J. Phycol. 47, 1292–1303 (2011)
2011 Phycological Society of America
DOI: 10.1111/j.1529-8817.2011.01078.x
INTERACTIVE EFFECTS OF IRRADIANCE AND CO2 ON CO2 FIXATION AND N2
FIXATION IN THE DIAZOTROPH TRICHODESMIUM ERYTHRAEUM (CYANOBACTERIA)1
Nathan S. Garcia, Fei-Xue Fu, Cynthia L. Breene
Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA
Peter W. Bernhardt, Margaret R. Mulholland
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, Virginia 23529, USA
Jill A. Sohm and David A. Hutchins2
Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA
The diazotrophic cyanobacteria Trichodesmium spp.
contribute approximately half of the known marine
dinitrogen (N2) fixation. Rapidly changing environmental factors such as the rising atmospheric partial
pressure of carbon dioxide (pCO2) and shallower
mixed layers (higher light intensities) are likely to
affect N2-fixation rates in the future ocean. Several
studies have documented that N2 fixation in laboratory
cultures of T. erythraeum increased when pCO2 was
doubled from present-day atmospheric concentrations
(380 ppm) to projected future levels (750 ppm).
We examined the interactive effects of light and pCO2
on two strains of T. erythraeum Ehrenb. (GBRTRLI101
and IMS101) in laboratory semicontinuous cultures.
Elevated pCO2 stimulated gross N2-fixation rates in
cultures growing at 38 lmol quanta Æ m)2 Æ s)1
(GBRTRLI101 and IMS101) and 100 lmol quanta Æ
m)2 Æ s)1 (IMS101), but this effect was reduced in both
strains growing at 220 lmol quanta Æ m)2 Æ s)1. Conversely, CO2-fixation rates increased significantly
(P < 0.05) in response to high pCO2 under mid- and
high irradiances only. These data imply that the stimulatory effect of elevated pCO2 on CO2 fixation and N2
fixation by T. erythraeum is correlated with light. The
ratio of gross:net N2 fixation was also correlated with
light and trichome length in IMS101. Our study suggests that elevated pCO2 may have a strong positive
effect on Trichodesmium gross N2 fixation in intermediate and bottom layers of the euphotic zone, but perhaps not in light-saturated surface layers. Climate
change models must consider the interactive effects of
multiple environmental variables on phytoplankton
and the biogeochemical cycles they mediate.
Abbreviations: DIC, dissolved inorganic carbon;
Fe, iron; N, nitrogen; P, phosphorus; pCO 2,
partial pressure of carbon dioxide; ppm, parts
per million
N2 fixation by marine diazotrophic cyanobacteria
such as Trichodesmium spp. contributes substantial
new nitrogen (N) to marine environments, including the North Atlantic, Pacific, and Indian oceans
(Carpenter et al. 1993, Capone et al. 1997, 2005,
Karl et al. 2002). This new nitrogen represents an
important sink for atmospheric carbon dioxide
(CO2), as organic carbon and nitrogen are drawn
down from surface layers to the deep ocean by the
biological carbon pump (Capone et al. 1997, Falkowski 1997, Karl et al. 1997). For this reason, interest in diazotrophic production has led to an
improved understanding of environmental factors
that control N2 fixation by marine diazotrophs
(Boyd et al. 2010). For instance, phosphorus (P)
and iron (Fe) have been identified as key factors
that control N2 fixation in the open ocean (Hutchins and Fu 2008). Studies in the North Atlantic
Ocean have demonstrated that P limits N2 fixation
by Trichodesmium spp. in this region (Wu et al. 2000,
Sanudo-Wilhelmy et al. 2001, Kustka et al. 2003,
Dyhrman et al. 2006, Webb et al. 2007, Sohm et al.
2008). Iron has been suggested as another potential
limiting factor for N2 fixation by marine diazotrophs
(Wu et al. 2000, Berman-Frank et al. 2001, Webb
et al. 2001, Fu and Bell 2003, Moore et al. 2009),
and in some regimes, Fe ⁄ P colimitation may be
important (Mills et al. 2004).
Recently, some studies have suggested that the
partial pressure of CO2 (pCO2) in the atmosphere
may be another possible limiting factor for N2 fixation and CO2 fixation by Trichodesmium (Barcelos e
Ramos et al. 2007, Hutchins et al. 2007, Levitan
et al. 2007, 2010, Kranz et al. 2009) and the widespread unicellular marine diazotroph Crocosphaera
Key index words: carbon dioxide; diazotrophy;
light; nitrogen fixation; ocean global change;
Trichodesmium
1
Received 3 August 2010. Accepted 23 March 2011.
Author for correspondence: e-mail [email protected].
2
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L I G H T A N D CO 2 E F F E C T S O N T R I C H OD E S M I U M N 2 F I X A T I O N
watsonii (Fu et al. 2008). This finding may be
related to the fact that the cyanobacterial RUBISCO
enzyme is relatively inefficient at fixing inorganic
carbon (C) compared to those of many other species of oxygenic photoautotrophs (Badger et al.
1998, Tortell 2000). Currently, atmospheric pCO2 is
35% higher than during the preindustrial era, and
continued anthropogenic CO2 emissions are
expected to double the current concentration
(385 ppm) before the turn of the century (IPCC
2007). The concomitant increase in pCO2 in the
surface of the world’s oceans could enhance global
N2-fixation rates, thereby augmenting current inputs
of new nitrogen and potentially increasing net CO2
drawdown (Hutchins et al. 2009). Such negative
feedback mechanisms between N2 fixation and
atmospheric CO2 have been hypothesized to be driven by iron supply to oligotrophic regions of the
world’s oceans and are thought to occur on glacialinterglacial timescales (Falkowski 1997, Michaels
et al. 2001). This new evidence suggests that both
short-term and geological timescale models of
ocean ⁄ atmosphere biogeochemical feedbacks may
also need to incorporate the effects of CO2 limitation of marine N2 fixation.
