Introduction
Dinitrogen (N2) fixation is the major external source of new
nitrogen (N) for the upper ocean
and particularly in
the southwestern Pacific Ocean
, which is recognized as
one of the highest N2 fixation area in the global ocean
. While N availability
primarily controls autotrophic plankton growth in low-nutrient low-chlorophyll (LNLC) ecosystems ,
the new N sources provided by N2 fixation may drive the planktonic
ecosystem from N limitation toward P limitation and may potentially affect
the magnitude of C fixation and eventually C export through the so called
N2-primed prokaryotic C pump
. Consequently, it is
important to quantify N fluxes to the ocean as well as to study the fate of N
newly fixed by diazotrophs (or diazotroph-derived N, hereafter referred to as
DDN) in order to understand how N2 fixation affects nutrient cycles and
productivity in the ocean.
Biogeochemical models including N2 fixation have been developed over the
last decades, some of them including diazotrophic organisms as state
variables as described below. In these models, Trichodesmium is the
most frequently represented organism since it is the most studied diazotroph
and its physiology is well documented in the literature
. In recent studies, other diazotrophs such as
unicellular Cyanobacteria (termed UCYN) or diatom–diazotroph associations
(termed DDAs) have been implemented in biogeochemical models. This was first
done by , who developed a diagnostic model to
assess the relative contribution of three distinct diazotrophs (i.e., Trichodesmium sp. and two UCYN from Group A and Group B – UCYN-A and
UCYN-B, respectively), at the tropical North Pacific station ALOHA. More
recently, other biogeochemical models including a more complex planktonic
food web and the contribution of Trichodesmium sp. DDAs, UCYN-A and
UCYN-B were developed
, together
with models representing Trichodesmium sp., and a general group of
UCYN . Although increasing numbers of
models include diazotrophic organisms as part of the food web, none of them
have yet focused on the fate of DDN throughout the ecosystem. Diazotrophs
release part of the recently fixed N2 as dissolved organic N (DON) and
ammonium (NH4+) in the dissolved pool
. The magnitude of this
release (10 to 80 %) is still under debate in the scientific community
and seems to depend on the physiological state of the cells
as well as on exogenous factors such as
viral lysis or sloppy feeding
. Nevertheless, recent methods coupling
15N2 isotopic labeling, cell sorting by flow cytometry and
high-resolution nanometer-scale secondary ion mass spectrometry (nanoSIMS)
analyses enable the quantification of the DDN transfer from diazotrophs
to specific groups of non-diazotrophic phytoplankton and bacteria, indicating
that the DDN released in the dissolved pool is available and actively used by
surrounding non-diazotrophic communities .
The aim of the VAHINE project was to investigate the fate of DDN in
oligotrophic ecosystems by deploying large-volume (∼ 50 m3)
mesocosms to isolate a water mass with diazotrophs and by combining both
field biogeochemical and planktonic diversity measurements and a mechanistic
modeling approach. The New Caledonian (Nouméa) lagoon is considered an
oligotrophic ecosystem influenced by oceanic waters inflowing from outside
the lagoon . It supports high N2 fixation
rates (235 µmol N m-2 d-1; ), high
Trichodesmium sp.
and UCYN abundances . This site therefore
represented an ideal location to investigate the fate of DDN.
The mesocosms were intentionally enriched with dissolved inorganic phosphate
(DIP) to enhance the potential development of N2 fixers in the mesocosms
and therefore amplify N2 fixation fluxes and facilitate the study of DDN
pathways in the planktonic ecosystem. Complementary field approaches were
used during the VAHINE project including a δ15N budget to assess
the dominant source of N (from NO3- and/or N2 fixation) fueling export
production throughout the experiment . explored the fate of DDN on shorter timescales, investigating the relative contribution of each
diazotroph phylotype to direct C export and quantifying the DDN release and
its subsequent transfer to different groups of plankton by using nanoSIMS. In
the present study, we developed a 1-D vertical biogeochemical model including
the representation of Trichodesmium and UCYN diazotrophs of Group C (UCYN-C), which developed extensively during the mesocosm experiment
. The goal of this study was
to enrich our understanding of the dynamics of the planktonic food web and
the associated biogeochemical fluxes during the mesocosm experiment by
providing information that could not be inferred through in situ
measurements. We also used the model to track the route of DDN into the
different compartments of the ecosystem (diazotrophs, non-diazotrophs,
dissolved pool, detrital pool and export).
Methods
The VAHINE experiment
The VAHINE experiment took place in January–February 2013 (austral summer) in
the oligotrophic New Caledonian lagoon. Three large-volume (∼ 50 m3,
15 m height) mesocosms equipped with sediment traps fixed at their bottom
were deployed, and the dynamics of the three mesocosms were monitored for 23
days. A full description of the design and deployment of the mesocosms,
including the selection of the study site and logistics, is provided in
. The mesocosms were enriched
with ∼ 0.8 µmol L-1 of orthophosphate (PO43-) on the
evening of day 4 to alleviate any potential DIP limitation, which is a
constant feature observed in the southwestern Pacific
, and to stimulate N2
fixation. Seawater was sampled daily in the three mesocosms (hereafter called
M1, M2 and M3) and outside (hereafter called lagoon waters) at three depths
(1, 6 and 12 m), and the sediment traps and the material they contained were
collected every 24 h by scuba divers. It should be noted that the term
“export” used hereafter does not correspond to the material exported
throughout the euphotic zone but to the sinking flux observed in the
experiment at 15 m depth. The methods used to measure the different variables (C,
N and P pools and fluxes, chlorophyll a stocks, and plankton abundances) used in
the present paper for comparison with the model simulations are detailed in
the companion papers of ,
and .
