numbers in the incubation tube were maximal,
meaning that high photosynthetic activity was rapidly consuming dissolved carbon dioxide.
Diazotrophs
Fig. 2. Summary of carbon isotope discrimination factor
(Δδ13C) during the growth of Agmenellum quadruplicatum. Cultures were constantly bubbled with 1.0% CO2 in air at 36.5°C.
FL-series were conducted using 1-L Erlenmeyer flasks. Instantaneous discrimination factor (Δδ13C) between time t1 and time t2
was calculated using a mass balance equation. TT-series were
cultured in a 100-mL test tube and harvested in the early stationary phase when algal cell density and photosynthetic activity
were high. The observed low Δδ13C in the FL-series might have
resulted from CO2 limitation with rather high growth and very
high algal cell density during the late logarithmic phase. Such
conditions were sometimes observed when bubbling of CO2 enriched air became insufficient during laboratory culturing.
in the flask cultures was estimated by the following isotope
mass balance equation:
δ13C Nt1 × f +δ13C NΔt (1－f ) = δ 13C Nt 2
(3)
where f=Nt1/Nt2 and Δt=t2−t1.
Equation (3) can be re-arranged to obtain δ13CNΔt, which
is the instantaneous discrimination factor during the interval between t1 and t2. This was done for the FL-1 and FL-2
incubation series in Fig. 2.
Results
Agmenellum quadruplicatum (PR-6)
Cyanobacterial cells with different growth rates under
different light intensities exhibited marked variation in
δ13C values (Fig. 2). The δ13C of cells ranged from −19.1 to
−10.7‰, relative to the CO2 used as the substrate. These
values correspond to discrimination factors of 19.8 and
11.1, respectively. The maximum discrimination factor was
observed in cells grown under low light intensities with
low growth rates. The variation in δ13C of the cells was divided into two groups: For one group there was a negative
The nitrogen isotope ratios (δ15N) of cultured cyanobacteria during N2 fixation ranged from −1.5 to −3.0‰ relative to atmospheric N2, with an average value of −1.74‰,
corresponding to a fractionation factor of 1.0017 (Table 1).
These values were constant regardless of algal species and
were comparable to those obtained for other N2-fixing microorganisms, including Azotobacter. Fractionation during
biological molecular nitrogen fixation was almost identical
among these organisms.
The partial rDNA sequence of cyanobacteria in the McMurdo Dry Valleys, Antarctica
Diazotrophic cyanobacteria closely related to the genera
Leptolyngbya, Oacillatoria, and Phormidium (N2-fixing
under anaerobic conditions, cf. Rippka & Herdman 1992)
were detected by partial sequencing of PCR-amplified 16S
rDNA using DNA extracted from the samples collected
from Antarctic saline ponds and lakes in the McMurdo
Dry Valleys.
Natural C and N isotope abundances of cyanobacteria
in McMurdo Dry Valleys
The natural C and N abundances of cyanobacteria collected from saline ponds and lakes in McMurdo Dry Valleys were precisely measured. δ15N values divided samples
into two groups with average values of −10 and −50‰,
respectively (Fig. 3 and Appendix). According to Wada et
al. (1981), the δ15N of soil nitrate in McMurdo Dry Valleys
is quite low, from −24 to −11‰. This can be considered a
result of the characteristic origins of nitrate associated
with atmospheric precipitation, including the production of
NOx by auroral activity. The former group (δ15N of −10‰)
reflects source nitrate δ15N, whereas the latter (low δ15N)
group probably arose under conditions of high nitrate concentration and low light intensity, with a significant occurrence of nitrogen isotope fractionation (up to 1.04) during
nitrate assimilation. In fact, the occurrence of a large nitrogen isotope fractionation was observed for several saline
ponds in the Labyrinth (Table 2). On one hand, for low
NO−
3 concentrations down to 1 μM, no nitrogen isotope
discrimination is expected during nitrate assimilation.
Consequently the δ15N of cyanobacteria in such ponds become close to the δ15N of nitrate (−20‰) used for growth.
However, the δ13C was highly variable, ranging from −25
to −3‰. High values resulted from the limitation of CO2
supply during growth. Algal mats are well known to show
139
C and N isotope ratios of cyanobacteria
Table 1. Nitrogen isotope ratios of N2-fixing cyanobacteria during N2 fixation.