Interactive effects of environmental factors that
control N2 fixation are important to consider
because some factors may act synergistically, while
others have independent effects (Boyd et al. 2010).
Hutchins et al. (2007) demonstrated that elevated
pCO2 enhanced CO2- and N2-fixation rates independently of changes in temperature or P limitation of
N2 fixation in Trichodesmium spp. In contrast, the
effects of pCO2 on CO2- and N2-fixation rates in
Crocosphaera watsonii (WH8501) were dependent on
Fe availability (Fu et al. 2008). Collectively, these
studies indicate that the interactive effects of multiple environmental factors, such as CO2, temperature, and nutrient concentrations, are important to
consider to accurately predict how CO2- and N2-fixation rates will change in diazotrophic cyanobacteria
within the next century.
Within the next 50–100 years, higher sea surface
temperature, increased precipitation, and ice melting are expected to create a more stratified water
column across much of the world’s oceans. The net
effect will decrease the average depth of the mixed
layer, thereby potentially increasing the average irradiance experienced by phytoplankton (Boyd and
Doney 2002, Sarmiento et al. 2004, Behrenfeld et al.
2006, Breitbarth et al. 2007, Boyd et al. 2010).
Despite this prediction, the interactive effects of
changing light environments and pCO2 on CO2and N2-fixation rates in marine diazotrophs have
only been examined in one other study (Kranz
et al. 2010 and Levitan et al. 2010), although several
studies have investigated the effects of irradiance
alone. In two different laboratory cultures of T. erythraeum, N2-fixation rates increased with increasing
light and reached a saturation point near 70 lmol
1293
quanta Æ m)2 Æ s)1 (Bell and Fu 2003) and 100 lmol
quanta Æ m)2 Æ s)1 (Staal et al. 2007). In natural
blooms of Trichodesmium in the tropical North Atlantic, however, N2-fixation rates Æ trichome)1 increased
with increasing irradiance with maximal rates near
the sea surface, where irradiance may be >1,000
lmol quanta Æ m)2 Æ s)1 (Capone et al. 2005). An
inverse relationship between mixed-layer depth and
colonial N2-fixation rates also suggested that
Trichodesmium diazotrophy was limited by light in
this region (Sanudo-Wilhelmy et al. 2001).
Irradiance may partially control the distribution of
Trichodesmium in the water column, such that colonies become negatively buoyant during light hours
following production of carbohydrates and positively
buoyant at night following carbohydrate exudation
or consumption (Villareal and Carpenter 2003,
Berman-Frank et al. 2007, Kranz et al. 2009). In a
recent study, Davis and McGillicuddy (2006) presented data suggesting that Trichodesmium may also
occupy deep water (>120 m depth).
This study examines the effects of present-day
and elevated pCO2 (near 100-year predicted levels)
on CO2 fixation and N2 fixation by T. erythraeum isolates from the Pacific (GBRRLI101) and Atlantic
(IMS101) oceans, grown under a range of irradiance. Regardless of geographic origin, both of these
Trichodesmium isolates showed similar responses of
CO2-fixation and gross N2-fixation rates, suggesting
that the interactive influence of irradiance and elevated pCO2 needs to be considered to accurately
predict how changing pCO2 could affect N2-fixation
rates in the future ocean.
MATERIALS AND METHODS
Cultures. Stock and experimental cultures of T. erythraeum
GBRTRL101 (GBR; from the Great Barrier Reef, Pacific Ocean,
Fu and Bell 2003) and IMS101 (IMS; from coastal North
Carolina, Atlantic Ocean, Prufert-Bebout et al. 1993) were
cultured at 24C (unless otherwise stated) in an artificial
seawater medium without fixed N using a modified version of
the YBCII recipe of Chen et al. (1996). Phosphate and tracemetal solutions were filtered (0.2 lm) and added in concentrations equivalent to the AQUIL recipe (Morel et al. 1979) to
microwave- (experiment with GBR) or autoclave-sterilized
(experiments with IMS) seawater. The AQUIL concentrations
of phosphate and trace metals are more than sufficient to
support maximal growth in our experiments, where medium is
renewed frequently through the semicontinuous dilutions.
Irradiance was supplied with cool-white fluorescent bulbs on a
12:12 light:dark (L:D) cycle. For all experiments, cultures were
grown in triplicate using a semicontinuous batch culturing
method to achieve steady-state exponential growth for 7–10
generations prior to sampling, to fully acclimatize cells to
treatment pCO2 and irradiance conditions. We monitored cell
density every 2–3 d using microscopic cell counts. When
the biomass reached 100–200 trichomes Æ mL)1 (100–
200 nmol C Æ mL)1), we diluted cultures with fresh medium
to 50–100 trichomes Æ mL)1 (50–100 nmol C Æ mL)1). In this
semicontinuous culturing method, the growth rate determines
the dilution rate; this culturing technique does not attempt to
control the growth rate with the dilution rate, as continuous
culturing methods do.
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NATHAN S. GARCIA ET AL.
Experimental design. IMS growth versus light experiment: This
experiment served as a reference for determining the range of
irradiance levels used in our CO2 and light experiments.
Cultures of IMS were cultured at 27C in fifteen 800 mL
culturing flasks on a 12:12 L:D cycle at 25, 50, 100, 180, and
300 lmol quanta Æ m)2 Æ s)1 irradiance. Once cells had
achieved steady state, we determined cellular growth rates
using microscopic cell counts between dilutions.
CO2 ⁄ light experiments: Two separate experiments were conducted with two T. erythraeum strains: GBR and IMS. Cultures of
Trichodesmium sp. (GBR) were grown in twelve 1 L polycarbonate bottles at 35 and 220 lmol quanta Æ m)2 Æ s)1 at two
concentrations of CO2 (see below). In the experiment with
IMS, cultures were grown in eighteen 1 L polycarbonate bottles
at 38, 100, and 220 lmol quanta Æ m)2 Æ s)1 at two concentrations of CO2. Both experiments were conducted on a 12:12 L:D
cycle at 24C.