Mesocosm modeling and hypothesis
The model used in the VAHINE project is embedded in the modular numerical
tool Eco3M , which uses mechanistic
formulations to describe the biogeochemical processes engaged in the dynamics
of marine pelagic ecosystems. Eco3M provides high flexibility by allowing its
users to remove or add variables or processes to better adapt the model to a
specific study. The VAHINE experiment consisted in the deployment of three
replicate mesocosms in New Caledonia. Each mesocosm was modeled through a 1-D
box model with 14 boxes of 1 m height each. Mass transfer between boxes is
only allowed through sinking of particulate matter. Until day 10, only the
detrital particles were allowed to sink but after this date, 10 % of all
the living and non-living dissolved and particulate compartments were allowed
to sink. The aim was to represent the setting of the aggregation process and
the subsequent intensification of the sinking process. The aggregation
process was indeed supposed to be favored, not only by the reduced eddy
fluxes due to the containment of water but also by the release of transparent
exopolymer particles (TEPs) as mentioned in
. At the bottom of the modeled mesocosms,
the sinking material was accumulated to be compared with the particulate
matter collected daily in the traps. Sinking velocities were not measured
during the experiment, and the matter collected daily in traps was used to
parameterize the sinking velocity . The latter is therefore set at a constant
0.7 m d-1 until day 10 and increases through the polynomial function
given by Eq. () to reach 10 m d-1 at the end of the
simulation:
V=α⋅t10+β,α=(Vmax-Vmin)tend10-tini10,β=Vmin-α⋅tini10,
where V is the sinking velocity, Vmin and Vmax are, respectively, the minimum and maximum sinking velocities (0.7 and 10 m d-1), t is time, tini is the moment at which the sinking rate starts
to increase (i.e., day 10) and tend is the final day of the run (i.e., day
25). reported that the VAHINE
data revealed that the water column inside the mesocosms was well mixed,
probably through natural convection at night. This feature is simply modeled
through a vertical homogenization of every concentration once a day (at
midnight), by imposing the vertically averaged concentrations in each box.
Light irradiance data from the nearest meteorological station (Nouméa
airport) were used for the surface irradiance in the model, and a vertical
gradient was simulated on the basis of a classical Beer–Lambert law using the
attenuation coefficient found in . When the total N
an P pools (Ntotal and Ptotal) were calculated from the model
outputs and compared to those obtained in situ, a significant
difference appeared regarding Ptotal, while the Ntotal fitted well
(data not shown). This gap was mainly due to a DIP concentration that was too
high compared to data, indicating a non-total consumption by organisms (not
shown). To deal with this DIP excess in the system, a loss of DIP was added
to the model. The main hypothesis to explain this DIP loss without a similar
loss in DIN is the formation of a biofilm of N2-fixing organisms on the
walls of the mesocosms (see , for
details and DIP consumption calculations by the biofilm). Based on the
calculations of , this loss was
estimated at 10 % d-1 and was assumed to have no influence on
primary, bacterial or export production.
The biogeochemical model
The biogeochemical model used in this study is based on the Eco3M-MED model used for the Mediterranean Sea .
The only modification made on this previous version lies in the addition of diazotrophs and N2 fixation process to adapt the model to the
VAHINE experiment. The model includes eight planktonic functional types (PFTs): four primary producers (autotrophic phytoplankton), three consumers
(zooplankton) and one decomposer (heterotrophic bacteria, BAC). All of them are represented in terms of several concentrations
(C, N and P and chlorophyll concentrations for phytoplankton) and abundances (cells or individual per liter; ).
Phytoplankton was originally divided into two size classes, namely the large
phytoplankton (≥ 10 µm; PHYL) and the small phytoplankton
(≤ 10 µm; PHYS). The two N2-fixing organisms are also
distinguished by their size, the large one representing
Trichodesmium sp. (TRI) and the small one Cyanothece sp.
(UCYN-C), which strongly developed in the mesocosms during the experiment
(Turk-Kubo et al., 2015). The zooplankton compartment is also divided into the
three size classes nano-, micro- and mesozooplankton, which, respectively, represent heterotrophic nanoflagellates (HNF), ciliates (CIL) and copepods
(COP). The latter is represented in terms of abundance and C, N and P
concentration. This differs from the model described in
, in which mesozooplankton is only
represented through an abundance and a C concentration. Three nutrients are
considered, namely nitrate (NO3-), ammonium (NH4+) and phosphate
(DIP). The dissolved organic pool (DOM) is composed of labile and semi-labile
fractions of DOC (LDOC and SLDOC) and labile fractions of DON and dissolved organic phosphate (DOP). The refractory organic pools are not represented. Finally, the
detrital particulate matter is represented in terms of C, N and P (DetC,
DetN and DetP). All the biogeochemical processes and interactions
between the state variables are described in Fig. .
Except for the new parameters associated with the new features of the model
as compared to the original one , the
parameters in common between the two model versions are identical.
Conceptual diagram of the biogeochemical model for the 1-D vertical model used in the VAHINE experiment.
Initial conditions
Initial values for the model state variables were derived from the in situ measurements averaged over the three mesocosms and the three sampling
depths (1, 6, 12 m).
