Introduction
Next to light, nitrogen (N) is the major limiting factor for primary
productivity in much of the low-latitude surface ocean (Falkowski, 1997;
Moore et al., 2013). Nitrate (NO3-) is the dominant form of fixed
nitrogen (N) in seawater and derives from the remineralization of sinking
organic N in the dark ocean. NO3- is supplied to photic waters by
upward mixing and transport, and constitutes the main source of fixed N for
photosynthetic organisms in the temperate and high-latitude ocean. In the
oligotrophic tropical and subtropical oceans, vertical mixing and transport
of NO3- is generally low and surface waters are often depleted in
NO3-.
In these ocean deserts, specialized organisms termed dinitrogen
(N2) fixers (or diazotrophs) are able
to use N in its simplest and most abundant form on Earth and in seawater,
namely dinitrogen (N2). Diazotrophs possess the nitrogenase enzyme,
which cleaves the strong triple bond of the N2 molecule to form
bioavailable ammonium (NH4+), which is assimilated as amino acids,
enabling biomass growth and division. N2 fixation thus introduces a
source of new bioavailable N to surface waters, and is considered to be the
most important external source of N to the ocean, more significant than
atmospheric and riverine inputs (Gruber, 2004).
The dynamics of microbial communities such as diazotrophs can change
abruptly in the ocean in response to small perturbations or environmental
stressors. In particular, N2 fixation has been described as a very
“patchy” process in the ocean (Bombar et al., 2015).
Many factors control the distribution and activity of diazotrophs such as
temperature (Bonnet et al., 2015; Moisander et al., 2010; Raveh et al.,
2015; Staal et al., 2003), nutrient availability (mainly phosphate and iron)
(e.g., Mills et al., 2004), pCO2 (e.g.,
Levitan et al., 2007), ambient concentrations of fixed N
(NO3- and NH4+) (e.g., Knapp et
al., 2012), and physical forcing (e.g., Fong et al., 2008). Most
studies dedicated to understanding the controls on marine N2 fixation
have been undertaken along large oceanic transects; these are particularly
valuable and have recently led to the compilation of a global ocean database
of diazotrophy (Luo et al., 2012). Spatial variability in N2
fixation is thus far better documented and understood than temporal
variability, despite the intimate connections between time and space scales
in the ocean. Time-series stations with near-monthly observations set up in
the late 1980s as part of the international JGOFS program in the subtropical
North Atlantic, Pacific, and Mediterranean Sea have provided valuable data
regarding the controls on N2 fixation and its role in biogeochemical
cycles on seasonal and interannual timescales (Dore et al., 2008; Garcia
et al., 2006; Grabowski et al., 2008; Karl et al., 2012; Knapp et al., 2005;
Orcutt et al., 2001), and have also revealed novel diazotrophic
microorganisms (Zehr et al., 2008) with unexpected
metabolic strategies such as UCYN-A cyanobacteria that lack the
oxygen-producing photosystem II complex (Tripp et al.,
2010). However, fairly little attention has been paid to sub-seasonal
variability in N2 fixation and its biogeochemical drivers and
consequences.
In the framework of the VAHINE (VAriability of vertical and tropHIc transfer
of diazotroph derived N in the south wEst Pacific) project, we deployed
three large-volume mesocosms (∼ 50 m3, Fig. 1) in the
tropical southwest Pacific coastal ocean, a region known to support
diazotrophy during the austral summer (Dupouy et al., 2000; Rodier and Le
Borgne, 2008, 2010). Our goal was to study the high-frequency temporal
dynamics of N2 fixation over short timescales (sampling every day for
23 days), in relation to hydrological parameters, biogeochemical stocks and
fluxes, and the dynamics of phytoplanktonic and bacterial communities in the
same water mass.
(a) Mesocosms (∼ 50 m3) deployed in the
framework of the VAHINE project. (b) Sediment traps screwed onto the base of
the mesocosms and were sampled daily by scuba divers.
The mesocosm approach allowed us to investigate the fate of the recently
fixed N2 and its transfer from diazotrophs to non-diazotrophic
organisms in this oligotrophic marine ecosystem. Diazotrophs can typically
release from 10 to 50 % of their recently fixed N2 (or diazotroph-derived N, hereafter called DDN) as dissolved organic N (DON) and
NH4+ (Glibert and Bronk, 1994; Meador et al., 2007;
Mulholland et al., 2006). This exudate is potentially available for
assimilation by the surrounding planktonic communities. However, such
transfer of DDN to the surrounding planktonic community and its potential
impact on export production is poorly understood and rarely quantified.
Over the course of this 23-day mesocosm experiment, diatom–diazotroph
associations (DDAs) were the most abundant N2 fixers during the
first half of the experiment (days 2 to 14), while a bloom of the unicellular
N2-fixing cyanobacteria from group C (UCYN-C) occurred during the second
half of the experiment (days 15 to 23) (Turk-Kubo et al., 2015). In the
VAHINE special issue, Berthelot et al. (2015b) described the evolution of the
C, N, and P pools and fluxes during the experiment and investigated the
contribution of N2 fixation and DON uptake to primary production and
particle export. They also explored the fate of the freshly produced
particulate organic N (PON), i.e., whether it was preferentially accumulated
and recycled in the water column or exported out of the system. Complementary
to this approach, Knapp et al. (2015) reported the results of a
δ15N budget performed in the mesocosms to assess the dominant
source of N (i.e., NO3- vs. N2 fixation) fueling export
production during the 23-day experiment. In the present study, we focus
specifically on the fate of DDN in the ecosystem during the UCYN-C bloom by
studying (i) the direct export of diazotrophs into the sediment traps and
(ii) the transfer of DDN to non-diazotrophic plankton using high-resolution
nanometer-scale secondary ion mass spectrometry (nanoSIMS) coupled with
15N2 isotopic labeling during a 72 h process experiment.