Within each irradiance treatment for both experiments,
cultures were bubbled with 0.2 lm filtered lab air (for the
experiment with IMS) or premixed air (prepared by Gilmore
Liquid Air Company, South El Monte, CA, USA) containing
present-day (380 ppm certified value for the experiment with
GBR) and elevated, 100-year predicted (750 ppm certified
value for both CO2 experiments) atmospheric CO2 concentrations. The rate of bubbling was visually monitored daily to
ensure that cultures were bubbled with sufficient positive gas
flow to keep the pH of the cultures at an appropriate level for
respective CO2 treatments. Based on rates of gas utilization
from the supply cylinders, estimated gas flow rates were
between 30 and 60 mL Æ min)1. Although we did not examine
trichomes for apical cells, we have not encountered detrimental effects of gentle bubbling on trichome length in our
previous experiments with culturing Trichodesmium in this
manner (Hutchins et al. 2007, 2009). Because bubbling rates
were approximately the same in all replicates, this factor did
not have a differential effect between treatments.
Due to incubator availability constraints in the laboratory,
the light manipulation and the CO2 ⁄ light manipulation
experiments were conducted at slightly different temperatures.
While we do not have growth and N2-fixation rates in response
to temperature for GBR, rates of Trichodesmium isolate IMS are
not significantly different from each other at 24C and 27C
(Breitbarth et al. 2007).
Analytical methods. Growth rates: Growth rates were estimated by measuring relative increases in cell number per unit
volume between dilutions (2–3 d periods) in steady-state
cultures.
N2 fixation: We estimated N2-fixation rates with two methods: the acetylene reduction method and the 15N2 isotope
tracer method. For the acetylene reduction method, described
in Capone (1993), two 10 mL samples from each experimental
replicate were incubated under treatment-specific conditions
of irradiance and temperature in air-tight vials for 10 h
(starting from the beginning of the light period) with 2 mL
acetylene in 16.75 mL of headspace. The amount of ethylene
accumulation was then estimated in 200 lL headspace gas with
a gas chromatograph (model: GC-8A, Shimadzu Scientific
Instruments, Columbia, MD, USA) at the 2nd, 4th, 6th, and 8th
hour of the light period yielding three 2 h rates of ethylene
accumulation. The accumulation of ethylene over 8 h was used
to calculate total gross N2-fixation rates (see explanation
below) using a conversion ratio of 3:1 for acetylene to N2
reduction. Maximum gross N2-fixation rates were determined
by finding the maximum rate of ethylene accumulation over a
2 h period. To calculate the concentration of ethylene in
seawater from the concentration in the vial headspace, we used
the Bunsen coefficient (0.088; from Breitbarth et al. 2004) for
ethylene in seawater at 24C and a salinity of 35. We also
estimated N2-fixation rates with the 15N2 isotope tracer method
(Mulholland et al. 2004, Mulholland and Bernhardt 2005) by
injecting 160 lL of highly enriched (99%) 15N2 gas into
combusted (4 h, 450C) 159 mL gas-tight bottles filled with
culture (without headspace) from a treatment replicate (triplicate samples for each experimental treatment). Culture
samples were then incubated for 12 h under treatment-specific
conditions of irradiance and temperature during the light
period only, and we terminated incubations by filtering
samples onto precombusted (450C, 4 h) Whatman GF ⁄ F
filters (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
pH was not determined in culture subsamples that were used to
estimate N2 fixation after the incubation period in our
experiments. Samples were stored frozen and dried before
analysis with a Europa 20 ⁄ 20 isotope ratio mass spectrometer
(originally manufactured by Europa Scientific Inc., Cincinnati,
OH, USA; refurbished by PDZ Europa Limited, Hill Street,
Elworth, Sandbach, Cheshire, UK) equipped with an automated nitrogen and carbon analyzer (ANCA). Estimates of N2
fixation made with the isotope tracer (15N2) method should
represent net N2-fixation rates because it estimates fixed N that
is retained within cells. We assumed that the acetylene
reduction method estimates gross N2-fixation rates because
this estimate includes N fixed regardless of fate (see Mulholland et al. 2004, Mulholland and Bernhardt 2005). We
estimated net 15N2-fixation rates in our experiment with IMS,
but not in our experiment with GBR.
Carbon fixation: Cell-specific CO2-fixation rates were determined as in Fu et al. (2008). Specifically, we inoculated two
30 mL samples from each treatment replicate with 25 or 50 lL
of 1 mCurie (mCi) stock solution of sodium bicarbonate
(H14CO3); 0.83–1.7 lCi Æ mL)1 final concentration). Samples
were incubated for 24 h under treatment-specific conditions of
irradiance and temperature and then filtered onto Whatman
GF ⁄ F filters and rinsed three times with 5 mL filtered
seawater to remove extracellular H14CO3). Nonphotosynthetically driven 14C incorporation was determined by incubating
replicate culture samples (30 mL) for 24 h in opaque bottles at
the experimental temperature with the same concentration of
H14CO3); these values were subtracted from measured total 14C
incorporation to estimate photosynthetic incorporation. The
total radioactivity of H14CO3) was determined by stabilizing 25
or 50 lL of the 1 mCi H14CO3) with 100 lL of a basic solution
of phenylethylamine (99%) before adding 4 mL of Ultima
Gold XR (PerkinElmer, Shelton, CT, USA) Radioactivity of
14
C was determined on a Multi-purpose Scintillation Counter
(model: LS-6500, Beckman Coulter, Fullerton, CA, USA).
Carbon-fixation rates (moles of CO2 fixed per unit time) were
estimated by calculating the ratio of the radioactivity of
photosynthetically driven 14C incorporation into cells over
24 h to the total radioactivity of H14CO3) and multiplying that
ratio by the total dissolved inorganic carbon concentration
(DIC, see method below).