Measured DOM values included the refractory organic matter, while the model
only represents the labile (and semi-labile for C) fraction. To extract the
labile fraction from the DON data, we assumed that the plateau reached by the
DON concentration at the end of the experiment (4 µmol L-1) was
equal to the concentration of the refractory DON in this study. Considering
that the refractory fraction of DON was stable throughout the experiment and
fixed at 4 µmol L-1, from an initial total concentration of 5 µmol L-1 at day 2, the initial labile fraction was therefore
estimated at 1 µmol L-1. The percentage of the labile portion over
the total DON was calculated and then applied to DOP to estimate the initial
concentration of labile DOP. The available DOC fraction (LDOC + SLDOC) was
evaluated at 5 µmol L-1 in the equatorial Pacific
. PHYL was initialized with diatom data and
PHYS with the sum of nanoeukaryotes, picoeukaryotes, Synechococcus
sp. and Prochlorococcus sp. The initial detrital particulate matter
was derived by subtracting the total living particulate matter considered in
the model form the total particulate matter measured in situ. Due to
the lack of data for nano- and microzooplankton, we initiated HNF and CIL
abundances using BAC / HNF and CIL / HNF abundance ratios from the literature, as
this was made with the Eco3M-MED model for the Mediterranean
. Several ratios were tested in the range of
those reported by and , and the ones
providing the best model outputs were used (Table ).
The standard value of 0.5 ind L-1 was used for adult COPind which
is consistent with the recent results of . The initial values of C, N and P and chlorophyll concentrations for the
planktonic compartments were derived from the initial cellular abundance data
and from arbitrarily fixed intracellular contents (Table ).
These intracellular contents were thus taken from the Eco3M-MED model
.
Initial conditions for the biogeochemical model.
State variable
Reference
Value
Unit
State variable
Reference
Value
Unit
BACcell
Data
4.75 × 108
Cell L-1
HNFcell
BACcell50
9.519 × 106
ind L-1
BACC
BACcell×QCmax
1.152
µmol C L-1
HNFC
HNFcell×QCmax
34.950
µmol C L-1
BACN
BACcell×QNmoy×0.7
0.107
µmol N L-1
HNFN
HNFcell×QNmoy×0.7
0.326
µmol N L-1
BACP
BACcell×QPmin
0.007
µmol P L-1
HNFP
HNFcell×QPmin
0.023
µmol P L-1
CILcell
HNFcell2500
3808
ind L-1
COPcell
Adapted data
0.5
ind L-1
CILC
CILcell×QCmax
1.538
µmol C L-1
COPC
COPcell×QCmax
0.350
µmol C L-1
CILN
CILcell×QNmoy×0.7
0.108
µmol N L-1
COPN
COPcell×QNmoy×0.7
0.042
µmol N L-1
CILP
CILcell×QPmin
0.005
µmol P L-1
COPP
COPcell×QPmin
0.002
µmol P L-1
PHYLcell
Data
4.48 × 104
Cell L-1
PHYScell
Data
8.11 × 107
Cell L-1
PHYLC
PHYLcell×QCmax
0.306
µmol C L-1
PHYSC
PHYScell×QCmax
1.664
µmol C L-1
PHYLN
PHYLcell×QNmoy×0.7
0.022
µmol N L-1
PHYSN
PHYScell×QNmoy×0.7
0.117
µmol N L-1
PHYLP
PHYLcell×QPmin
9.634 × 10-4
mol P L-1
PHYSP
PHYScell×QPmin
0.005
µmol P L-1
PHYLChl
PHYLC25
0.012
µg Chl L-1
PHYSChl
PHYSC12
0.138
µg Chl L-1
UCYN-Ccell
Data
210
Cell L-1
TRIcell
Data
180
trich L-1
UCYN-CC
UCYN-Ccell×QCmax
4.308
pmol C L-1
TRIC
TRIcell×QCmax
0.123
µmol C L-1
UCYN-CN
UCYN-Ccell×QNmax
0.650
pmol N L-1
TRIN
TRIcell×QNmax
0.018
µmol N L-1
UCYN-CP
UCYN-Ccell×QPmin
0.013
pmol P L-1
TRIP
TRIcell×QPmin
0.388
nmol P L-1
UCYN-CNase
TRINase33300
1.9 × 1020
mol N cell-1 s-1
TRINase
Rabouille et al. (2006)
7.5 × 1016
molN trich-1 L-1
UCYN-CChl
UCYN-CC12
0.359
pg Chl L-1
TRIChl
TRIC25
0.005
µg Chl L-1
Labile DOC
Data
0.25
µmol C L-1
POCDet
POC Tot-POCLiving
3.791
µmol C L-1
Semi-labile DOC
Labile DOC ×19
4.75
µmol C L-1
PONDet
PONTot-PONLiving
0.188
µmol N L-1
Labile DON
Data
1.0
µmol N L-1
POPDet
POPTot-POPLiving
0.012
µmol P L-1
Labile DOP
Data
0.0132
µmol P L-1
NO3-
Data
53
nmol L-1
NH4
Data
36
nmol L-1
PO4
Data
30
nmol L-1
Modeling N2 fixation
The mathematical formulation (see Eq. ) used to represent
N2 fixation was adapted from in order to
be compatible with the formal features of the present model. It describes the
N2 fixation flux as a function of the nitrogenase (i.e., the enzyme
catalyzing N2 fixation) activity (Nase) and the diazotroph abundance
(DIAZOcell, where DIAZO either refers to TRI or UCYN-C). The N2
fixation flux is regulated by the intracellular C quota and the N : C and P : C
ratios (Eq. ) and by the intracellular N quota and N : C ratio
(Eq. ). Intracellular N quota controls the net N2 fixation rate
through a quota function (1-fQN, Eq. ), the N excess exuded
being equally distributed into the DON and NH4+ pools. As in
, the nitrogenase activity (Nase, in
mol N cell-1 s-2) is a state variable, the dynamics of which are described in Eq. (). The nitrogenase activity results from the
balance between the increase and the decrease in its activity. The increase
in the potential nitrogenase activity is assumed to be controlled by the N
intracellular quota (Eq. ) and by the NO3- concentration in the field (Eq. ). Trichodesmium are non-heterocystous
filamentous cyanobacteria with differentiated cells, called diazocysts, located in the center
part of the colony , where
N2 fixation occurs. This spatial segregation mechanism is used by the
organism to protect the nitrogenase enzyme from oxygen inactivation produced
by photosynthesis . In
addition, Trichodesmium combines spatial and temporal segregation to
maximize the protection of the nitrogenase. This therefore allows the cells
to fix N2 for only a few hours in the daytime at around noon
.
