Plankton Benthos Res 7(3): 135–145, 2012

Ecological aspects of carbon and nitrogen isotope ratios of cyanobacteria

EITARO WADA1,*, KAORI 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 Marine-Earth 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, Japan3 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 abun-dances 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 implica-tions are discussed. Our results suggest that the δ13C of marine phytoplankton is positively correlated with sea sur-face 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 mi-croorganisms in aquatic ecosystems. In photosynthetic or-ganisms, carbon isotope fractionation is generally recog-nized 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 correla-tion 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

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 Goer-icke & Fry (1994) emphasized that geographic differences in kinetic isotope effects associated with plankton biosyn-thesis and metabolism were important factors causing lati-tudinal 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 fraction-ation factor under light- and nitrogen-limited conditions. The same trend was also reported for nitrate-uptake pro-cesses 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.* Corresponding author: Eitaro Wada; E-mail, eitaro1939@yahoo.co.jp

Plankton & Benthos Research

© The Plankton Society of Japan

136 E. Wada et al.

In the open ocean, the nitrogen isotope ratios of plank-ton are closely correlated with the chemical forms of inor-ganic 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 molec-ular 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, nitro-gen fixation sometimes occurs at low rates relative to the total nitrogen budget (Ohki et al. 1991, Capone et al. 1997). Unicellular diazotrophic cyanobacteria contribute signifi-cantly 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 pro-ducers 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 precipi-tated nitrate is present as a powdered crystal of NaNO3, which is the nitrogen source used for growth by the cyano-bacteria 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 pond-derived organic matter in the McMurdo Dry Valleys. Re-cently, Hopkins et al. (2009) reported isotopic evidence for the provenance and turnover of organic carbon by soil mi-croorganisms in the McMurdo Dry Valleys.

In this study, we investigated the effects of cyanobacte-rial growth rate on carbon isotope fractionation in a ma-rine 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. Cyano-bacteria collected from saline lakes and ponds in the Mc-Murdo Dry Valleys, Antarctica, were also studied in detail, with an emphasis on cyanobacterial growth physiology. Finally, we summarized the characteristics of cyanobacte-ria on a δ15N vs. δ13C map and discuss some ecological im-plications of algal isotope ratios based on our data in com-bination 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 or-ganism 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 liq-uid cultures was followed turbidimetrically and the spe-cific growth rate constant (μ) was estimated using the method of Kratz and Myers (1955), where μ=0.301 corre-sponds to a generation time of 1.00 d.

Cells for isotopic studies were harvested at the late loga-rithmic phase or early stationary phase for test tube cul-tures. In large-scale cultures, cells were harvested at inter-vals 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 cul-tured in a synthetic medium as described in Ohki & Fujita (1982) and Ohki et al. (1986). Five unicellular and one fila-mentous 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 Oceano-graphic Institution, USA, were maintained in a liquid mod-ified Aquil medium without combined nitrogen (Ohki et al. 1986). Two freshwater heterocystous cyanobacteria in sec-tions IV and V (Nostoc sp. PCC7120 and Fischerella sp1ATCC29114, respectively) were cultured in BG0 (Rip-pka & 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, cen-trifuged at 3,000×g for 4 min at 4°C, and lyophilized for stable isotope measurements.

Cyanobacteria from the McMurdo Dry Valleys, Ant-arctica

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 Laby-rinth, 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

C and N isotope ratios of cyanobacteria 137

Valley (Fig. 1).Samples were frozen and brought back to Japan. Prelim-

inary 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 fea-tures of the McMurdo Dry Valley lakes and ponds were re-viewed 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 ampli-fied using cyanobacteria-specific primers as follows: 8F (forward, Wilmotte et al. 1993) and PISTE-Cyano-R (re-verse, CTCTGTGCCAAGGTATC, Harverkamp, unpub-lished 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 pro-gram 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‰.

13 15 3sample standardC or N ( ) = ( / 1) ×10R Rd d ‰ － (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 ex-pressed as a difference in the δ13C value between source and product (Takahashi et al. 1991):

13 1313

13

C (source) C (product)C =

1 C (source) /1000d∆

+δ －δ

δ (2)

Spontaneous discrimination factor at biomass Nt1 and Nt2

Fig. 1. Locations of sampling sites in the McMurdo Dry Valleys of southern Victoria Land, Antarctica (revised from Matsu-moto 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 cyanobacte-ria), 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).

138 E. Wada et al.

in the flask cultures was estimated by the following isotope mass balance equation:

1 2

13 13 13Nt NΔt NtC × f + C (1 f ) Cδ=δ δ － (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 inter-val 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 di-vided into two groups: For one group there was a negative

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 loga-rithmic phase or early stationary phase. Under those condi-tions, cell numbers in the incubation tube were maximal, meaning that high photosynthetic activity was rapidly con-suming dissolved carbon dioxide.

Diazotrophs

The nitrogen isotope ratios (δ15N) of cultured cyanobac-teria during N2 fixation ranged from －1.5 to －3.0‰ rela-tive 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 mi-croorganisms, including Azotobacter. Fractionation during biological molecular nitrogen fixation was almost identical among these organisms.