Seawater carbonate system estimates: Total DIC was preserved
in whole water samples (5–70 mL; stored at 4C) with a 5%
HgCl2 solution (final concentration diluted to 0.5% HgCl2) as
described in Fu et al. (2007), and estimated by acidifying 5 mL
and quantifying the CO2 trapped in an acid sparging column
(model: CM 5230) with a carbon coulometer (model: CM 140,
UIC Inc., Joliet, IL, USA) as described in Beman et al. (2010)
and King et al. (2011). Reference material for the DIC analysis
was prepared by Andrew Dickson at Scripps Institute of
Oceanography. pH was measured with a pH meter (model:
Orion 5 star Thermo Scientific, Beverly, MA, USA) and was
monitored to ensure that perturbations of the seawater with
the either air or certified premixed air (Gilmore Liquid Air
Company, 750 ppm) resulted in the desired target pH of either
8.2 or 7.95. For the CO2 ⁄ light manipulation experiment
with GBR, samples for total DIC were taken from cultures at the
same time CO2- and N2-fixation rates were estimated. For the
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L I G H T A N D CO 2 E F F E C T S O N T R I C H OD E S M I U M N 2 F I X A T I O N
CO2 ⁄ light manipulation experiment with IMS, measurements
of pH were paired with total DIC samples 5–6 d prior to
measuring rates of CO2 and N2 fixation and were used to
calculate pCO2 at 24C using the CO2sys program provided by
Lewis and Wallace (1998) with K1 and K2 constants from
Mehrbach et al. (1973) refit by Dickson and Millero (1987).
Cellular C, N, and P quotas: Particulate N and C were
estimated in cells filtered onto combusted GF ⁄ F filters.
Samples were dried at 80C–90C for 2 d and compressed into
pellets, and the amounts of C and N were determined using an
elemental analyzer (model: 4010, Costech Analytical Technologies Inc., Valencia, CA, USA). Particulate P was estimated by
filtering 20–30 mL of the cultures onto combusted GF ⁄ F filters.
Filters were rinsed twice with 2 mL of a 0.17M sodium sulfate
solution and dried in a combusted glass vial with 2 mL of a
0.017M magnesium sulfate solution, as described in Fu et al.
(2005). Samples were then combusted for 2 h at 450C to
volatilize organic compounds bound to P. Residual P was
estimated using the spectrophotometric method of Fu et al.
(2005) with a spectrophotometer (model: SP-830, Barnstead ⁄ Turner, Dubuque, IA, USA) at a wavelength of 885 nm.
Trichome length: In our light ⁄ CO2 manipulation experiment
with IMS, trichome length was measured in samples collected
from treatment-specific acclimated cultures 2 d prior to and on
the final sampling day. Length data from the two time points
were averaged, and the resulting mean value from each
experimental replicate was used in our analyses.
Statistics. With JMPIN 4.0.3 statistical software (SAS Institute Inc., Cary, NC, USA), we used a one-way and a two-way
analysis of variance (ANOVA) test combined with a Tukey
analysis of multiple comparisons to determine statistical
differences (P < 0.05) between treatments. We report the SE
associated with the mean of treatment replicates.
measured DIC concentrations were close to values
expected based on respective pCO2 treatments
(Table 1).
Growth rates. For both GBR and IMS, growth was
limited by light in our low-light treatments (irradiance of 25–35 lmol quantaÆm)2Æs)1; growth rates of
0.13–0.16 d)1) compared to light-saturated maximum growth rates of 0.25–0.45 d)1 at irradiance
‡100 lmol quanta Æ m)2 Æ s)1 (Fig. 1, a–c). Within
pCO2 treatments for IMS, growth rates at 220 lmol
quanta Æ m)2 Æ s)1 were not significantly different
from those at 100 lmol quanta Æ m)2 Æ s)1 (P >
0.15). The whole model effect of the two-way
RESULTS
Seawater carbonate estimates. In the experiment
with IMS101, total DIC concentrations and pH were
close to those documented for present-day atmospheric pCO2 (Table 1). For example, Bates (2001)
reported a seasonal range of 2,010–2,070 lmol total
CO2 Æ kg)1 seawater in the Sargasso Sea for the years
1988–1998. Riebesell et al. (2010) provide reference
values of seawater pH for present-day and 100-year
predicted concentrations of atmospheric CO2. Using
the certified value of the pre-mixed air, and assuming that the air treatment was 380 ppm, we backcalculated pH with our estimates of total DIC. Our
pH electrode measurements were close to the backcalculated pH values. We did not measure pH in
our experiment with GBR because of instrument
problems and therefore we do not have calculated
values of pCO2 for this experiment; however, the
FIG. 1. Cellular growth rate of Trichodesmium erythraeum
(IMS101 and GBRRLI101) in response to irradiance (a) and present-day and elevated pCO2 (b–c). Growth rates were estimated
from changes in cell number per unit volume over time. The SE
is reported on the means of triplicate samples.
Table 1. Estimates of measured* total dissolved inorganic carbon (DIC) were paired with measured* pH data to give the
calculated** partial pressure of CO2, with SE in steady-state semicontinuous cultures of Trickodesmium erythraeum. Our measured estimates of total DIC were also paired with certified* pCO2 values to give calculated** pH. Calculations were done
using the CO2sys program at 24C. No pH data (n.d.) were acquired for the experiment with GBR. The pCO2, of air was
not measured.
Strain
pCO2 treatment
DIC* (lM)
SE
pH*
SE
pH**
SE
IMS
Present-day
100-year predicted
Present-day
100-year predicted
2018
2116
2037
2165
27
15
9
9
8.23
8.00
n.d.
n.d.
0.01
0.00
8.28
8.01
8.27
8.01
0.00
0.00
0.00
0.00
GBR
pCO2* (ppm)
Air (380)
750
380
750
pCO2**(ppm)
SE
435
771
n.d.
n.d.
9
8
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NATHAN S. GARCIA ET AL.
ANOVA indicated that elevated pCO2 had a significant positive effect on cellular growth in IMS
(P = 0.05, F1,17 = 4.6; Fig. 1b) but not in GBR
(P = 0.37, F1,11 = 0.88; Fig. 1c).