In contrast, have shown that UCYN-C can only
use a temporal strategy to separate N2 fixation and photosynthesis
processes and thus need to fix N2 to protect the nitrogenase from O2,
released by photosynthesis during the day. The inhibition of N2 fixation
during the day for UCYN-C and during the night for TRI is simulated by the
finhib function which controls the nitrogenase activity
(Eq. ; 12 h lagged between TRI and UCYN-C). The decrease in
nitrogenase activity is regulated by a saturation function involving
Nasedecmax and a coefficient of nitrogenase degradation (see
Eq. () and Table ). Both the increase and
decrease in nitrogenase are energy dependent and controlled by the
intracellular C quota (Eq. ).
Parameters added for the diazotroph organisms.
Parameter
Definition
TRI value
UCYN-C value
Unit
QCmin
Minimum cell quota of C
2.28 × 10-10
6.84 × 10-15
mol C cell-1
QCmax
Maximum cell quota of C
6.84 × 10-15
2.05 × 10-14
mol C cell-1
QNmin
Minimum cell quota of N
3.44 × 10-11
1.03 × 10-15
mol N cell-1
QNmax
Maximum cell quota of N
1.03 × 10-10
3.09 × 10-15
mol N cell-1
QPmin
Minimum cell quota of P
3.44 × 10-11
1.03 × 10-15
mol N cell-1
QPmax
Maximum cell quota of P
1.03 × 10-10
3.09 × 10-15
mol N cell-1
QCNmin
Minimum cell C : N ratio
5.0
5.0
mol C mol N-1
QCNmax
Maximum cell C : N ratio
19.8
19.8
mol C mol N-1
QCPmin
Minimum cell C : P ratio
35.33
35.33
mol C mol P-1
QCPmax
Maximum cell C : P ratio
318.0
318.0
mol C mol P-1
μmax
Maximum growth rate
2.08 × 10-6
3.2 × 10-5
s-1
km
Specific natural mortality rate
1.16 × 10-6
1.16 × 10-6
s-1
KNO3-
Half-saturation constant for NO3-
1.85 × 10-6
7.6 × 10-6
mol L-1
VNO3-max
Maximum uptake rate for NO3-
3.16 × 10-15
9.91 × 10-20
mol cell-1 s-1
KNH4+
Half-saturation constant for NH4+
7.0 × 10-6
1.69 × 10-6
mol L-1
VNH4+max
Maximum uptake rate for NH4+
3.16 × 10-15
9.91 × 10-20
mol cell-1 s-1
KPO43-
Half-saturation constant for PO43-
1.4 × 10-6
2.62 × 10-7
mol L-1
VPO43-max
Maximum uptake rate for PO43-
1.98 × 10-16
6.19 × 10-21
mol cell-1 s-1
KDON
Half-saturation constant for DON
4.32 × 10-5
1.05 × 10-5
mol L-1
VDONmax
Maximum uptake rate for DON
3.16 × 10-15
9.91 × 10-20
mol cell-1 s-1
KDOP
Half-saturation constant for DOP
3.4 × 10-6
6.57 × 10-7
mol L-1
VDOPmax
Maximum uptake rate for DOP
3.16 × 10-15
6.19 × 10-21
mol cell-1 s-1
Naseprodmax
Maximum rate of increase
1.17 × 10-21
3.51 × 10-26
mol cell-1 s-2
of nitrogenase activity
Nasedecrmax
Maximum rate of decay
9.36 × 10-22
2.83 × 10-26
mol cell-1 s-2
of nitrogenase activity
KNase
Coefficient of nitrogenase degradation
9.44 × 10-16
1.92 × 10-20
mol cell-1 s-1
COSTDIAZO
Respiration cost for nitrogen fixation
1.5
1.5
mol mol-1
EXUDDON
Exudation part of N2 fixed towards DON
0.5
0.5
EXUDNH4
Exudation part of N2 fixed towards NH4
0.5
0.5
FluxN2fix︸mol NL-1s-1=Nase︸mol Ncell-1s-1×DIAZOCELL︸cellL-1×fQC×(1-fQN)dNasedt=Naseprodmax︷Maximum
rate of increase×min(fNase,fNO3-)×fQC×finhib︸Increase in Nitrogenase activity-Nasedecmax︷Maximum
rate of decrease×NaseNase+KNase×fQC︸Decrease in Nitrogenase activityfNase=minmaxQNCmax-QNCQNCmax-QNCmin0.06,0,QNmax-QNQNmax-QNmin0.06,1fNO3-=11+NITKNO3-fQC=maxQNCmax-QNCQNCmax-QNCmin0.06,0,maxQPCmax-QPCQPCmax-QPCmin0.06,0,QC-QCminQCmax-QCmin0.06,1fQN=0 siQN≤QNminmin1+QNmax-QNQNmax-QNmin0.06,2ifQN≥QNmax1-QNCmax-QNCQNCmax-QNCmin0.06ifQN∈[QNmin,QNmax]andQNC≤QNCmaxmin1+QNCmax-QNCQNCmax-QNCmin0.06,2elsefinhib=exp(3.7(cos(2πt-π)-1))
Parametrization of diazotrophs and diazotrophs activity
Trichodesmium sp. and unicellular cyanobacteria (Group C and especially Cyanothece sp.) exhibit distinct physiologies, sizes and
morphologies. Regarding the parametrization of diazotrophs and the processes
they undertake that they have in common with non-diazotrophs, it has arbitrarily been
considered that Trichodesmium cells are equivalent to PHYL cells, and
the TRI state variable was therefore parameterized like 100 PHYL cells
(assuming that a trichome includes 100 cells; ), and
UCYN-C were parameterized like PHYS. For the diazotrophy process, parameters
for TRI were configured following the work of
. TRI was also hypothesized as not being
grazed in the field. Its main predator is the copepods of the Harpacticoida
order (mostly Macrosetella and Miracia; ), which
are not found in significant numbers in the study area as reported in
. To our knowledge,
were the first to propose a dynamic model to
depict the N2 fixation by unicellular cyanobacteria (UCYN-C,
Crocosphaera watsonii). Nevertheless, since this formulation of
N2 fixation was different from that of , we
were unable to use the parameters provided in .