Results
N2 fixation rates in the mesocosms
Bulk N2 fixation rates averaged 18.5 ± 1.1 nmol N L-1 d-1
throughout the 23 days of the experiment in the three mesocosms
(all depths averaged together) (Table 1). The variance between the three
mesocosms was low, and the temporal dynamics of the rates were similar
(Fig. 2, Table 1), indicating good replicability between the mesocosms. Based on
our data on N2 fixation dynamics, we could identify three main periods
during the experiments. These three periods were also defined by
Berthelot et al. (2015b) based on biogeochemical
characteristics and by Turk-Kubo et al. (2015) based on
changes in abundances of targeted diazotrophs. During the first period (P0;
from day 2 to 4, i.e., prior to the DIP fertilization), the average bulk
N2 fixation rate for the three mesocosms was
17.9 ± 2.5 nmol N L-1 d-1 (Fig. 2a). These N2 fixation rates decreased
significantly (p < 0.05) by ∼ 40 % from day 5 to
∼ 15 (hereafter called P1) to
10.1 ± 1.3 nmol N L-1 d-1 and then increased significantly (p < 0.05) from day 15 until
the end of the experiment (day 15 to 23, hereafter called P2) to an average
of 27.3 ± 1.0 nmol N L-1 d-1 (Fig. 2a). Maximum rates were
reached during P2 (between days 18 and 21) with 69.7, 67.7, and
60.4 nmol N L-1 d-1 in M1 (12 m), M2 (6 m), and M3 (12 m), respectively. From
day ∼ 15 to 21, N2 fixation rates were higher at 12 m
depth than in the surface. The difference was significant in M2 and M3
(p < 0.05), but not in M1 (p > 0.05). Size fractionation
experiments indicate that 37 ± 7 % of the measured N2 fixation
was associated with the < 10 µm size fraction (Fig. 2b), and
N2 fixation rates in this fraction followed the same temporal trend as
bulk N2 fixation. These data indicate that, for the experiment as a
whole, the majority (∼ 63 %) of the N2 fixation was
associated with the > 10 µm fraction. N2 fixation
rates measured in the lagoon waters were half those measured in the
mesocosms, and were on average 9.2 ± 4.7 nmol N L-1 d-1 over
the 23 days of the experiment.
N2 fixation rates (nmol N L-1 d-1) measured in the
mesocosms and in lagoon waters. Table shows the range, median, mean,
contribution of the < 10 µm fraction to total rates
(%), and number of samples analyzed (n). NA – not available.
Range
Median
Mean
% < 10 µm
n
M1
0.5–69.7
15.9
19.7
38
61
M2
3.0–67.7
15.1
18.1
43
57
M3
2.9–60.4
14.2
17.7
29
59
Average mesocosms
2.1–65.9
15
18.5
37
177
Lagoon waters
1.9–29.3
8.7
9.2
NA
61
(a) Horizontal and vertical distributions of bulk N2
fixation rates (nmol N L-1 d-1) in M1, M2, M3 and lagoon waters,
and (b) < 10 µm N2 fixation rates
(nmol N L-1 d-1) in M1, M2, and M3. Note that N2 fixation
rates in the < 10 µm fraction were not measured in lagoon
waters.
The Spearman correlation matrix (Table 2) indicates that N2 fixation was
positively correlated with seawater temperature in the mesocosms, which was
not the case in lagoon waters, although temperature was exactly the same
inside and outside the mesocosms (from 25.4 to 26.8 ∘C) (Bonnet et
al., 2016b). N2 fixation in the mesocosms was also positively correlated
with particulate organic carbon (POC), nitrogen (PON), and phosphorus
(POP) (except in M2) concentrations; Chl a concentrations; primary
production; bacterial production; alkaline phosphatase activity (APA); and
Synechococcus, picoeukaryote, and nanoeukaryote (except in M2)
abundances. N2 fixation was negatively correlated with NO3-,
DIP, DON, dissolved organic phosphorus (DOP) (except in M2) concentrations,
and DIP turnover time.
Spearman correlation matrix of N2 fixation
rates and hydrological parameters, biogeochemical stocks and fluxes, and
planktonic communities (n= 66). The significant correlations
(p < 0.05) are indicated in bold. NA – not available.