δ15N (‰)
Organisms
−3.5
−1.9
−4.1
Azotobacter Azotobacter agile
Azotobacter chroococcum
Azotobacter vinelandii
Blue green
algae
Anabaenacylindrica
Trichodesmium spp.
Gloeothece. sp. 68DGA (unicellular)
Gloeothece. sp. 11DG (unicellular)
Gloeothece. sp. 30D1 (unicellular)
Gloeothece. sp. 20B5 (unicellular)
Gloeothece. sp. 38U6 (unicellular)
Crocosphaera watsonii WH8501 (unicellular)
Lyngbya sp. 10B (Filamentous)
Nostoc sp. PCC7120
Fischerella sp1ATCC29114
Trichodesmium spp.
−3.0
−0.6
−2.2
−1.3
−3.7
−1.5
−1.6
−1.5
−1.4
−1.8
−2.2
−1.7
−1.8
−2.0
−1.3 and −3.7
mean
Fig. 3.
−1.7
References
Hoering & Ford (1960)
ibid.
ibid.
Wada & Hattori (1974)
Minagawa & Wada (1986)
This work
Carpenter et al. (1997)
Carpenter et al. (1997)
strain: Ohki et al. (2008)
strain: Ohki et al. (2008)
strain: Ohki et al. (2008)
strain: Ohki et al. (2008)
strain: Ohki et al. (2008)
strain: Waterbury et al. (1989)
Reported by Carpenter et al. (1997)
Only for cyanobacteria except data by Carpenter et al. (1997)
δ15N–δ13C map for cyanobacteria in the McMurdo Dry Valleys, Antarctica. For details, see Fig. 1 and Appendix.
F1
F2
→ CO 2 
CO 2 ←
→ Organic-C
F3
such high δ13C values.
Discussion
Theoretical consideration of steady-state C/N kinetic
isotope effects
Isotope fractionation during photosynthetic CO2 fixation
and nitrate assimilation can be described in the following
way (Farquhar et al. 1989, Takahashi et al. 1991).
The first step is CO2 transport into the cell through bidirectional passive diffusion (F1 and F3), and the next is assimilation (F2) into organic carbon by ribulose bisphosphate carboxylase (RuBPCase).
Under the steady state condition during photosynthetic
carbon fixation, we obtain
∆δ 13C = b + (a －b) × F2/ F1
(4)
140
E. Wada et al.
Table 2. Nitrogen isotope fractionation during cyanobacterial growth in saline ponds of the Labyrinth in the McMurdo Dry Valleys,
Antarctica. Nitrogen isotope fractionation factors up to 1.04 were observed during nitrate-uptake processes under high nitrate concentrations.
Pond
NH4+−N
(mgN L−1)
L-11
L-12
L-13
L-14
S-1 Valley
4.5
10.3
6.6
8.3
14.2
+
NO−
3 +NH4
−1
(mgN L )
455
1,370
1,290
1,370
14.2
δ15N (NH4+)
(‰)
δ15N (NO−
3)
(‰)
δ15N (cyanobacteria)
(‰)
δ13C (cyanobacteria)
(‰)
−22.4
−21.2
n.d.
−8.0
+2.0
−11.6
−10.0
−1.6
−5.6
−13.2
−50.0
−51.4
−52.7
−63.4
−19.8
−4.55
−6.55
−7.76
−13.30
−1.50
where a and b are the discrimination factors (DF) associated with the processes of CO2 diffusion (F1) and carboxylation reaction (F2), respectively (Takahashi et al. 1991).
For this simple model, we can obtain DF of 4.4 and 30 for
the diffusion process and carboxylation reaction, respectively. Then we obtain
Δδ 13C = 30 + (4.4−30) × F2/ F1
(5)
Here we predict that the quotient F2/F1 in the above equation is proportional to μ, because F1 seems to be independent of μ and kept constant under constant ambient pCO2.
Thus, a constant F1 flux implies a negative relationship
between Δδ13C and either F2 or μ. In fact, during algal
photosynthesis, ΔD has a negative relationship with the
growth rate constant μ (Takahashi et al. 1991). The following equation was obtained by Takahashi et al. (1991) for
Chlamydomonas reinhardtii Dangeard (IAM C-238):
∆δ 13C = －35.4 µ + 25.3 (r =
－0.92)
(6)
The nitrogen isotopic fractionation factor is inversely correlated with the growth rate constant in the assimilation of
nitrate by the marine diatom Phaeodactylum tricornutum.