The latter were therefore derived from that of TRI, not only on the basis of
cell size considerations but to obtain overall agreement with N2 fixation
fluxes measured during the experiment. All the parameters added for both TRI
and UCYN-C new compartments are detailed in Table .
Conceptual diagram of the DDN pathway with compartments and processes engaged in the DDN transfer within the food web.
The fate of fixed N2
The main purpose of the DIP enrichment was to enhance diazotrophy in the
mesocosms and facilitate the measurement of the DDN transfer. To monitor the
pathways of DDN throughout the food web, a post-processing treatment was
realized since the model itself does not allow a distinction to be made
between the DDN and other N sources. The aim of the post-processing treatment
was to dynamically calculate the DDN proportion in each compartment of the
biogeochemical model . At the beginning of the simulation, we assumed that
DDN was equal to zero in each compartment. We further assumed that the ratio
DDN / N in each N flux leaving a given compartment was the same as that within
this compartment. DDN transfer starts with N exudation by diazotrophs. This
DDN release fueled the DON and NH4+ compartments, which are then taken up
by autotrophs and heterotrophs. Grazing by zooplankton on the lower trophic
levels will then transfer part of the DDN by excretion, sloppy feeding and
egestion of fecal pellets. Finally, remineralization and natural mortality
will also contribute to the transfer of DDN through the planktonic food web.
Figure illustrates the different processes involved in the
DDN transfer within the ecosystem.
Results
Two simulations of the mesocosm experiment were run: the first includes the
representation of the DIP enrichment (SIME), while the second does not
consider this enrichment (SIMC). SIMC outputs were compared to the data
from the surrounding waters where DIP concentration remained very low and
constant throughout the experiment. Since mesocosms do not include
hydrodynamic processes, this is merely an approximation. However, this
comparison provided the opportunity to further validate the model under
dramatically different nutrient conditions. For the sake of clarity and
better readability, the prefixes “m” and “o” will be used to refer to model
and observations, respectively, and a * will be used to signify data measured
outside the mesocosms. Vertical homogeneity was observed in the mesocosms
during the experiment for most of the biogeochemical and physical
characteristics . We thus used the average of the three sampling depths to plot both
model results and observations. Three periods (namely P0, P1 and P2) were
distinguished during the experiment based on biogeochemical characteristics
as detailed in and on changes
in the diazotroph community composition . P0 stands for the few days before the DIP enrichment, P1 is the
period when diatom–diazotroph associations dominate the diazotrophic community
(i.e., from day 5 to day 14), and P2 is the period when UCYN-C dominate the
diazotrophic community (i.e., from day 15 to day 23).
Patterns of change
over time in (a) dissolved inorganic phosphate (DIP),
(b) nitrate (NO3-), (c) dissolved organic phosphate
(DOP), (d) dissolved organic nitrogen (DON),
(e) particulate organic phosphorus (POP), (f) particulate
organic nitrogen (PON), (g) total phosphorus (Ptotal) and
(h) total nitrogen (Ntotal) concentrations
(µmol L-1) in model outputs (solid lines: SIME – blue;
SIMC – black) averaged over depth superimposed on data observations
averaged over depth in the three mesocosms (M1 – red; M2 – blue; M3 –
green) and in surrounding waters (black). Red vertical lines distinguish the
three periods P0 (before the DIP enrichment), P1 (diatom–diazotroph
associations dominate the diazotrophic community) and P2 (unicellular
N2-fixing cyanobacteria (Group C) dominate the diazotrophic
community).
Dynamics of the different N and P pools
During P0, mDIP in SIME decreases slowly from 47 to 24 nmol L-1
(Fig. a). In response to the DIP enrichment at the end of day 4,
mDIP reached 830 nmol L-1, before gradually decreasing to the low
concentrations observed before the enrichment (Fig. a). During
the experiment, the DIP enrichment led to three different oDIP in the
three mesocosms with 740, 780 and 990 nmol L-1 in M1, M2 and M3,
respectively, reflecting the slightly different volumes of the mesocosms
. oDIP then decreased below the
quantification limit of 50 nmol L-1 in the three mesocosms, but the
consumption of oDIP in M1 was the fastest and those in M2 the slowest.