Parameter
M1
M2
M3
Lagoon waters
Hydrological parameters
Temperature
0.394
0.319
0.347
0.228
Salinity
0.211
0.213
0.266
-0.122
Biogeochemical stocks and fluxes
NO3-
-0.539
-0.302
-0.341
0.145
NH4+
0.152
0.103
0.006
0.197
DIP
-0.613
-0.569
-0.482
-0.116
DON
-0.329
-0.413
-0.235
-0.180
DOP
-0.563
-0.157
-0.316
-0.243
PON
0.575
0.293
0.494
0.077
POP
0.514
0.001
0.439
0.036
POC
0.399
0.352
0.356
-0.061
Chl a
0.660
0.656
0.656
0.220
Primary production
0.443
0.498
0.445
0.268
Bacterial production
0.708
0.408
0.471
0.189
T-DIP
-0.670
-0.603
-0.564
-0.190
APA
0.575
0.568
0.273
-0.062
Planktonic communities
HNA
0.317
-0.043
0.458
NA
LNA
0.262
-0.021
0.000
NA
Prochlorococcus
0.429
-0.122
0.138
NA
Synechococcus
0.699
0.434
0.499
NA
Picoeukaryotes
0.614
0.563
0.414
NA
Nanoeukaryotes
0.477
0.002
0.442
NA
Diatoms
-0.099
0.456
-0.200
NA
Dinoflagellates
0.242
-0.392
-0.321
NA
UCYN-A1
0.545
-0.521
-0.503
0.200
UCYN-A2
0.127
-0.631
0.248
0.333
UCYN-B
0.083
0.696
0.467
0.101
UCYN-C
0.373
0.621
0.515
-0.167
Trichodesmium
-0.145
0.147
0.285
-0.117
DDAs
-0.036
-0.264
-0.527
0.262
γ-24774A11
0.327
0.497
-0.750
0.733
The intercomparison between the bubble and dissolution methods performed on
day 11 in M2 indicates that rates determined for the six replicates were
7.2 ± 0.8 and 6.4 ± 2.0 nmol N L-1 d-1
for the dissolution method and the bubble method, respectively,
demonstrating that, at least in this study, N2 fixation rates were not
significantly different (p > 0.05) between the two methods.
Phenotypic characterization of UCYN by microscopy
The average size of the UCYN-C cells present in the mesocosms was
5.7 ± 0.8 µm (n= 17). Both free-living and aggregated UCYN-C cells were
observed in the water columns of the mesocosms. However, the detailed
microscopic analysis performed on day 17 and day 19 in M2 (during the bloom
of UCYN-C) (Fig. 3) indicates that the proportion of free-living cells (ROI
characterized by one cell or two cells defined as dividing cells) was low
(< 1 % on day 17 and < 5 % on day 19). The average
number of UCYN-C cells per aggregate increased with depth (Fig. 3a), with
the size of the aggregates reaching 50–100 µm at 6 m and
100–500 µm at 12 m depth. On day 17, the number of cells per aggregate
averaged 162, 74, and 1273 at 1, 6, and 12 m, respectively. On day 19, the
aggregates were much smaller (∼ 50 µm) with only 4, 11,
and 19 cells per aggregate. The sediment traps contained extremely high
densities of UCYN-C cells, with the average number of cells per aggregate 60
to 50 000 times higher than that measured in the water column aggregates
(Fig. 3b–e).
(a) UCYN-C cells per aggregate in M2 on day 17 and 19.
(b–e) Green excitation (510–560 nm) epifluorescent replicate micrographs of UCYN-C
on day 17 taken at 1 m depth (×40) (b), 6 m depth (×40) (c), 12 m depth
(×40) (d), and in the sediment traps (×10) (e). Scale bar 20 µm (b–d)
and 100 µm (e).
Quantification of diazotrophs in sediment traps
qPCR analysis confirmed that UCYN-C was the most abundant diazotroph in the
sediment traps on days 17 and 19, with abundances reaching 2.7 × 108 to
4 × 109 nifH copies L-1 (Fig. 4a). UCYN-C accounted for
97.4 to 99.2 % of the total nifH pool quantified in the traps. Abundances were higher in
M2 and M3 (1.8 × 109 in M2 and 3 × 109 nifH copies L-1 in M3)
compared to M1 (2.5 × 108 nifH copies L-1) on day 19. Het-1 and het-3
were always recovered in the sediment traps, albeit at lower abundances (1.8
to 8.6 × 106 nifH copies L-1 for het-1 and 4.9 × 106 to
2.8 × 107 nifH copies L-1 for het-3) (Fig. 4b). They represented between
0.1 and 1.8 % of the targeted nifH pool. UCYN-B was detected in
all mesocosm traps on both days (except
in M1 on day 19), and UCYN-A2 and Trichodesmium were detected in M2
on day 17 but at low abundances (0.05 % of the total nifH pool)
compared to the other phylotypes. Het-2 was never detected in the traps, and
neither was γ24774A11 or UCYN-A1.
(a) Abundance of UCYN-C (nifH copies L-1)
and (b) other nifH phylotypes (UCYN-A2, UCYN-B,
Trichodesmium, het-1, het-3) (nifH copies L-1)
recovered in the sediment trap on day 17 and 19. (c) Proportion of
POC export associated with diazotrophs in the sediment traps on day 17 in M2
(height of UCYN-C bloom).
Using the volume of each mesocosm (Bonnet et al., 2016b) and
the total nifH copies for each diazotroph phylotype in the sedimenting material
and in the water column the day before the collection of the sediment traps
(Turk-Kubo et al., 2015) (assuming a sinking velocity of
the exported material of ∼ 10 m d-1; Gimenez
et al., 2016), we estimated the export efficiency for each phylotype. For
UCYN-C, 4.6 and 6.5 % of the cells present in the water column were
exported to the traps per 24 h on day 17 and 19, respectively (assuming one
nifH copy per cell). For het-1, 0.3 and 0.4 % of cells were exported into
the traps on day 17 and 19; for het-3, 15.5 and 10.5 % were
exported; and for UCYN-B, 37.1 and 15.5 % of UCYN-B were exported on
day 17 and 19, respectively.