The partial rDNA sequence of cyanobacteria in the Mc-Murdo 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 col-lected from saline ponds and lakes in McMurdo Dry Val-leys 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 con-centration and low light intensity, with a significant occur-rence of nitrogen isotope fractionation (up to 1.04) during nitrate assimilation. In fact, the occurrence of a large nitro-gen isotope fractionation was observed for several saline ponds in the Labyrinth (Table 2). On one hand, for low NO3

－ concentrations down to 1 μM, no nitrogen isotope discrimination is expected during nitrate assimilation. Consequently the δ15N of cyanobacteria in such ponds be-come 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

Fig. 2. Summary of carbon isotope discrimination factor (Δδ13C) during the growth of Agmenellum quadruplicatum. Cul-tures were constantly bubbled with 1.0% CO2 in air at 36.5°C. FL-series were conducted using 1-L Erlenmeyer flasks. Instanta-neous 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 station-ary 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 en-riched air became insufficient during laboratory culturing.

C and N isotope ratios of cyanobacteria 139

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).

2 2CO CO Organic-C→ →←F1 F2

F3

The first step is CO2 transport into the cell through bidi-rectional passive diffusion (F1 and F3), and the next is as-similation (F2) into organic carbon by ribulose bisphos-phate carboxylase (RuBPCase).

Under the steady state condition during photosynthetic carbon fixation, we obtain

13C = ( ) /δ∆ + ×b a b F2 F1－ (4)

Table 1. Nitrogen isotope ratios of N2-fixing cyanobacteria during N2 fixation.

Organisms δ15N (‰) References

Azotobacter Azotobacter agile －3.5 Hoering & Ford (1960)Azotobacter chroococcum －1.9 ibid.Azotobacter vinelandii －4.1 ibid.

Blue green algae

Anabaenacylindrica －3.0 Wada & Hattori (1974)－0.6 Minagawa & Wada (1986)

Trichodesmium spp. －2.2 This work－1.3 Carpenter et al. (1997)－3.7 Carpenter et al. (1997)

Gloeothece. sp. 68DGA (unicellular) －1.5 strain: Ohki et al. (2008)Gloeothece. sp. 11DG (unicellular) －1.6 strain: Ohki et al. (2008)Gloeothece. sp. 30D1 (unicellular) －1.5 strain: Ohki et al. (2008)Gloeothece. sp. 20B5 (unicellular) －1.4 strain: Ohki et al. (2008)Gloeothece. sp. 38U6 (unicellular) －1.8 strain: Ohki et al. (2008)Crocosphaera watsonii WH8501 (unicellular) －2.2 strain: Waterbury et al. (1989)Lyngbya sp. 10B (Filamentous) －1.7Nostoc sp. PCC7120 －1.8Fischerella sp1ATCC29114 －2.0Trichodesmium spp. －1.3 and －3.7 Reported by Carpenter et al. (1997)

mean －1.7 Only for cyanobacteria except data by Carpenter et al. (1997)

Fig. 3. δ15N–δ13C map for cyanobacteria in the McMurdo Dry Valleys, Antarctica. For details, see Fig. 1 and Appendix.

140 E. Wada et al.

where a and b are the discrimination factors (DF) associ-ated with the processes of CO2 diffusion (F1) and carbo-xylation 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, respec-tively. Then we obtain

13C = 30 (4.4 30) F2/ F1Δ + ×－δ (5)

Here we predict that the quotient F2/F1 in the above equa-tion is proportional to μ, because F1 seems to be indepen-dent 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 follow-ing equation was obtained by Takahashi et al. (1991) for Chlamydomonas reinhardtii Dangeard (IAM C-238):

13C= 35.4 25.3 ( 0.92)δ µ∆ + =r－ － (6)

The nitrogen isotopic fractionation factor is inversely cor-related 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 fluc-tuation 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 in-crease in its growth rate, similar to Chlamydomonas rein-hardtii Dangeard (IAM C-238) described above. We can conclude that the δ13C content of PR-6 decreases with in-creasing 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 lim-itation on algal photosynthetic activity lowered the appar-ent 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 esti-mated by the above eq. (5).

Diazotrophs

Molecular nitrogen has a very strong triple bond be-tween nitrogen atoms. Theoretically, the cleavage of nitro-gen atoms under a reversible reaction results in a very large nitrogen isotope fractionation. However, the ob-served fractionation was small for biological molecular ni-trogen 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 mo-lecular 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 dis-tinguished by the nitrogen isotope ratio and where N2-fix-ing cyanobacteria exhibit low δ15N and high δ13C. These results support the findings of other studies that have dem-onstrated 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 cy-anobacteria can be explained by the kinetic isotope effects

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 concentra-tions.