Without the DIP enrichment (SIMC), mDIP is quickly consumed and the
concentrations remained close to zero until the end of the simulation,
consistent with oDIP*, which was <50 nmol L-1 throughout the
experiment. As well as oNH4+, mNH4+ remained low and stable around
15 nmol L-1 throughout the simulation (not shown here). mNO3- also
fits in well with oNO3-, with nearly constant concentrations close to the
quantification limit of 50 nmol L-1 (Fig. b) over the
whole simulation.
oDOP and oDON remained relatively stable throughout the experiment, with values around 5 and
0.14 µmol L-1, respectively, with a slight decrease in P2 at the end (Fig. c and d).
A slight increase in mDOP in SIME from 0.14 to 0.18 µmol L-1 and a slight decrease in both mDOP and
mDON were observed during P2. For SIMC, mDOP and mDON remained stable throughout the simulated period (Fig. c and d).
In the mesocosms, the trend was similar for oPOP (particulate organic
phosphorus) and oPON (particulate organic nitrogen), with constant
concentrations or a slight decrease during P1, followed by a strong increase
during P2 (by a factor of 1.5, 1.5 and 2 in M1, M2 and M3, respectively, in
oPON and by a factor of 1.4, 1.4 and 2.4 in M1, M2 and M3, respectively, in
oPOP; Fig. e and f). SIME results are in good agreement with
data for mPON, which starts at 1 µmol L-1 and then increases
to a maximum of 1.5 µmol L-1 during P2. While oPOP decreased
slightly at the beginning of P1 and increased during P2 during the
experiment, mPOP in SIME remains constant (0.08 µmol L-1)
from day 5 to 10 and increases after day 10. The increase in mPOP up to the
0.14 µmol L-1 peak is stronger and occurs earlier than oPOP,
before decreasing as in the observed data at the end of P2. In SIMC, the
total particulate organic matter dropped throughout the entire simulation,
from 0.06 to 0.02 µmol L-1 for mPOP and from 1 to
0.4 µmol L-1 for mPON.
In the mesocosms, oNtotal averaged 6.2 µmol L-1
during P1 and started to decrease at the end of P2 (Fig. h).
mNtotal in both SIME and SIMC was quite similar and in the
same range as that observed in the data, with a slightly sharper decrease for
SIMC at the end of P2. SIME showed an immediate and strong increase in
mPtotal (1–1.2 µmol L-1) on day 5, corresponding
to the DIP enrichment, while mPtotal in SIMC was constant (250 nmol L-1) throughout the simulation (Fig. g). After the
enrichment, mPtotal started to decline down to
0.2–0.25 µmol L-1 on day 22. In the mesocosms, oChl remained
stable during P1 and increased during P2 by a factor of 5 up to a maximum of
1 µg L-1 in M3 (Fig. a). oChl a was lower (0.6 µg L-1) in M1 and M2 at the end of P2. mChl a calculated by
SIME was similar to oChl a in M1 and M2, with a decrease a little more
marked during P1 and a maximum of 0.5 µg L-1 in P2. While
mChl a increased during P2 in SIME, mChl a in SIMC remained stable
(∼0.1 µg L-1) until the end of the simulation.
Patterns of change over time in (a) dinitrogen fixation
(N2 fixation) rates (nmol N L-1 d-1), (b) dissolved
inorganic phosphate turnover time (TDIP, days),
(c) chlorophyll a (Chl a, µg L-1),
(d) particulate organic C exported (POCexp, dry matter in
mmol C), (e) primary production (PP) rates
(µmol C L-1 d-1), (f) particulate organic
nitrogen exported × 16 (PONexp, dry matter in mmol N),
(g) bacterial production (BP) rates
(µmol C L-1 d-1) and (h) particulate organic
phosphate exported × 106 (POPexp, dry matter in mmol N) in model outputs (solid lines: SIME –
blue; SIMC – black) averaged over depth superimposed on data observations
averaged over depth in the three mesocosms
(M1, red; M2, blue; M3, green) and in surrounding waters (black). Red vertical lines distinguish the three periods
P0 (before the DIP enrichment), P1 (diatom–diazotroph associations dominate the diazotrophic community) and P2
(unicellular N2-fixing cyanobacteria (Group C) dominate the diazotrophic community).
Dynamics of the different fluxes
The biogeochemical fluxes relative to the main processes such as primary and
bacterial productions (PPs and BPs), N2 fixation (N2fix),
turnover time of DIP (TDIP), and particulate matter export fluxes
(particulate organic C (POCexp), PONexp and
POPexp) have been calculated by the model and compared to the
measured values (Fig. b to h).
At the beginning of P0, oN2fix as well as mN2fix (both in SIME
and SIMC) were about 17 nmol N L-1 d-1 and declined gradually
during P1 down to 10 nmol N L-1 d-1. While mN2fix in SIMC
continued to decrease during P2, mN2fix in SIME increased during P2
by a factor of 4, consistent with oN2fix and reaching a maximum of 42 nmol N L-1 d-1 on day 23. Primary and bacterial production (PP and
BP) exhibited the same temporal dynamics in both data and SIME results.