Carbon fixation. Irradiance and pCO2 had a significant impact on C-specific CO2-fixation rates in
both strains of T. erythraeum (P < 0.02) (Figs. 2a and
3a). Elevated pCO2 enhanced C-specific CO2-fixation rates at irradiances ‡100 lmol quanta Æ
m)2 Æ s)1 in both IMS (P < 0.001, F1,12 > 40) and
GBR (P = 0.01, F1,8 = 10.7), but not under low light
GBR
P > 0.36,
(IMS
P = 0.92,
F1,12 < 0.01;
F1,8 = 0.94). Cell-normalized CO2-fixation rates
yielded similar results, with significantly different
rates between pCO2 treatments at irradiances
‡100 lmol quanta Æ m)2 Æ s)1 in both strains
(P < 0.03), but not at low light in either strain
(P > 0.09) (Figs. 2b and 3b). Thus, elevated pCO2
had no effect on CO2 fixation at low light, but had
a positive effect at high light.
N2 fixation. The effects of light and pCO2 on
gross N2-fixation rates in IMS and GBR were exam-
ined in four ways; maximum and total gross N2-fixation rates (see ‘‘Materials and Methods’’) were
normalized to both cellular N and cell density. Net
N2-fixation rates were also normalized to cellular N
and cell density.
Not surprisingly, light had a highly significant
impact on gross N2-fixation rates in IMS and GBR,
and on net N2-fixation rates in IMS (P < 0.001)
(Figs. 2 c–h; 3, c–f). Total and maximum gross
N2-fixation rates at 220 lmol quanta Æ m)2 Æ s)1 were
not significantly higher than rates measured in
treatments maintained at 100 lmol quanta Æ m)2 Æ
s)1 in either pCO2 treatment (P > 0.10, F1,12 < 3.2;
Fig. 2, c–f). Conversely, net N2-fixation rates were
significantly higher in IMS cultures maintained at
220 lmol quanta Æ m)2 Æ s)1 than at 100 lmol
quanta m)2 Æ s)1 in both pCO2 treatments (P < 0.01,
F1,12 > 9.6; Fig. 2, g and h). Thus, while steady-state
gross N2-fixation rates were saturated near 100 lmol
quanta Æ m)2 Æ s)1 at both pCO2 levels investigated,
net N2-fixation rates may not have been saturated,
even at 220 lmol quanta Æ m)2 Æ s)1.
FIG. 2. CO2- and N2-fixation rates by Trichodesmium erythraeum (IMS101) at present-day (air) and elevated pCO2 (750 ppm) under variable
irradiance. C-specific CO2 fixation (a) and cell-specific CO2 fixation (b). Total N-specific gross N2 fixation (c), total cell-specific gross N2 fixation (d), maximum N-specific gross N2 fixation (e), maximum cell-specific gross N2 fixation (f), net N-specific N2-fixation rates (g), and net
cell-specific N2-fixation rates (h). C- and N-specific rates are in units of h)1, and cell-specific rates are in units of fmol C or N Æ cell)1 Æ h)1. The
SE is reported on the means of triplicate samples.
L I G H T A N D CO 2 E F F E C T S O N T R I C H OD E S M I U M N 2 F I X A T I O N
1297
FIG. 3. Response of CO2 and N2 fixation in Trichodesmium erythraeum (GBRRLI101) to present-day and elevated pCO2 under variable
irradiance. CO2 fixation was normalized to cellular C (a, h)1) and cell density (b, fmol C Æ cell)1 Æ h)1). Total (8 h rate, c–d) and maximum (highest 2 h rate, e–f) gross N2-fixation rates were normalized to cellular N (h)1) and cell density (fmol N Æ cell)1 Æ h)1). The SE is
reported on the means of triplicate samples.
Elevated pCO2 had a highly significant effect on
total N-specific gross N2-fixation rates in IMS, according to the whole two-way ANOVA model (P < 0.001,
F1,12 = 62, Fig. 2c), but in GBR, increases were smaller, variability between replicates was higher, and differences were not significant (P > 0.71, F1,8 = 0.15,
Fig. 3c). When normalized to cell density, total gross
N2-fixation rates were stimulated by high pCO2 in
IMS only at irradiance ‡100 lmol quanta Æ m)2 Æ s)1
(P < 0.01, F1,12 > 12.8, Fig. 2d), and not in the low- or
high-light treatments with GBR (P = 0.12, F1,8 = 3.0,
Fig. 3d). Likewise, elevated pCO2 significantly
increased maximum N-specific gross N2-fixation rates
in IMS at irradiance ‡100 lmol quanta Æ m)2 Æ s)1
(P < 0.003, F1,12 > 14, Fig. 2e). Maximum cell-specific
gross N2-fixation rates in IMS, however, were significantly higher in the elevated pCO2 treatment compared with the present-day pCO2 treatment at all
irradiances examined (P < 0.01, F1,12 > 9.6, Fig. 2f).
Elevated pCO2 did not significantly affect maximum
gross N2-fixation rates in GBR when N2 fixation was
normalized to cellular N (P > 0.06, F1,8 < 4.6, Fig. 3e)
but had a significant positive effect at low light when
normalized to cell numbers (P = 0.48, F1,8 = 5.4,
Fig. 3f). There were no significant differences in
N-specific net 15N2-fixation rates between pCO2 treatments in IMS (P = 0.68, F1,12 = 0.18, Fig. 2g), but
pCO2 had a significant positive effect on net N2-fixation rates (15N2 uptake) when normalized to cell density at 220 lmol quanta Æ m)2 Æ s)1 (P = 0.01,
F1,11 = 9.9, Fig. 2h).
Cellular C, N, and P quotas. IMS cellular C quotas
varied as a function of light (P = 0.01, F2,11 = 7.3), but
not as a function of pCO2 (P = 0.7, F1,11 = 0.16,
Fig. 4a). Average cellular N concentrations were
higher in the high pCO2 treatments compared with
present-day pCO2 treatments at irradiance ‡100 lmol
quanta Æ m)2 Æ s)1, although not significantly (P > 0.05,
FIG. 4. Cellular quotas of carbon (a), nitrogen (b), and phosphorus (c), in present-day (closed symbols) and elevated pCO2
(open symbols) cultures of Trichodesmium erythraeum IMS101. The
SE is reported on the means of triplicate samples.