DDN transfer experiment performed on day 17
Net 15N2 uptake was 24.1 ± 2.8 nmol N L-1 during the
first 24 h of the DDN transfer experiment performed from days 17 to 20
(Fig. 5a). As expected, integrated 15N2 uptake increased over the course
of the experiment to reach 28.8 ± 4.3 nmol N L-1 at T48 h and
126.8 ± 35.5 nmol N L-1 at T72 h. The DDN quantified in the TDN
pool ranged from 6.2 ± 2.4 nmol N L-1 at T24 h to 9.6 ± 1.6 nmol N L-1
at T72 h. Considering gross N2 fixation as the sum of
net N2 fixation and DDN release (Mulholland et al., 2004),
the DDN released to the TDN pool accounted for 7.1 ± 1.2 to 20.6 ± 8.1 % of gross N2 fixation.
During the 72 h targeted experiment (Fig. 5b) the diazotroph assemblage
reflected that of the mesocosms from which they were sampled: UCYN-C
dominated the diazotrophic community, comprising on average 62 % of the
total nifH pool. The other most abundant phylotypes were UCYN-A2 and het-2,
which represented 18 and 13 % of the total nifH pool, respectively. UCYN-A1,
UCYN-B, het-1, het-3, and Trichodesmium were also detected but together comprised
less than 8 % of the total targeted community. Phylotype abundances
remained relatively stable throughout the 72 h of the experiment.
Results from the DDN transfer experiment performed from day 17 to 20
in M2. (a) Temporal changes in 15N2 uptake
(white, nmol N L-1) and quantification of DDN in the dissolved pool
(grey) over the course of the experiment. Error bars represent the standard
deviation of three independent replicate incubations. (b) Temporal
changes in diazotroph abundance determined by qPCR
(nifH gene copies L-1) during the same experiment. Error
bars represent the standard deviation of triplicate incubations.
(c) Summary of the nanoSIMS analyses. Measured 13C and
15N at. % values of non-diazotrophic diatoms (white) and
picoplankton (grey) as a function of incubation time. The horizontal dashed
line indicates the natural abundance of 15N (0.366 at. %), and the
error bars represent the standard deviation for the several cells analyzed by
nanoSIMS.
NanoSIMS analyses performed on individual UCYN-C at 24 h (Fig. 6) revealed
significant (p < 0.05) 13C (1.477 ± 0.542 at. %,
n= 35) and 15N (1.515 ± 0.370 at. %, n= 35) enrichments
relative to natural abundance, indicating that UCYN-C were actively
photosynthesizing and fixing N2. The correlation between 13C
enrichment and 15N enrichment was significant (r= 0.85,
p < 0.01, Fig. 6b). NanoSIMS analyses performed on diatoms and
picoplankton (Fig. 5c) also revealed significant (p < 0.05)
15N enrichment of non-diazotrophic plankton, demonstrating a transfer of
DDN from the diazotrophs to other phytoplankton. Both diatoms and
picoplanktonic cells were significantly (p < 0.05) more enriched
at the end of the experiment (T72 h) (0.489 ± 0.137 at. %,
n= 12 for diatoms; 0.457 ± 0.077 at. %,
n= 96 for picoplankton) than after the first 24 h (0.408 ± 0.052 at. %,
n= 23 for diatoms; 0.389 ± 0.014 at. %, n= 63 for
picoplankton). Finally, the 15N enrichment of picoplankton and diatoms
was not significantly different (p > 0.05) during the DDN
experiment.
Discussion
The bubble vs. the dissolution method: an intercomparison
experiment
The intercomparison experiment performed on day 11 reveals slightly lower,
yet insignificantly different (p > 0.05), average N2
fixation rates when using the bubble method compared to the dissolution
method. This result is in accordance with some comparisons made by Shiozaki
et al. (2015) in temperate waters of the North Pacific. However, a lower
degree of dissolution of the 15N2 bubble may occur in warm tropical
waters such as those near New Caledonia compared to the cooler, temperate
North Pacific waters. In calculating N2 fixation rates using the
dissolution method, we used the value of 2.4 ± 0.2 at. % for the
15N enrichment of the N2 pool as measured by MIMS. For the bubble
method, we used the theoretical value of 8.4 at. % calculated for
seawater with a temperature of 25.5 ∘C and salinity of 35.3 (as was
the case on day 11). If we assume that equilibration was incomplete in our
experiment using the bubble method, i.e., 75 % instead of 100 % as
shown by Mohr et al. (2010), we calculate higher, albeit still insignificant
(p > 0.05), N2 fixation rates for the bubble method
(8.3 ± 2.8 nmol N L-1 d-1) compared to the dissolution
method (7.2 ± 0.8 nmol N L-1 d-1), confirming that
equivalent results are obtained with both methods in this ecosystem.
(a) Green excitation (510–560 nm) epifluorescent
micrographs of UCYN-C, (b) 13C and 15N isotopic enrichment
(at. %) in individual UCYN-C cells on day 17 in M2, and (c, d) nanoSIMS images showing the 13C (c) and
15N (d) enrichment of individual UCYN-C cells after 24 h of
incubation. The white outlines show regions of interest (ROIs), which were
used to estimate the 13C / 12C and 15N / 14N
ratios.