They first slightly decreased before the DIP enrichment, remained stable
during P1 and increased during P2 by a factor of 4.4 and 2.7 for PP and BP,
respectively (Fig. c and e). During P2, mPP (SIME) rose
to 2 µmol C L-1 d-1, which is in the range of the oPP
measured in the three mesocosms. M3 exhibited higher values of oPP than those
in M1 and M2 during P2 (around 4 µmol C L-1 d-1 on day
22). Even if mBP (in SIME and SIMC) started at a higher rate than oBP
measured in the three mesocosms, it decreased rapidly from day 2 to 4 to reach
the in situ value before the enrichment. The increase in mBP from day 11 to
day 17 in SIME was somewhat overestimated compared to data. BP better
fitted the data measured at the end of P2 and especially in M3 (1 µmol C L-1 d-1). In SIMC, the increase in mBP and mPP
during P2 did not occur and these rates remained constant around 0.5 µmol C L-1 d-1 for mPP and 0.4 µmol C L-1 d-1
for mBP throughout the 25 days of the
simulation. mBP values in SIMC were lower than those measured in the three
mesocosms and consistent with the oBP values measured in lagoon waters
(Fig. e).
TDIP is a relevant indication of DIP availability in the water column.
After a slight decline in oTDIP during P0 to values lower than 1 day,
TDIP increased dramatically up to 30 days (oTDIP) and 21 days
(mTDIP) after the DIP enrichment. mTDIP then decreased linearly in
SIME as well as oTDIP, in the three mesocosms. mTDIP in SIMC
showed the same trend as the oTDIP measured in the lagoon waters
(Fig. g).
The fluxes of exported matter – POCexp,
PONexp and POPexp for C, N and P, respectively – are represented in
Fig. d, f and h in terms of dry matter measured in the
sediment traps . During P1, the daily export
remained relatively stable and averaged 18, 1.13 and 0.09 mmol for
mPOCexp, mPONexp and mPOPexp, respectively. oPOCexp,
oPONexp and oPOPexp gradually increased during P2 (from day 15 to
25) to reach a maximum of 57 mmol C d-1, 5 mmol N d-1 and 0.5 mmol P d-1, respectively. In SIME, mPOCexp, mPONexp and
POPexp fitted well with data with a slight overestimation of
mPOPexp at the end of P2, which reaches a maximum of 0.75 mmol P d-1.
There was no significant difference between SIME and SIMC for
mPOCexp and mPONexp, from the beginning of the experiment to the
middle of P2 (day 18). From day 19, the increase in mPOCexp and
mPONexp is less important in SIMC than in SIME. For mPOPexp,
the increase in SIME occurs earlier (day 15) and the discrepancy between
SIME and SIMC was wider at the end of the simulation.
Patterns of change in abundances over time of (a) Trichodesmium (TRI, trichom L-1), (b) unicellular N2-fixing
cyanobacteria (UCYN-C, cell L-1), (c) large phytoplankton (PHYL,
cell L-1), (d) small phytoplankton (PHYS, cell L-1),
(e) heterotrophic bacteria (BAC, cell L-1), (f) hetero-nanoflagellates (HNF,
ind L-1), ciliates (CIL, ind L-1) and copepods (COP, ind L-1)
in model outputs (solid lines: SIME – blue; SIMC – black) averaged
over depth superimposed on data observations averaged over depth in the three
mesocosms (M1 – red; M2 – blue; M3 – green) and in surrounding waters
(black). Red vertical lines
distinguish the three periods P0 (before the DIP enrichment), P1 (diatom–diazotroph associations
dominate the diazotrophic community) and P2 (unicellular N2-fixing cyanobacteria (Group C) dominate the diazotrophic community).
Evolution of planktonic abundances
The model also simulated the abundances of organisms in cell L-1 for
single cells, in trichome L-1 for TRI and in ind L-1 for
zooplankton, besides being represented in terms of biomass (C, N and P and Chl for
phytoplankton).
mTRI remained constant in SIME around 250 trichomes L-1. By contrast, a strong development of UCYN-C occurred
during P2, with mUCYN-C reaching 5.107 cell L-1
(Fig. a and b). This increase in mUCYN-C is consistent
with the observed dynamics, though the mUCYN-C increase is overestimated in
SIME compared to oUCYN-C. mPHYL decreased over time in both SIME and
SIMC (Fig. c). In the three mesocosms, oPHYL increased
from day 10 to 15 reaching 105 cell L-1 before decreasing back to
values close to that of mPHYL. During P0, mPHYS decreased slightly like
oPHYS. During P1, the decrease in mPHYS (down to 0.1 × 108 cell L-1)
was stronger than that of oPHYS, which increased from day 10 and reached the
same range of values as oPHYS at the beginning of P2. During P2, mPHYS and
oPHYS increased up to 1.5 × 108 cell L-1 for mPHYS in SIME and
1.3–2.9 × 108 cell L-1 for oPHYS. While mPHYS was similar in SIME
and SIMC from day 2 to day 8, the increase in mPHYS after day 8 and until
the end of P2 was lower in SIMC than in SIME (Fig. d). As for PHYS, there was a slight decrease in mBAC and oBAC during P0.
The DIP enrichment on day 4 led to a strong decline from day 5 to day 8,
which was more marked in mBAC (9.5 × 107 cell L-1 in SIME) than in
oBAC (2.3–3.1 × 108 cell L-1). From day 8 to the end of the simulation,
mBAC increased up to a maximum of 1.1 × 109 cell L-1, while oBAC
reached a maximum of 6.8–8.5 × 108 cell L-1 at the end of the
experiment. In the same way, mBAC was similar in SIME and SIMC from
day 2 to day 8 and then increased until the end of the simulation but to a
lesser extent in SIMC than in SIME. Since no zooplankton data that
could be used for comparison with the model results were available, only the
dynamics of SIME and SIMC are presented (Fig. g to f). mHNF and mCIL showed the same trends though they are time-shifted. mCOP
was similar in SIMEand SIMC, with a decline from 0.5 ind L-1 at
the beginning to less than 0.1 ind L-1 at the end of the simulation.