The nitrogen isotope fractionation factor is 1.001 at high
growth rates of P. tricornutum, and goes up to 1.023 under
light-limited conditions (Wada & Hattori 1978). In view of
these facts, variation in cellular δ13C was examined for the
cyanobacterium PR-6.
Agmenellum quadruplicatum (PR-6)
The enzymatic carboxylation reaction is the primary
factor contributing to carbon isotope fractionation during
photosynthesis (Christeller et al. 1976, Estep et al. 1978).
The temperature-dependent isotope exchange equilibrium
between CO2 and bicarbonate, pCO2, CO2 diffusion, and
diffusion/carboxylation partitioning (F2/F1 in the eq. (5))
relating to the growth physiology of organisms are also
principal components governing the magnitude of carbon
isotope fractionation (O Leary 1981). Of these factors, the
growth physiological condition is most likely to cause fluctuation or deviation in the 13C content of phytoplankton in
the surface waters of the ocean. The variation in δ13C in
PR-6 in the present study also suggests that carbon isotope
fractionation is regulated by the kinetics of photosynthetic
carbon fixation pathways. The differential or instantaneous
carbon isotope discrimination factor decreased with an increase in its growth rate, similar to Chlamydomonas reinhardtii Dangeard (IAM C-238) described above. We can
conclude that the δ13C content of PR-6 decreases with increasing growth rate, as indicated by the shaded broad line
in Fig. 2. In contrast, the low Δδ13C for the data within the
circle in Fig. 2 strongly suggests that the effect of CO2 limitation on algal photosynthetic activity lowered the apparent fractionation factor at the rather high growth rates of
dense populations within the small incubation tubes. In
summary, we conclude that algal δ13C is governed by two
major factors: growth rate and the supply of CO2 as estimated by the above eq. (5).
Diazotrophs
Molecular nitrogen has a very strong triple bond between nitrogen atoms. Theoretically, the cleavage of nitrogen atoms under a reversible reaction results in a very
large nitrogen isotope fractionation. However, the observed fractionation was small for biological molecular nitrogen fixation, ca. 1.002. Such a small nitrogen isotope
fractionation factor strongly suggests that triple-bond
cleavage takes place suddenly under irreversible conditions
between substrate N2 and an activated substrate. Judging
from the present results and from data in the literature, we
conclude that isotope fractionation during biological molecular nitrogen fixation is 1.002 in general. This means
that the δ15N supplied via molecular nitrogen fixation is ca.
−2‰, a fairly low value in marine environments and
eutrophic lakes, where the N2-fixing process is easily distinguished by the nitrogen isotope ratio and where N2-fixing cyanobacteria exhibit low δ15N and high δ13C. These
results support the findings of other studies that have demonstrated the significance of molecular nitrogen fixation in
the western North Pacific Ocean (Saino & Hattori 1987,
Wada & Hattori 1991).
Epibenthic cyanobacteria from McMurdo Dry Valleys
The carbon and nitrogen isotope ratios of epibenthic cyanobacteria can be explained by the kinetic isotope effects
C and N isotope ratios of cyanobacteria
described above during cyanobacterial growth in McMurdo Dry Valleys. The nitrogen and carbon isotope ratios
of microalgae closely depend on the isotope ratios of the
substrate used for their growth as well as on their uptake
kinetics.
Under substrate-limiting conditions, isotope fractionation factors become close to 1.000 because the diffusion
of the substrate into algal cells is the rate-limiting step during the assimilation of inorganic nitrogen, whereas large
fractionation factors can occur when enzymatic processes
limit the overall reaction under low-light conditions with
slow growth rates. As reported by Wada et al. (1984), the
δ15N of sodium nitrate in the McMurdo Dry Valleys is as
low as −20‰. Species of cyanobacteria found in the
McMurdo Dry Valleys by Matsumoto et al. (1993, 1996)
included Lyngbya murrayi, Oscillatoria priestley, and
Phormidium priestley. According to Matsumoto, who participated in field surveys in this area from 1976 through
1985, our samples are consistent with these organisms (GI
Matsumoto, pers. comm.). The results of our 16S rDNA sequence analysis support the observations of Matsumoto et
al. (1993, 1996).