The temporal dynamics of N2 fixation in the mesocosms
Average N2 fixation rates measured in the lagoon waters (outside the
mesocosms, 9.2 ± 4.7 nmol N L-1 d-1, Table 1) are of the same
order of magnitude as those reported for the Noumea lagoon during austral
summer conditions (Biegala and Raimbault, 2008). They are
within the upper range of rates reported in the global ocean database
(Luo et al., 2012). Indeed, open ocean cruises performed offshore of New
Caledonia in the Coral and Solomon seas (e.g., Bonnet et al., 2015;
Garcia et al., 2007) also suggest that the southwest Pacific Ocean is one
of the areas with the highest N2 fixation rates in the global ocean.
Averaged over the 23 days of the experiment, N2 fixation rates in the
mesocosms were ∼ 2-fold higher (18.5 ± 1.1 nmol N L-1 d-1)
than those measured in lagoon waters (9.2 ± 4.7 nmol N L-1 d-1).
The maximum observed rates of > 60 nmol N L-1 d-1
from days 18 to 21 are among the highest reported for marine
waters (Luo et al., 2012). DIP concentration was the predominant
difference between the ambient lagoon waters and those of the mesocosms. The
mesocosms were fertilized with DIP on day 4, reaching ambient concentrations
of ∼ 0.8 µmol L-1 compared to lagoon waters in
which DIP concentrations were typically < 0.05 µmol L-1.
According to our experimental assumption, diazotrophy would be promoted by
high concentrations of DIP. Yet, in all three mesocosms, N2 fixation
rates were negatively correlated with DIP concentrations and DIP turnover
time and positively correlated with APA (Table 2). Below, we describe the
scenario that likely occurred in the mesocosms, which likely explains these
correlations.
During P0 (day 2 to 4), N2 fixation rates were higher in the mesocosms
than in the lagoon waters, possibly due to the reduction of turbulence in
the water column facilitated by the closing of the mesocosms
(Moisander et al., 1997) and/or to the reduction of the grazing
pressure in the mesocosms as total zooplankton abundances were slightly
lower (by a factor of 1.6) in the mesocosms compared to the lagoon waters
(Hunt et al., 2016). The most abundant diazotrophs in the
mesocosms at P0 were het-1 and Trichodesmium, which were probably the most competitive
groups under the initial conditions, i.e., NO3- depletion
(concentrations were 0.04 ± 0.02 µmol L-1, Table 3) and low
DIP concentrations (0.03 ± 0.01 µmol L-1, Table 3).
Trichodesmium is able to use organic P substrates (DOP pool) under conditions of DIP
deficiency (Dyhrman et al., 2006; Sohm and Capone,
2006). Twenty-four hours after the DIP fertilization (day 5), N2 fixation rates in
the mesocosms decreased by ∼ 40 %, reaching rates
comparable to those measured in lagoon waters during P1 (days 5 to 14).
Enhanced DIP availability likely enabled non-diazotrophic organisms with
lower energetic requirements and higher growth rates to outcompete the
diazotrophs in the mesocosms via utilization of recycled N derived from
recent N2 fixation. This is supported by the observation that
nanoeukaryotes and non-diazotrophic cyanobacteria such as Prochlorococcus sp. increased in
abundance during P1 (Leblanc et al., 2016) in the three
mesocosms when N2 fixation rates declined (Fig. 2).
Average NO3-, DIP, DON, and DOP concentrations (µmol L-1)
measured over the P0, P1, and P2 periods. NO3- and
DIP concentrations were determined using a segmented flow analyzer according
to Aminot and Kerouel (2007). The detection limit was 0.01 and 0.005 µmol L-1
for NO3- and DIP, respectively. DON and DOP
concentrations were determined according to the wet oxidation procedure
described in Pujo-Pay and Raimbault (1994) and
Berthelot et al. (2015b).
Average P0
Average P1
Average P2
NO3-
0.04 ± 0.02
0.03 ± 0.01
0.02 ± 0.01
DIP
0.03 ± 0.01
0.48 ± 0.20
0.08 ± 0.05
DON
5.19 ± 0.37
5.22 ± 0.54
4.73 ± 0.49
DOP
0.14 ± 0.01
0.16 ± 0.03
0.12 ± 0.02
During P2 (day 15 to 23), N2 fixation rates increased dramatically in
all three mesocosms. This period was defined by a high abundance of UCYN-C,
which were present in low numbers in the lagoon and within the mesocosms
during P0 and P1 (Turk-Kubo et al., 2015). The increase in
UCYN-C abundance was synchronous with a decrease in DIP concentrations in
the mesocosms (Turk-Kubo et al., 2015): UCYN-C abundance
first increased in M1 (day 11), then in M2 (day 13), and finally in M3 (day
15). In all cases, the increase in UCYN-C abundance coincided with low DIP
turnover time, indicative of DIP deficiency
(Berthelot et al., 2015b; Moutin et al., 2005).
Under NO3- depletion and low DIP availability, UCYN-C appeared to
be the most competitive diazotroph in the mesocosms, as they exhibited the
highest maximum growth rates compared to those calculated for the other
diazotrophic phylotypes for the same period (Turk-Kubo et
al., 2015). Some Cyanothece strains possess the genes required for utilization of
organic P substrates such as phosphonates (Bandyopadhyay et al.,
2011). Thus, UCYN-C, which were the major contributors to N2 fixation
during P2 (see below), may have used DOP as a P source during this period,
consistent with the negative correlation observed between N2 fixation
rates and DOP concentrations (except in M2, Table 2), and driving the
significant decline in DOP concentrations observed in all three mesocosms
during P2 (Berthelot et al., 2015b; Moutin et al.,
2005).