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NATHAN S. GARCIA ET AL.
FIG. 5. Total (8 h rate) gross:net (12 h rate) N2 fixation (a)
and trichome length (lm, b) in present-day and elevated
pCO2 cultures of Trichodesmium erythraeum IMS101 as a function
of irradiance. The SE is reported on the means of triplicate
samples.
Fig. 4b). Cellular P quotas were significantly higher in
the high pCO2 treatment relative to the present-day
pCO2 treatment (P > 0.03, Fig. 4c) but did not vary as
a function of light (P > 0.05).
Gross:net N2 fixation. The ratio of total gross:net
N2 fixation gives an estimate of cellular N release
(Mulholland et al. 2004, Mulholland 2007). For IMS,
this ratio was significantly higher in the high-pCO2
treatment compared to the low-pCO2 treatment
under low irradiance (P < 0.001, F1,12 = 38), but not
at irradiance ‡100 lmol quanta Æ m)2 Æ s)1 (P > 1.8,
F1,12 < 2.0) (Fig. 5a). Total gross:net N2 fixation was
negatively correlated with light (r = )0.85).
Trichome length. The partial pressure of CO2 had
a strong effect on trichome length in IMS
(P = 0.007, F1,12 = 10). The low-light (P = 0.003,
F1,12 = 14) and mid-light (P = 0.02, F1,12 = 6.6) treatments contributed to the pCO2 treatment variation,
with trichomes being longer under the present-day
pCO2 treatment compared with high pCO2
(Fig. 5b); pCO2 did not affect trichome length at
high light (P = 0.5, F1,12 = 0.5). In addition, the
effect of elevated pCO2 on the percent increase in
trichome length was negatively correlated with the
effect of elevated pCO2 on the percent increase in
total gross:net N2-fixation rates (r = 0.72, n = 3,
Fig. 5), and highly correlated with light (r = 0.98).
DISCUSSION
The main finding of our study is that the effect
of elevated pCO2 on gross and net N2-fixation rates
from T. erythraeum was dependent on light intensity.
The results from the CO2 ⁄ light experiments with
two different strains of T. erythraeum were similar. In
both strains of T. erythraeum, our data indicate that
FIG. 6. Postulated effect of elevated pCO2 on N2 fixation in
Trichodesmium erythraeum IMS101 and GBRRLI101 in a water column setting expressed as a percent increase from present-day
pCO2. Estimates of the effect were normalized to cell density and
cellular N and averaged for the maximum (highest 2 h rate) and
total (8 h rate) gross N2-fixation rates and the net 15N2-fixation
(12 h) rates. *Effect of elevated pCO2 (present-day to 750 ppm
pCO2) on chl a–normalized maximum N2-fixation rate of GBR
from Hutchins et al. (2007) at 100 lmol quanta Æ m)2 Æ s)1. The
SE is reported on means. Light data were paired with depth data
from Breitbarth et al. (2008). Question marks estimate published
(net 15N2 fixation) and unpublished (gross N2 fixation) results
from field experiments with Trichodesmium colonies collected near
surface waters (see Hutchins et al. 2009).
the positive effect of elevated pCO2 on gross N2 fixation was large (50% increase) under mid and ⁄ or
low irradiances compared with that at high light
(20% increase; Figs. 2 and 6). Data from Kranz
et al. (2010), Levitan et al. (2010), and our unpublished field experiments (see below) corroborate
our laboratory data describing the combined effects
of light and elevated pCO2 on N2-fixation rates by
IMS and GBR. We speculate that the declining
effect of elevated pCO2 on N2 fixation with increasing light could have been a negative feedback interaction caused by enhanced retention of cellular N
under high-light conditions.
We determined the effect of elevated pCO2 on
maximum and total gross N2-fixation rates in IMS
and GBR by calculating the percent increase in rates
in high-pCO2 treatments over rates in present-day
pCO2 treatments (Fig. 6). To factor in both cellular
nitrogen and cell density as separate estimators of
biomass, we averaged the effect of pCO2 on gross N2
fixation (% change) when normalized to cell number and the effect when normalized to cellular N.
We supplemented our GBR data set on the effect of
elevated pCO2 on gross N2-fixation rates with data
from two experiments reported by Hutchins et al.
(2007). In those experiments, the maximum chl a–
normalized gross N2-fixation rates were 54% and
63% higher in elevated-pCO2 treatments relative to
L I G H T A N D CO 2 E F F E C T S O N T R I C H OD E S M I U M N 2 F I X A T I O N
current-day pCO2 treatments in cultures growing
under conditions similar to those used in our experiments (24C, 100 lmol quanta Æ m)2 Æ s)1; Fig. 6).
These data indicate that the effect of elevated pCO2
on maximum and total gross N2-fixation rates was
negatively correlated with light in both IMS and light
was negatively correlated with maximum gross N2fixation rates in GBR (IMS r = )0.98, )0.68, respectively; GBR r = )0.85), decreasing with increasing
light intensity. We also calculated the effect of elevated pCO2 on net 15N2-fixation rates in the same
way in IMS. When normalized to both cell number
and cellular N, the effect was positively correlated
with irradiance, increasing with increasing light
intensity (r = 0.85 and 0.84, respectively; Fig. 6).
Three field experiments also indicated that net
N2-fixation rates (15N2 incorporation) increased in
response to elevated pCO2 at high irradiance in a
deck-board incubation of natural Trichodesmium colonies collected from the Gulf of Mexico (Hutchins
et al. 2009). However, gross N2-fixation rates (acetylene reduction) in these experiments did not
increase with pCO2 (unpublished data). In that
shipboard experiment, colonies were collected from
near the surface, and the incubation irradiance was
high. Both the net and gross estimates of N2-fixation rates follow our conceptual model in Figure 6
if the trends were extended to near surface irradiance. However, our lab experimental data extend
only as high as 220 lmol quanta Æ m)2 Æ s)1, so the
general trends in these parameters at very high surface light intensities (>1,000 lmol quanta Æ
m)2 Æ s)1) remain speculative.