In the present work, we detected two algal groups based
on nitrogen isotope ratios, i.e., −20‰ and −80 to −50‰
(Fig. 3). The former group might grow under substratelimited conditions caused by low nitrate concentrations
and/or the aggregation of algal mats at the bottom of saline
ponds. However, very low δ15N values could also occur
when algal growth is limited by light intensity under high
nitrate concentrations, up to 1,000 mg nitrate-N L−1. In
such cases, we expect the occurrence of a high nitrogen
isotope fractionation factor (up to ca. 1.04) during nitrate
assimilation processes. In McMurdo Dry Valleys, there are
several saline ponds with the conditions listed in Table 2.
Relationship between the δ13C of marine plankton and
sea surface temperature (SST)
The reddish brown Trichodesmium erythraeum
(Marumo & Asaoka 1974) has the highest δ13C among the
pelagic plankton samples documented in the literature
(Fig. 4). Nitrogen supply via molecular nitrogen fixation is
responsible for the low δ15N values of Trichodesmium
(Wada 1980, Minagawa & Wada 1986). Therefore, Trichodesmium holds a unique position among marine phytoplankton with regard to its carbon and nitrogen isotopic
composition (Fig. 4), as described by Carpenter et al.
(1997).
The relationship between δ13C and growth rate constant
was examined, based on all marine phytoplankton δ13C
data collected from the Pacific Coast off Japan (Fig. 4).
Generally, phytoplankton δ13C depends on pCO2, growth
rate, SST, nutrient concentration, and the cell geometry of
each species (Popp et al. 1998). According to Eppley
(1972), seawater temperature and nutrient concentrations
are two major factors affecting phytoplankton growth. By
accounting for the combined effects of temperature and
141
Fig. 4. Relationship between the δ13C of total organic carbon
in pelagic plankton and surface water temperature (SST). Data
sources: Wada and Hattori (1991), Aita et al. (2011). Unpublished
data collected from the Pacific Coast of Japan are included in the
figure by their sampling location name.
concentration on nitrate uptake as measured in shipboard
experiments, Smith (2010) found evidence of greater temperature sensitivity than that reported by Eppley (1972) for
laboratory measurements of growth rate. The lowest δ13C
values (ca. −30‰) were those of plankton from high-latitude areas (Rau et al. 1982, Goericke & Fry 1994). We
could thus expect to see a positive relationship between the
growth rate constant and either δ13C in phytoplankton
(POC) or SST. In fact, we obtained a linear relationship for
phytoplankton: δ13C=0.25 SST−26.2 (r2=0.59), as indicated in Fig. 4. This linear relationship observed in the
western North Pacific off Japan is quite similar to that reported by Goericke and Fry (1994), whose linear regression of δ13CPOC on SST was δ13CPOC=0.17 [±0.04]×SST−
24.6‰ (r=0.72, >5°C in the northern hemisphere).
Therefore, phytoplankton δ13C is a possible parameter
for deducing in situ growth rate constants in the marine
environment. Thus, the variability in δ13C content among
various types of particulate organic matter in certain ocean
waters suggests that stable carbon isotope studies could
provide unique information about the growth physiology
as well as the carbon dynamics of the plankton community.
142
E. Wada et al.
Fig. 5. δ15N–δ13C map of marine phytoplankton and cyanobacteria with an emphasis on diazotrophs. (a) δ15N–δ13C map of
marine phytoplankton (×; Wada & Hattori, 1991) and Trichodesmium spp. (□; this study), cyanobacteria in Lake Suwa, Japan
(●), cyanobacteria from the McMurdo Dry Valleys (⃝), algae from human-impacted Brazilian lakes (▽), and Microcystis from
Lake Barra Bonite under sewage loading from São Paulo, Brazil (▼; Wada et al. 1991). The values for marine phytoplankton
and cyanobacteria from Lake Suwa almost overlap, as indicated by the shaded area. The cyanobacteria from the McMurdo Dry
Valleys had extremely low δ15N and a wide range of δ13C values. (b) δ15N–δ13C map of cyanobacteria in Lake Suwa, Japan. Numerical values in circles correspond to the months when samples were collected. Data were obtained from Yoshioka et al.
(1994). (c) δ15N–δ13C map of marine phytoplankton from the western North Pacific, following Wada and Hattori 1991. Data
from the Oyashio current area are from Aita et al. (2011).
An isotopic map of cyanobacteria and plankton
Lake Suwa (36°03′N, 138°05′E) is a typical eutrophic
shallow lake in Nagano prefecture, in central Japan, with a
surface area of 13.3 km2 and a maximum depth of 6.8 m.