While temperature was not correlated with N2 fixation in the lagoon, we
observed a significant positive correlation between these parameters in the
mesocosms (Table 2), probably because some diazotrophic phylotypes present
in the mesocosms and absent in the lagoon waters were particularly sensitive
to seawater temperature. UCYN-C reached high abundances inside the
mesocosms, but was virtually absent in the lagoon waters outside the
mesocosms. Turk-Kubo et al. (2015) showed that UCYN-C
abundance was positively correlated with seawater temperature, suggesting
that the optimal temperature for UCYN-C growth is above 25.6 ∘C.
This result is consistent with culture studies performed using three UCYN-C
isolates from the Noumea lagoon that are closely related to the UCYN-C
observed here, indicating maximum growth rates at around 30 ∘C and
no growth below 25 ∘C (Camps, Turk-Kubo, Bonnet, personal communication, 2015).
Temperatures above 25.6 and up to 26.7 ∘C were
reached on day 12 and were maintained through to the end of the mesocosm
experiment, possibly explaining why UCYN-C was not observed during P0 (when
temperature was 25.4 ∘C) even though DIP turnover time was low
(below ∼ 1 day) (Berthelot et al.,
2015b; Moutin et al., 2005).
If low DIP concentrations and seawater temperatures greater than 25.6 ∘C are prerequisites for UCYN-C growth, an obvious question is
why they did not thrive (despite being present at low abundances) in the
lagoon waters during P2, when similar conditions prevailed. We consider three
possible explanations that are discussed extensively in
Turk-Kubo et al. (2015): first, it is possible that UCYN-C
are sensitive to turbulence, which was likely reduced in the mesocosms
compared to the lagoon waters that are susceptible to trade winds and tides.
Second, grazing pressures on UCYN-C may have been reduced as total
zooplankton abundances were slightly lower (by a factor of 1.6) in the
mesocosms compared to those in the lagoon waters (Hunt et al.,
2016). Third, the water masses outside the mesocosms changed with tides and
winds; thus, it is possible that UCYN-C were absent from the water mass
encountered outside the mesocosms when we sampled for this experiment.
In the mesocosms, the cell-specific 15N2 fixation rate measured on
day 17 (M2) for UCYN-C was
6.3 ± 2.0 × 10-17 mol N cell-1 d-1.
Multiplying this rate by the abundance of UCYN-C indicates that UCYN-C
accounted for 90 ± 29 % of bulk N2 fixation during that
period. This is consistent with the positive correlation observed between
N2 fixation rates and UCYN-C abundances in M2 (Table 2). In M1 and M3,
the correlation was also positive yet insignificant. This may have been due
to the low number of UCYN-C data points, thus decreasing the sensitivity of
the statistical test. Coupling between UCYN-C 13C and 15N
incorporation was significant (r= 0.85, p < 0.01) (Fig. 6b)
and contrasts with results reported by Berthelot et al. (2016) for UCYN-C, in
which 13C and 15N enrichment (and thus inorganic C and N2
fixation) was uncoupled in the cells. Based on their observations, these
authors suggest that the heterogeneity in the 15N and 13C
enrichments can be explained by a specialization of some cells that induces
variability in cell-specific 15N enrichment, e.g., diazocytes that contain the nitrogenase enzyme as in
the colonial filamentous Trichodesmium sp. Spatial partitioning of
N2 and C fixation by colonial unicellular types was also evidenced for
diazocyte-like formation in colonial Crocosphaera watsonii-like
(UCYN-B) cells (Foster et al., 2013). Here, UCYN-C cells fixed both 13C
and 15N proportionally, which suggests they did not utilize diazocytes
to separate diazotrophy from photosynthesis in our experiments.
Summary of the simplified pathways of N transfer in the
first trophic level of the food web and the potential impact on the sinking
POC flux at the height of the UCYN-C bloom in the VAHINE mesocosm
experiment.
UCYN aggregation and export
Throughout the 23 days of the experiment, the majority of N2 fixation
(63 %) occurred in the > 10 µm size fraction, even during
P2 when the small (5.7 ± 0.8 µm) unicellular UCYN-C dominated
the mesocosm diazotrophic community. These findings can be explained by the
aggregation of UCYN-C cells into large (> 10 µm)
aggregates (Fig. 7) that were retained on 10 µm filters (Fig. 3).
These large UCYN-C aggregates probably formed in part due to the presence of
sticky transparent exopolymer particles (TEP) (Berman-Frank et al., 2016) or other
extracellularly released proteins, and were characterized by a high sinking
velocity due to their large size (up to 500 µm in diameter) and a
density greater than that of seawater (Azam and Malfatti, 2007). Their
aggregation and subsequent sinking within the mesocosms likely explains why
volumetric N2 fixation rates were higher at 12 m than at the surface
during P2, as well as why the size of the aggregates increased with depth,
and why numerous large-size aggregates and extremely high abundances of
UCYN-C were recovered in the sediment traps. Aggregation processes may have
been favored by the low turbulence in the mesocosms, and it would be
necessary to confirm that such processes also occur in the open ocean.