Kranz et al. (2010) reported similar combined
effects of irradiance and elevated pCO2 on gross
N2-fixation rates in IMS 101. Under low light
(50 lmol quanta Æ m)2 Æ s)1), gross N2-fixation rates
were 200% higher in a high-pCO2 treatment
(900 latm) compared with a low-pCO2 treatment
(150 latm), whereas under high light (200 lmol
quanta Æ m)2 Æ s)1), gross N2-fixation rates were only
112% higher under elevated pCO2 compared with a
low-pCO2 treatment. Collectively, all of these studies
indicate that the effect of elevated pCO2 on gross
N2-fixation rates in Trichodesmium is high under low
irradiance and that this effect decreases as light
increases.
At elevated pCO2, reduced demand for active
transport of inorganic carbon across the cell membrane may shift additional energy to N2 fixation in
Trichodesmium. Several authors have hypothesized
that elevated pCO2 may indirectly enhance N2-fixation rates in diazotrophic cyanobacteria by this
mechanism (Barcelos e Ramos et al. 2007, Hutchins
et al. 2007, Levitan et al. 2007, Fu et al. 2008, Kranz
et al. 2009). Badger et al. (2006) and Price et al.
(2008) report that Trichodesmium actively transports
HCO3) across the cell membrane with low- to medium-affinity transporters, and lacks high-affinity
carbon transporters. Kranz et al. (2009), however,
1299
indicated that K1 ⁄ 2 values for HCO3) changed in
response to pCO2, perhaps due to differential
expression or posttranslational modification of medium- and low-affinity HCO3) transporters. Our
experiments appear to support the energy shift
hypothesis at mid- and low irradiances, but our laboratory and field data suggest that this effect on gross
N2-fixation rates diminishes at high light.
One possible explanation for the declining effect
of elevated pCO2 on gross N2-fixation rates as irradiance increased is that oxygen production was higher
in the high-light, high-pCO2 treatment relative to
the high-light, present-day pCO2 treatment. Declining N2-fixation rates could then be attributed to the
well-documented inhibitory effect of oxygen on the
nitrogenase enzyme (Gallon 1981, Zehr et al. 1993).
We discount this explanation, however, as Kranz
et al. (2010) reported convincing evidence that O2
production did not increase with pCO2 in their
high-light treatments with IMS 101.
Another possible cause for this declining effect of
elevated pCO2 on gross N2-fixation rates at high
light is differences in intracellular N concentration
between pCO2 treatments. This hypothesis assumes
that intracellular N concentration exerts an influence on gross N2-fixation rates through a negative
feedback response. Indeed, the cellular N content
in our experiment with IMS increased with increasing light (Fig. 4b), with the highest cellular N concentration in the high-light, high-pCO2 treatment.
We speculate that this higher intracellular N concentration may have caused the reduced gross
N2-fixation rates in this treatment.
At high light, our cellular N content data corroborate findings by Kranz et al. (2010) showing that
particulate organic nitrogen (PON) production
increased as a function of pCO2. We speculate that
the relatively high accumulation of N in the highpCO2 treatment probably had an inhibitory effect
on N2 fixation, and data from Levitan et al. (2010)
and Kranz et al. (2010) seem to support this hypothesis. In their experiment, low concentrations of the
nitrogenase Fe protein (NifH) were associated with
high PON production rates in the high-light, highpCO2 treatment, whereas high NifH concentrations
were associated with low PON production rates in a
low-pCO2 treatment. Those data suggest that high
PON levels at high irradiance may influence a negative feedback response on NifH at the transcription
level. At low irradiance, our gross N2-fixation data
are similar to the high effect of elevated pCO2 on
gross N2 fixation reported by Kranz et al. (2010). As
discussed above, the reason for the high gross:net
ratio in our high-pCO2 treatment at low light with
IMS remains unknown. The lack of a pronounced
effect of elevated pCO2 on net 15N2 uptake rates we
documented at low light (Fig. 2g) does not seem to
be consistent with the increased PON production
rates at elevated pCO2 at low irradiance reported by
Kranz et al. (2010).
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NATHAN S. GARCIA ET AL.
Interestingly, both trichome length and irradiance
seemed to be related to cellular N retention in IMS
(Fig. 5). For instance, when trichome length did not
change as a function of light, as in the high-pCO2
treatment, the gross:net ratio declined with increasing irradiance, implying that cellular N retention
increased with increasing light (see Mulholland
et al. 2004, Mulholland 2007). In the present-day
pCO2 treatment, trichome length varied dramatically
with light, increasing with decreasing irradiance. In
this case, the gross:net ratio remained low at all light
levels, implying that cellular N retention did not
change as a function of light. Although we are not
aware why trichome length was so dramatically different between pCO2 treatments at low irradiance in
our experiment with IMS, these data suggest that
trichome length and cellular N retention might be
related in some way. Conversely, Levitan et al.
(2007) reported an increase in average trichome
length with increasing pCO2 in IMS, although differences were not significant between pCO2 treatments,
and that experiment was conducted at 80 lmol
quanta Æ m)2 Æ s)1. The largest impact of elevated
pCO2 on trichome length in our experiment with
IMS was at a considerably lower irradiance of
38 lmol quanta Æ m)2 Æ s)1. Although the gross:net
N2-fixation rate ratio is correlated with trichome
length data in our CO2 ⁄ light experiment with IMS,
the physiological reason for this relationship remains
unclear. Unfortunately, we do not have estimates of
gross:net N2-fixation rates for GBR, and we did not
measure trichome length in that experiment. Future
experiments are needed to investigate this relationship further, particularly at low irradiance.