Blooms of Microcystis are often observed from spring to
summer in this highly eutrophic lake. Microcystis utilizes
nitrate from domestic sewage in spring, then use regenerated ammonium in summer. After that the N2 fixing cyanobacteria Anabaena typically appears under nitrogen-de-
143
C and N isotope ratios of cyanobacteria
ficient and phosphate rich conditions in August. The pattern of succession in this cyanobacterial community provided a useful comparison with the regional differences
between marine phytoplankton when plotted on the δ15N–
δ13C map (Fig. 5a).
Data from a 1985 bloom (Yoshioka et al. 1994) are
shown in Fig. 5b, together with plankton data from the pelagic ocean in Fig. 5c (Wada & Hattori 1991). Furthermore,
data from human-impacted lakes in tropical areas in Brazil
were also included in Fig. 5a to demonstrate how human
activity such as dam construction and sand mining
changed δ15N and δ13C values of phytoplankton (Wada et
al. 1991). Coincidentally, the δ15N of inorganic nitrogenous
compounds in Lake Suwa was quite similar to that of pelagic nitrate (ca. 6‰). The isotopic map of pelagic marine
phytoplankton, Microcystis, and Anabaena cylindrica, the
N2 fixing cyanobacteria that appear in August in Lake
Suwa, Japan, can be divided into three groups (nitrate-rich,
nitrate-poor, and N2-fixing communities) depending on nutrient availability, from the nitrate-rich spring season to nitrate poor-phosphate rich summer season (Fig. 5b).
As shown in Fig. 5a, cyanobacteria and phytoplankton
were found in almost the same areas of the isotopic map,
strongly suggesting that the isotopic compositions of algae
are governed by a growth physiology common among marine algae and cyanobacteria. In the figure, a dramatic increase can be observed in the δ15N values of phytoplankton
in tropical lakes in Brazil affected by human impacts such
as sand mining, dam construction, and sewage loading
(Wada et al. 1991). Compared to data for marine phytoplankton (the shaded areas in Fig. 5a), the isotopic data for
cyanobacteria varied with habitat structure. The lowest
value, a δ15N of −76.5‰, was obtained from cyanobacteria
collected at Lake Bonney, in the McMurdo Dry Valleys
(Fig. 3).
Conclusion
The relationship between growth rates and carbon isotope fractionation during photosynthesis was investigated
using a marine cyanobacterium (A. quadruplicatum).
Under light-limiting growing conditions, rapid growth of
the organism was accompanied by low isotope fractionation factors (μ=1.6, α=1.017; μ=0.20, α=1.024); that is,
δ13C was positively correlated with the growth rate. Our
results also suggest that the δ13C of marine phytoplankton
may be useful for estimating in situ growth rates in the
open ocean as a first approximation. We also suggest that
the isoscape of phytoplankton δ13C and δ15N over the
oceans could help to verify marine ecosystem models by
their distributions.
Various marine N2-fixing cyanobacteria were cultured
under N2-fixing conditions to elucidate nitrogen isotope
fractionation values during N2 fixation. δ15N ranged from
−2.27 to −1.48‰, with an average value of −1.74‰. The
highest δ13C among pelagic plankton (−13.6‰) was found
for T. erythraeum.
The natural abundances of cyanobacteria collected from
Antarctic saline ponds in the McMurdo Dry Valleys were
measured precisely. Their δ15N values were divided into
two groups, with average values of −10 and −50‰, respectively. δ13C was highly variable, from −25 to −3‰.
The algal growth physiology of these cyanobacteria is discussed on the basis of their carbon and nitrogen isotope ratios.
An isotopic map of cyanobacteria and marine plankton
was presented that summarizes data appearing in the present work and in the literature. The isotope ratios of cyanobacteria in natural aquatic ecosystems have extremely
wide ranges. Marine phytoplankton and the cyanobacteria
of Lake Suwa were divided into three groups (nitrate-rich,
nitrate-poor, and N2-fixing communities) based on nutrient
availability and growth rate.
Funding
This work was supported by Grant-in-Aid for Scientific
Research (C) number 20580208 to KO and a Grant-in-Aid
for Challenging Exploratory Research (22651007) to MNA
from the Japan Society for the Promotion of Science.