Colonial phenotypes of UCYN (UCYN-B) have been observed in the water column
of the northern tropical Pacific (ALOHA station) (Foster et al.,
2013), but to our knowledge, this is the first time that UCYN have been
detected in sediment traps. Contrary to published data (e.g., White et al., 2012), here we demonstrate a greater
export efficiency of UCYN (∼ 10 % exported to the traps
within 24 h) compared to the export of DDAs (efficiency of 0.24 to 4.7 %).
Diatoms sink rapidly and DDAs have been found in sediment traps at
station ALOHA (Karl et al., 1997, 2012; Scharek et al.,
1999a, b), in the Gulf of California
(White et al., 2012), and in the Amazon River plume
(Subramaniam et al., 2008). In our study, we observed limited
export of het-1 (Richelia in association with Rhizosolenia) and het-3 (Calothrix) during P2, while het-2
(Richelia associated with Hemiaulus) was never recovered in the sediment traps. This is
likely because Hemiaulus has a lower sinking rate than Rhizosolenia due do its smaller size, or
may be more easily grazed by zooplankton than Rhizosolenia or Calothrix, which are known to be
toxic to crustaceans (Höckelmann et al., 2009). We observed
only rare occurrences of Trichodesmium export in this study probably due to its extremely
limited presence and low growth rates in the mesocosms. Direct comparisons
of our export results with findings from open ocean studies should be made
cautiously as our mesocosms were shallower (15 m) than typical oceanic
export studies (> 100 m) and were also probably characterized by
reduced turbulence (Moisander et al., 1997).
We estimate that the direct export of UCYN-C accounted for 22.4 ± 5.5 %
of the total POC exported in each mesocosm at the height of the UCYN-C
bloom (day 17) and decreased to 4.1 ± 0.8 % on day 19 (Figs. 4c and 7). This calculation is based on the total POC content measured in the
sediment traps (Berthelot et al., 2015b), our Ccon for
UCYN-C estimated as described above, and published Ccon for other
diazotrophs. The corresponding export of het-1, het-3, Trichodesmium, and UCYN-B on day
17 based on published Ccon (Leblanc et al., 2012; Luo et al., 2012),
and using an average of three Richelia and Calothrix symbionts per diatom, accounted for
6.8 ± 0.5, 0.5 ± 0.02, 0.3 ± 0.3, and 0.1 ± 0.01 % of
the POC export on day 17, respectively, and for 4.2 ± 1.7, 0.04 ± 0.03
of the POC export on day 19 (the contribution of Trichodesmium and UCYN-B to POC
export on day 19 was negligible). Thus, our data emphasize that, despite
their small size relative to DDAs, UCYN-C are able to directly export
organic matter to depth by forming densely populated aggregates that can
rapidly sink. This observation is further confirmed by the e ratio, which
quantifies the efficiency of a system to export POC relative to primary
production (e ratio = POC export/PP) and was significantly higher
(p < 0.05) during P2 (i.e., during the UCYN-C bloom; 39.7 ± 24.9 %)
than during P1 (i.e., when DDAs dominated the diazotrophic community;
23.9 ± 20.2 %) (Berthelot et al., 2015b). It is also
consistent with the significantly (p < 0.05) higher contribution of
N2 fixation to export production during P2 (56 ± 24 %, and up
to 80 % at the end of the experiment) compared to P1 (47 ± 6 %,
and never exceeded 60 %) as estimated by Knapp et al. (2015)
using a δ15N budget for the mesocosms. Our calculated
contribution of N2 fixation to export production is very high compared
to other tropical and subtropical regions where diazotrophs are present (10
to 25 %; e.g., Altabet, 1988; Knapp et al., 2005). However,
it is consistent with the high rates of N2 fixation measured in the
enclosed mesocosms compared to those from the lagoon and other tropical
pelagic studies (Luo et al., 2012). The direct export of UCYN-C and other
diazotrophs cannot solely explain the high e ratio estimated for P2. We thus
hypothesize that a fraction of the DDN export that occurred during P2 was
transferred indirectly via primary utilization by non-diazotrophic plankton
cells that were eventually exported to the sediment traps (Fig. 7).
DDN transfer to non-diazotrophic phytoplankton and ecological
implications
The amount of DDN measured in the TDN pool during the 72 h DDN transfer
experiment is higher than that reported for culture studies of Cyanothece populations
(1.0 ± 0.3 to 1.3 ± 0.2 % of gross N2 fixation;
Benavides et al., 2013; Berthelot et al., 2015a). The
DDN measured in the TDN pool reflects the DDN release by diazotrophs during
N2 fixation and is likely underestimated here as a fraction of this DDN
has been taken up by surrounding planktonic communities. In our experiment,
other diazotrophs were present in addition to Cyanothece, and they may have also
contributed to the dissolved pool. Moreover, unlike in culture studies,
field experiments are also impacted by other exogenous factors such as viral
lysis (Fuhrman, 1999) and sloppy feeding (O'Neil and
Roman, 1992; Vincent et al., 2007), which may enhance N release.