Pond NH4+－N

(mgN L－1)NO3

－+NH4+

(mgN L－1)δ15N (NH4

+)(‰)

δ15N (NO3－)

(‰)δ15N (cyanobacteria)

(‰)δ13C (cyanobacteria)

(‰)

L-11 4.5 455 －22.4 －11.6 －50.0 －4.55L-12 10.3 1,370 －21.2 －10.0 －51.4 －6.55L-13 6.6 1,290 n.d. －1.6 －52.7 －7.76L-14 8.3 1,370 －8.0 －5.6 －63.4 －13.30

S-1 Valley 14.2 14.2 +2.0 －13.2 －19.8 －1.50

C and N isotope ratios of cyanobacteria 141

described above during cyanobacterial growth in Mc-Murdo 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 fraction-ation factors become close to 1.000 because the diffusion of the substrate into algal cells is the rate-limiting step dur-ing 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 par-ticipated 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 se-quence 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 substrate-limited 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, Tricho-desmium holds a unique position among marine phyto-plankton 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

concentration on nitrate uptake as measured in shipboard experiments, Smith (2010) found evidence of greater tem-perature 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-lati-tude 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 indi-cated in Fig. 4. This linear relationship observed in the western North Pacific off Japan is quite similar to that re-ported by Goericke and Fry (1994), whose linear regres-sion 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 commu-nity.

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.

142 E. Wada et al.

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 regener-ated ammonium in summer. After that the N2 fixing cya-nobacteria Anabaena typically appears under nitrogen-de-

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. Nu-merical 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).

C and N isotope ratios of cyanobacteria 143

ficient and phosphate rich conditions in August. The pat-tern of succession in this cyanobacterial community pro-vided 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 pe-lagic 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 pe-lagic 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 nu-trient availability, from the nitrate-rich spring season to ni-trate 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 ma-rine algae and cyanobacteria. In the figure, a dramatic in-crease 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 phyto-plankton (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 iso-tope 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 fraction-ation 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‰, re-spectively. δ13C was highly variable, from －25 to －3‰. The algal growth physiology of these cyanobacteria is dis-cussed on the basis of their carbon and nitrogen isotope ra-tios.

An isotopic map of cyanobacteria and marine plankton was presented that summarizes data appearing in the pres-ent work and in the literature. The isotope ratios of cyano-bacteria 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.

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Appendix. Carbon and nitrogen isotope ratios of cyanobacteria collected from the McMurdo Dry Valleys, Antarctica.

Site Sample no. δ13C (‰)

δ15N (‰) Remarks

Wright Valley Lake Vanda V-A-1 －17.5 －10.5 Dried cyanobacteria under a stoneLake Vanda V-A-2 －18.5 －10.3 Under sand at 5 cm depthLake Vanda V-A-2″ －17.7 －9.5 Under the ice at 5 cm depth Lake Vanda V-A-3 －18.1 －10.3 Degraded cyanobacteriaLake Vanda V-A-5 －7.9 －14.7 Wet cyanobacteria at the edge of the cape at 50 cm depthLake Vanda V-A-5′ －6.1 －13.4 ibid.Lake Vanda V-A-7 －20.7 －12.3 Attached to a rock at the river mouthLake Vanda X1 －16.0 －17.7 Wet cyanobacteriaLake Vanda X4 －15.6 －17.4 Dried cyanobacteriaLake Vanda X5 －15.6 －15.9 Dried cyanobacteriaLake Canopus C2 －18.5 －4.7 Dried cyanobacteriaLake Canopus C3 －9.6 －3.1 Wet cyanobacteriaLake Canopus C4 －11.7 －11.8 Wet cyanobacteriaLake Canopus C8 －11.0 －1.9 Wet cyanobacteriaLake Canopus C9 －14.7 －3.0 Dried cyanobacteriaDon Juan Pond D1 －1.8 －19.7 Dried cyanobacteriaDon Juan Pond D2 －13.0 －19.1 Dried cyanobacteriaDon Juan Pond D3 －15.1 －20.2 Dried cyanobacteriaUnnamed pond in the Labyrinth L11-A －4.5 －50.0 Dried oneUnnamed pond in the Labyrinth L11 Black －5.6 －46.7 Wet cyanobacteria under the ice at 5 cm depth Unnamed pond in the Labyrinth L12 －6.5 －51.3 Wet cyanobacteriaUnnamed pond in the Labyrinth L13 －7.7 －52.6 Wet cyanobacteriaUnnamed pond in the Labyrinth L13 －6.5 －62.5 Wet cyanobacteria under the ice at 3 cm depthUnnamed pond in the Labyrinth L13 10 cm black －17.1 －9.3 Wet cyanobacteria under iceUnnamed pond in the Labyrinth L14 －13.3 －63.4 Wet cyanobacteriaUnnamed pond in the Labyrinth L20 lichen A －25.2 －17.3 lichenUnnamed pond in the Labyrinth L20 lichen B －25.2 －20.4 Degraded lichen

Taylor Valley East L. Bonney B1 －9.3 －76.5 Wet cyanobacteria near river mouthMelt-stream from Hughes Glacer B2 －12.7 －10.0 Wet cyanobacteriaMelt-stream from Hughes Glacer B3 －12.7 －9.4 Wet cyanobacteria