References
Aita MN, Tadokoro K, Ogawa NO, Hyodo F, Ishii R, Smith SL,
Saino T, Kishi MJ, Saitoh S, Wada E (2011) Linear relationship
between carbon and nitrogen isotope ratios along simple food
chains in marine environments. J Plankton Res 33: 1629–1642.
Capone DG, Zehr JP, Paerl HW, Bergman B, Carpenter EJ
(1997) Trichodesmium, a globally significant marine cyanobacterium. Science 276: 1221–1229.
Carpenter EJ, Harvey HR, Fry B, Capone DC (1997) Biogeochemical tracers of the marine cyanobacterium Trichodesmium. Deep-Sea Res I: Oceanographic Research Papers 44:
127–138.
Chen YB, Zehr JP, Mellon M (1996) Growth and nitrogen fixation of the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. IMS101 in defined media: evidence for a circadian rhythm. J Phycol 32: 916–923.
Christeller JT, Loing WA, Troughton JH (1976) Isotope discrimination by ribulose 1,5-diphosphatecarboxylase. Plant Physiol
57: 580–582.
Craig H (1957) Isotopic standards for carbon and oxygen and
correction factors for mass-spectrometric analysis of carbon
dioxide. Geochim Cosmochim Acta 12: 133–149.
Eppley RW (1972) Temperature and phytoplankton growth in the
sea. Fish Bull 70: 1063–1085.
Estep MF, Tobita FR, Parker PL, Van Baalen C (1978) Carbon
isotope fractionation by ribulose-1,5-biphosphatecarboxylase
from various organisms. Plant Physiol 61: 680–687.
Farquhar GD, Ehlernger JR, Hubick KT (1989) Carbon isotope
discrimination and photosynthesis. Annu Rev Plant Physiol
Plant Mol Biol 40: 503–537.
Finzi-Hart JA, Pett-Ridge J, Eeber PK, Popa R, Fallon SJ,
144
E. Wada et al.
Gunderson T, Hutcheon ID, Nealson KH, Capone DG (2009)
Fixation and fate of C and N in the cyanobacterium Trichodesmium
using nanometer-scale secondary ion mass spectrometry. Proc
Natl Acad Sci USA 106: 6345–6350.
Goericke R, Fry B (1994) Variations of marine plankton δ13C
with latitude, temperature, and dissolved CO2 in the world
ocean. Glob Biogeochem Cycl 8: 85–90.
Hage MM, Uhle ME, Macko SA (2007) Biomarker and stable
isotope characterization of coastal pond-derived organic matter, McMurdo Dry Valleys, Antarctica. Astrobiology 7: 645–
661.
Hoering T, Ford HT (1960) The isotope effect in the fixation of
nitrogen by Azotobacter. J Am Chem Soc 82: 376–378.
Hopkins DW, Sparrow AD, Gregorich EG, Elberling B, Novis P,
Fraser F, Scrimgeour C, Dennis PG, Meier-Augenstein W,
Greenfield LG (2009) Isotopic evidence for the provenance
and turnover of organic carbon by soil microorganisms in the
Antarctic dry valleys. Environ Microbiol 11: 597–608.
Kratz W, Myers J (1955) Nutrition and growth of several bluegreen algae. Am J Botany 42: 282–287.
Marumo R, Asaoka O (1974) Distribution of pelagic blue-green
algae in the North Pacific Ocean. J Oceanogr Soc Jap 30: 77–
85.
Matsumoto G, Torii T, Hanya T (1982) High abundance of algal
24-ethylcholesterol in Antarctic lake sediment. Nature 299:
52–54.
Matsumoto GI (1993) Geochemical features of the McMurdo Dry
Valley lakes. In: Physical and Biogeochemical Processes in
Antarctic Lakes (Antarct. Res. Ser. 59) (eds. Green W, Friedman EI). American Geophys Union, Washington DC, pp. 95–
118.
Matsumoto GI, Akiyama M, Watanuki K, Torii T. (1990) Unusual distributions of long-chain n-alkanes and n-alkenes in
Antarctic soil. Org Geochem 15: 403–412.
Matsumoto GI, Ohtani S, Hirota K (1993) Biogeochemical features of hydrocarbons in cyanobacterial mats from the McMurdo Dry Valleys, Antarctica. Proc NPIR Symp Polar Biol 6:
98–105.