This DDN release plays a critical role in the N transfer between diazotrophs
and non-diazotrophs. The cell-specific uptake rates of DDN during the DDN
transfer experiment were calculated for each cell analyzed by nanoSIMS
(diatoms and cells from the 0.2–2 µm fraction). By multiplying
cell-specific N uptake rates by the cellular abundance of each group on a
particular day, we could identify the specific pool (diazotrophs, dissolved
pool, non-diazotrophs) into which the DD15N was transferred after 24 h,
as well as the extent to which this 15N2 accumulated. The results are
summarized in Fig. 7. After 24 h, 52 ± 17 % of the newly fixed
15N2 remained in the UCYN-C biomass, 16 ± 6 % had
accumulated in the dissolved N pool, and 21 ± 4 % had been
transferred to non-diazotrophic plankton. In addition, 11 % of the newly
fixed 15N2 accumulated in a pool that we refer to as “others”
(corresponding to diazotrophs other than UCYN-C and potential
non-diazotrophs to which 15N2 was transferred; these cells were
not analyzed by nanoSIMS due to their very low abundance). Uncertainties
take into account both the variability of the 15N enrichment determined
on ∼ 25 cells per group by nanoSIMS, and the uncertainty in
the N content per cell measured or taken from the literature.
Within the fraction of DDN transferred to the non-diazotrophs after 24 h
(21 %), we calculate that 18 ± 4 % was transferred to picoplankton,
and only 3 ± 2 % was transferred to diatoms (Fig. 7). The 15N
enrichment of picoplankton and diatoms was not significantly different
(p > 0.05) in this study, but as picoplankton dominated the
planktonic community in the mesocosms at the time of the DDN transfer
experiment, they were the primary beneficiaries of the DDN. This is
consistent with the positive correlation between N2 fixation rates,
Synechococcus, and picoeukaryote abundances in the mesocosms (Table 2), as well as with
the observed dramatic increase in Synechococcus and picoeukaryote abundances (by a
factor of > 2 between P1 and P2) (Leblanc et al.,
2016). Diatom abundances also increased in the mesocosms by a factor of 2
between P1 and P2 (largely driven by Cylindrotheca closterium), but this increase occurred earlier
than the picoplankton increase, i.e., at the end of P1 (days 11–12). Maximum
diatom abundances were reached on day 15–16 at the very beginning of P2, and
then declined by day 18 to reach abundances similar to those observed during
P1. These results suggest that diatoms were the primary beneficiaries of DDN
in the mesocosms at the start of P2, when N2 fixation rates and UCYN-C
abundances increased dramatically. This is consistent with a previous DDN
transfer study performed in New Caledonia (Bonnet et al.,
2016a) during which diatoms (mainly Cylindrotheca closterium) advantageously competed and
utilized DDN released during Trichodesmium blooms. When the present DDN transfer
experiment was performed (days 17 to 20), diatom abundances had already
declined, likely due to DIP limitation (DIP turnover time was low, i.e below
1 day). We hypothesize that picoplankton were more competitive for DDN under
low-DIP conditions as small cells with high surface to volume ratios are
known to outcompete larger cells for the available DIP (Moutin
et al., 2002). Moreover, some prokaryotes from the 0.2–2 µm
size fraction can utilize DOP compounds (Duhamel et al., 2012).
In this study, we could not discriminate the DDN transfer to picoautotrophs
from that to picoheterotrophs, but it is likely that both communities took
advantage of the DDN, as both primary production (Berthelot
et al., 2015b) and bacterial production (Van Wambeke et al.,
2015) were positively correlated with N2 fixation rates (Table 2) and
increased dramatically following the increase in N2 fixation during P2.
The standing stocks of POC, PON, and POP were also positively correlated
with N2 fixation rates, suggesting that DDN sustained productivity in
the studied system.
Conclusions
While studies on the fate of DDN in the ocean are rare, the contribution of
DDN to particle export based on the δ15N signatures of exported
material indicates that N2 fixation can efficiently contribute to export
production in the oligotrophic ocean (Dore et al., 2008).
The export of DDN may be either direct, through the sinking of diazotrophs,
or indirect, through the transfer of DDN to non-diazotrophic plankton in the
photic zone that are subsequently exported.
Trichodesmium is rarely recovered in sediment traps (Walsby, 1992) and most of
the research dedicated to the export of diazotrophs has focused on DDAs
(Karl et al., 2012) due to their high sinking velocity. Here,
we demonstrate for the first time that UCYN can efficiently contribute to
POC export in oligotrophic systems, predominantly due to the aggregation of
small (5.7 ± 0.8 µm) UCYN-C cells into large aggregates, which
increase in size (up to 500 µm) with depth. Our results suggest that
these small (typically 3–7 µm) organisms should be considered in
future studies to confirm whether processes observed in mesocosms are applicable
to open-ocean systems.
Moreover, the experimental and analytical approach used in this study allowed
for the quantification of the actual transfer of DDN to different groups of
non-diazotrophic plankton in the oligotrophic ocean. Our nanoSIMS results
coupled with 15N2 isotopic labeling revealed that a significant
fraction of DDN (21 ± 4 %) is quickly (within 24 h) transferred to
non-diazotrophic plankton, which increased in abundance simultaneously with
N2 fixation rates. A similar nanoSIMS study performed during a
Trichodesmium bloom (Bonnet et al., 2016a) revealed that diatoms
were the primary beneficiaries of DDN and developed extensively during and
after Trichodesmium.
blooms. Diatoms are efficient exporters of organic matter to depth (Nelson et
al., 1995). These studies show that plankton grown on DDN in the oligotrophic
ocean drive indirect export of organic matter out of the photic zone, thus
revealing a previously unaccounted for conduit between N2 fixation and
the eventual export to depth of DDN from the photic zone.