Elevated pCO2 did not enhance net cellular CO2fixation rates at low light, but did at higher light levels in our light ⁄ CO2 experiment with IMS (Fig. 2, a
and b). Similarly, elevated pCO2 acted to increase
cellular growth rates of IMS only under mid- and
high irradiance (Fig 1b); growth rates were not different between treatments of pCO2 under low light.
In contrast, Kranz et al. (2010) reported that the
highest effect of elevated pCO2 on PON production
and growth rates was at low irradiance, which was
somewhat proportional to the effect of elevated
pCO2 on gross N2-fixation rates. In contrast, for the
same IMS strain, we found the largest growth rate
enhancement by pCO2 at high light (Fig. 1b). Our
growth rate data for GBR, however, corroborate the
combined effects of light and elevated pCO2 on
growth rates in IMS reported by Kranz et al. (2010)
(Fig. 1c). Growth rates of GBR were higher in the
elevated-pCO2 treatment at low light but were not
different between pCO2 treatments at high light.
Differences in cellular N retention in response to
light and pCO2 might explain differences in the
response of gross N2-fixation rates to elevated pCO2
between our experiments with IMS and GBR. For
example, elevated pCO2 had a significant positive
effect on gross N2-fixation rates by IMS at all light
levels when normalized to cellular N (Fig. 2, c and
e). This is not the case for GBR, however; elevated
pCO2 only had a significant positive effect on maximum gross N2-fixation rates under low light when
normalized to cell density (Fig. 3f). Differences in
cellular N retention could have influenced the cellular N content between strains and may be responsible for the differing effects of elevated pCO2 on
gross N2-fixation rates.
We assume that the ratio of acetylene reduction
to N2 reduction did not vary substantially between
our treatments of light and pCO2. Various studies
have used different ratios for Trichodesmium and
other cyanobacteria (Carpenter and Price 1977, Mague et al. 1977, Montoya et al. 1996). For consistency, we used the acetylene:nitrogen reduction
ratio of 3:1, which has been used in our previous
studies involving Trichodesmium IMS and GBR
(Hutchins et al. 2007). While the precision of this
ratio might be important for determining N retention and exudation when examining the gross:net
N2-fixation rates in field studies, we were mostly
interested in making comparisons between culture
experimental treatments. The assumption that the
ratio of acetylene:nitrogen reduction does not
change between treatments of pCO2 and light may
or may not be valid, and this question needs to be
examined in future studies.
Even though our estimates of gross and net N2fixation rates were calculated over different amounts
of time (10 h for gross and 12 h for net estimates),
incubation times for both estimates were centered
on the middle of the light period, where N2-fixation
rates are highest (Berman-Frank et al. 2001, Kranz
et al. 2009). For example, our gross N2-fixation rates
probably accounted for >90% of the total N2 fixed.
Still, our gross rates are probably slightly higher
than they would have been if they were documented
over a 12 h period, thus affecting our calculated
gross:net N2-fixation rate ratios. Minor differences
in this ratio would however probably not affect our
interpretation of the data.
Steady-state cell-specific growth rates in our
experiments with IMS were saturated near 100 lmol
quanta Æ m)2 Æ s)1 (Fig. 1, a and b), while Goebel
et al. (2008) and Breitbarth et al. (2008) report an
IMS growth saturation point near 140 lmol
quanta Æ m)2 Æ s)1 and 180 lmol quanta Æ m)2 Æ s)1,
respectively. One important distinction that separates our study from those other two studies is that
growth kinetic data in our study were collected from
steady-state, semicontinuous batch cultures, whereas
those studies used batch cultures in non-steady-state
growth. Although we do not have enough growth
rate data in response to light for GBR to predict a
light saturation point for growth, our data suggest
that the maximum steady-state growth rate for GBR
is near 0.45 d)1 (Fig. 1). Similar to results reported
by Staal et al. (2007), gross N2-fixation rates were
saturated near 100 lmol quanta Æ m)2 Æ s)1 in our
L I G H T A N D CO 2 E F F E C T S O N T R I C H OD E S M I U M N 2 F I X A T I O N
CO2 ⁄ light experiment with IMS in both low- and
high-pCO2 cultures. In addition, the impact of
elevated pCO2 on gross N2-fixation rates in response
to elevated pCO2 was greatest at 100 lmol quanta Æ
m)2 Æ s)1.
The colonial form often dominates natural Trichodesmium blooms rather than free trichomes. Our
laboratory cultures did not contain colonies, but
instead consisted of free trichomes. The physiological responses of colonial Trichodesmium to elevated
pCO2 and changing irradiance might be different
from the responses of free trichomes, and further
studies with intact natural colonies are needed.
Future field studies should examine the influence
of elevated pCO2 on CO2 and N2 fixation at higher
light intensities. Our results suggest that elevated
pCO2 may not have a positive effect on gross N2-fixation rates at surface irradiance, which would yield
a lower net effect of rising pCO2 on global N2-fixation rates than previously estimated. Instead, the
effect of elevated pCO2 on gross N2-fixation rates
may be more important at lower light intensities as
suggested in our conceptual model in Figure 6. Capone et al. (1994) suggested that a major pathway
of N and C transfer from Trichodesmium through trophic levels might be by amino acid leakage from
Trichodesmium cells, followed by assimilation by heterotrophs. If elevated pCO2 and light can influence
cellular N retention in the open ocean, this finding
may have consequences for the ecology and biogeochemistry of future oceans. For instance, models
predict higher average irradiance experienced by
phytoplankton circulating within shallower mixed
layers and modest increases in primary productivity
(Boyd and Doney 2002, Sarmiento et al. 2004, Breitbarth et al. 2007, Boyd et al. 2010). Finally, it is evident that future studies investigating the impacts of
elevated pCO2 on CO2 fixation and N2 fixation in
Trichodesmium should include field experiments that
encompass the entire mixed layer.
We thank Eric Webb for allowing us to use his gas chromatograph for the acetylene reduction assay, and Doug Capone
for allowing us to use some of his general lab equipment.
Grant support was provided by NSF OCE 0722337 to D.
Hutchins, NSF OCE 0850730 to F.-X. Fu, and NSF OCE
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