Matsumoto GI, Watanuki K, Torii T (1987) Total organic carbon
in ponds from the Labyrinth of Southern Victoria Land in the
Antarctic. Antarc Rec 31: 171–176.
Matsumoto GI, Yamada S, Ohtani S, Broady PA, Nagashima H
(1996) Biogeochemical features of hydrocarbons in cultured
cyanobacteria and green algae from Antarctica. Proc NPIR
Symp Polar Biol 9: 275–282.
Minagawa M, Wada E (1986) Nitrogen isotope ratios of red tide
organisms in the East China Sea: a characterization of the biological nitrogen fixation. Mar Chem 19: 245–259.
Mizutani H, Wada E (1982) Effect of high atmospheric CO2 concentration on 13C of algae. A possible cause for the average depletion of 13C in Precambrian reduced carbon. Origin of Life
12: 377–390.
Ohki K (2008) Intercellular localization of nitrogenase in a nonheterocystous cyanobacterium (Cyanophytes), Trichodesmium
sp. NIBB1067. J Oceanogr 64: 211–216.
Ohki K, Fujita Y (1982) Laboratory culture of the pelagic bluegreen alga Trichodesmium thiebautii: conditions for unialgal
culture. Mar Ecol Prog Ser 7: 185–190.
Ohki K, Rueter JG, Fujita Y (1986) Cultures of the pelagic cyanophytes Trichodesmium erythraeum and T. thiebautii in
synthetic medium. Mar Biol 91: 9–13.
Ohki K, Rueter JG, Fujita Y (1988) Aerobic nitrogenase activity
measured as acetylene reduction in the marine non-heterocystous cyanobacterium Trichodesmium spp. grown under artificial conditions. Mar Biol 98: 111–114.
Ohki K, Honda D, Kumazawa S, Ho KK (2008) Morphological
and phylogenetic studies on unicellular diazotrophic cyanobacteria (Cyanophytes) isolated from the coastal waters
around Singapore. J Phycol 44: 142–151.
Ohki K, Zehr JP, Fujita Y (1991) Trichodesmium: Establishment
of culture and characteristics of N2-fixation. In: Marine Pelagic Cyanobacteria: Trichodesmium and other Diazotrophs
(eds Carpenter EJ, Capone DG, Rueter JG). Kluwer Academic
Publishers, Dordrecht, pp. 307–316.
O Leary MH (1981) Carbon isotope fractionation in plants. Phytochemistry 20: 553–567.
Popp B, Laws EA, Bidigare RR, Dore JE, Hanson KL, Wakeham
SG (1998) Effect of phytoplankton cell geometry on carbon
isotopic fractionation. Geochim Cosmochim Acta 62: 69–77.
Rau GH, Sweeney ES, Kaplan IR (1982) Plankton 13C : 12C ratio
changes with latitude: differences between northern and
southern oceans. Deep Sea Res 29: 1035–1039.
Rippka R, Herdman M (1992) Pasteur culture collection of cyanobacterial Strains in axenic culture. Catalogue and taxonomic
handbook, Vol. 1: catalogue of strains 1992/1993. Institut Pasteur, Paris, France, pp. 1–103.
Saino T, Hattori A (1987) Geographical variation of the water
column distribution of suspended particulate organic nitrogen
and its 15N abundance in the Pacific and its marginal seas.
Deep Sea Res 34: 807–827.
Smith LS (2010) Untangling the uncertainties about combined
effects of temperature and concentration on nutrient uptake
rates in the ocean. Geophys Res Lett 37, L11603, doi:10.1029/
2010GL043617, 2010
Takahashi K, Wada E, Sakamoto M (1991) Relation between carbon isotope discrimination and the specific growth rate of
green alga, Chlamydomonas reinhardtii. Jpn J Limnol 52:
105–112.
Van Baalen C (1962) Studies on marine blue-green algae. Botanica Marina 4: 129–139.
Wada E (1980) Nitrogen isotope fractionation and its significance
in biogeochemical processes occurring in marine environments. In: Isotope Marine Chemistry (eds Goldberg ED,
Horibe Y, Saruhashi K). Uchida Rokkakuho Co., Tokyo,
Japan, pp. 375–398.
Wada E, Hattori A (1978) Nitrogen isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms.
Geomicrobiol 1: 85–101.
Wada E, Hattori A (1991) Nitrogen in the sea: forms, abundances, and rate processes. CRC Press, Boca Raton, Florida,
208 pp.
Wada E, Imaizumi R, Nakaya S, Torii T (1984) 15N abundance in
the Dry Valley Area, South Victoria Land, Antarctica: ecophysiological implications of microorganisms. Mem Natl Inst
Polar Res Spec Iss 32: 130–139.
Wada E, Lee JA, Kimura M, Koike I, Reeburgh WS, Tundisi JG,
Yoshinar T, Yoshioka T, Vuuren MI (1991) Gas exchange in
ecosystems: framework and case studies. Jpn J Limnol 52:
263–281.
Wada E, Shibata R, Torii T (1981) 15N abundance in Antarctica:
origin of soil nitrogen and ecological implications. Nature 292:
327–329.
Waterbury JB, Rippka R (1989) Subsection I. Order Chroococcales Wettstein 1924, emend. Rippka et al., 1979. In: Bergy s
Manual of Systematic Bacteriology, Vol. 3 (eds Staley JT, Bryant MP, Pfennig N, Holt JG). Williams and Wilkins, Baltimore, pp. 1728–1746.
Wilmotte AG, Van der Auwera, DeWachter R (1993) Phylogenic
Appendix.
145
C and N isotope ratios of cyanobacteria
relationships among the cyanobacteria based on 16S rRNA sequences. In: Bergey s Manual of Systematic Bacteriology. 2nd
ed. Vol. 1 (eds Boone D, Castenholz RW, Garrity GM).
Springer, New York, pp. 439–514.
Yoshioka T, Wada E, Hayashi H (1994) A stable isotope study on
seasonal food web dynamics in a eutrophic lake. Ecology 75:
835–846.
Zehr, JP, Waterbury JB, Turner PJ, Montoya JP, Omoregie E,
Steward GF, Hansen A, Karl DM (2001) Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean. Nature 412:
635–637.
Carbon and nitrogen isotope ratios of cyanobacteria collected from the McMurdo Dry Valleys, Antarctica.
Site
Wright Valley Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Vanda
Lake Canopus
Lake Canopus
Lake Canopus
Lake Canopus
Lake Canopus
Don Juan Pond
Don Juan Pond
Don Juan Pond
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Unnamed pond in the Labyrinth
Taylor Valley East L. Bonney
Melt-stream from Hughes Glacer
Melt-stream from Hughes Glacer
Sample no.
δ13C
(‰)
δ15N
(‰)
V-A-1
V-A-2
V-A-2″
V-A-3
V-A-5
V-A-5′
V-A-7
X1
X4
X5
C2
C3
C4
C8
C9
D1
D2
D3
L11-A
L11 Black
L12
L13
L13
L13 10 cm black
L14
L20 lichen A
L20 lichen B
B1
B2
B3
−17.5
−18.5
−17.7
−18.1
−7.9
−6.1
−20.7
−16.0
−15.6
−15.6
−18.5
−9.6
−11.7
−11.0
−14.7
−1.8
−13.0
−15.1
−4.5
−5.6
−6.5
−7.7
−6.5
−17.1
−13.3
−25.2
−25.2
−9.3
−12.7
−12.7
−10.5
−10.3
−9.5
−10.3
−14.7
−13.4
−12.3
−17.7
−17.4
−15.9
−4.7
−3.1
−11.8
−1.9
−3.0
−19.7
−19.1
−20.2
−50.0
−46.7
−51.3
−52.6
−62.5
−9.3
−63.4
−17.3
−20.4
−76.5
−10.0
−9.4
Remarks
Dried cyanobacteria under a stone
Under sand at 5 cm depth
Under the ice at 5 cm depth
Degraded cyanobacteria
Wet cyanobacteria at the edge of the cape at 50 cm depth
ibid.
Attached to a rock at the river mouth
Wet cyanobacteria
Dried cyanobacteria
Dried cyanobacteria
Dried cyanobacteria
Wet cyanobacteria
Wet cyanobacteria
Wet cyanobacteria
Dried cyanobacteria
Dried cyanobacteria
Dried cyanobacteria
Dried cyanobacteria
Dried one
Wet cyanobacteria under the ice at 5 cm depth
Wet cyanobacteria
Wet cyanobacteria
Wet cyanobacteria under the ice at 3 cm depth
Wet cyanobacteria under ice
Wet cyanobacteria
lichen
Degraded lichen
Wet cyanobacteria near river mouth
Wet cyanobacteria
Wet cyanobacteria