Except for mCOP and mPHYL, the DIP enrichment had a strong impact on the
plankton dynamics as significant differences between the results of SIME
and SIMC in mTRI, mUCYN-C, mPHYS, mBAC, mHNF and mCIL are observed.
Overall, SIMC presented abundances 3 to 680 times lower than those
simulated by SIME, though the temporal trends were similar between the two
simulations.
Patterns of change over time in specific (green) and population
(blue) growth rates function of (a) unicellular N2-fixing cyanobacteria
(UCYN-C, cell s-1) and (b) Trichodesmium (TRI,
trichome s-1) and carbon (C, blue), nitrogen (N, green) and phosphorus
(P, red) relative intracellular quota in (c) unicellular N2-fixing
cyanobacteria (Group C; UCYN-C, %) and (d) Trichodesmium
(TRI, %) in model outputs in SIME. Red vertical lines distinguish the
three periods P0 (before the DIP enrichment), P1 (diatom–diazotroph
associations dominate the diazotrophic community) and P2 (unicellular
N2-fixing cyanobacteria (Group C) dominate the diazotrophic
community).
DIP enrichment and diazotrophs growth
The model also gives additional information not provided by the data
regarding the growth of the organisms or their intracellular content. The
population growth rate (in cell L-1 s-1) for TRI and UCYN-C, as
well as the specific (i.e., per cell) growth rates (in s-1) of TRI and
UCYN-C are plotted in Fig. a and b, while the relative
intracellular C, N and P quotas (i.e., QC, QN and QP) are plotted in
Fig. c and d. The DIP enrichment at the end of day 4 had
a direct impact on QP for both TRI and UCYN-C, with an instantaneous
increase in QP up to 100 % on day 5. While QC, QN and QP for
TRI remained at their maximum value until the end, QN and QC of UCYN-C
decreased as soon as QP increased on day 5. During P2, QP gradually
declined for TRI and faster for UCYN-C. The reverse process then occurred with
an increase in QN and QC for UCYN-C when QP decreased from day 15,
whereas this was not observed for TRI. Throughout the simulation, the trends
of both population and specific growth rates for TRI were similar, with a
sudden increase on day 5 followed by rather constant and then decreasing
values (Fig. b). By contrast, the increase in the specific
growth rate of UCYN-C after the DIP enrichment (day 5) was not observed in
the UCYN-C population growth rate, namely on the population scale
(Fig. a). The population growth rate of UCYN-C increased
10 days later, i.e., during P2, up to a maximum of 200 cell L-1 s-1
on day 22.
Patterns of change over time of DDN proportion (%) in
(a) Trichodesmium (TRI), (b) unicellular N2-fixing cyanobacteria
(Group C) (UCYN-C), (c) dissolved organic nitrogen (DON), (d) ammonium
(NH4+), (e) nitrate (NO3-), (f) detrital nitrogen (DETN),
(g) heterotrophic
bacteria (BAC), (h) small phytoplankton (PHYS), (i) large phytoplankton (PHYL), (j) hetero-nanoflagellates
(HNF), (k) ciliates (CIL), (l) copepods (COP) and (m) in traps (TRAP) in SIME. Red vertical lines
distinguish the three periods P0 (before the DIP enrichment), P1
(diatom–diazotroph associations dominate the diazotrophic community) and P2
(unicellular N2-fixing cyanobacteria (Group C) dominate the diazotrophic community).
Fate of DDN in the ecosystem
The fate of the N that was fixed at the beginning of the simulation (DDN) was
examined using the post-processing treatment described in the “Methods” section. In short, the proportion of the total DDN present in each living and
non-living compartment of the water column and in the traps was calculated
throughout the simulation period (Fig. ). At the start of the
experiment, DDN was nearly exclusively in TRI (the proportion of DDN in
UCYN-C was negligible), but this proportion decreased throughout the
simulation. Until day 10, most of the DDN was transferred to the DON pool, which contained about 35 % of the total DDN on day 10, followed by, in
their order of importance, NH4+ (up to 10 % on day 5), DET (12 % on
day 10) and the components of the microbial loop. Until day 10, the
proportion of DDN in each compartment except TRI, either increased with time or reached a maximum around day 5, consistent with the decrease in N2
fixation rates during that period. After day 10, the proportion of DDN
increased in all living organisms, thereby indicating the transfer of DDN to
non-diazotrophic organisms. The proportion of DDN increased almost until the
end of the simulation in CIL and HNF but only until day 18 in BAC, PHYS and COP before decreasing again. In the non-living compartments, the proportion
of DDN decreased after day 10 (day 12 for DON) until the end of the
simulation. Finally, the proportion of DDN in traps was almost zero during
the first 10 days of the experiment, before increasing and then stabilizing
around 4 %. The percentage of DDN with respect to total particulate N
contained in the traps was also plotted (Fig. ). This
percentage increased quite linearly with time from 0 to nearly 0.4 %
between day 2 and day 10. On day 10, the percentage increased much more
rapidly until day 12 and then rose gradually to a plateau around 1.2 %
before increasing again at the very end of the experiment.
Discussion
Nitrogen (N) input by N2 fixation in the upper SW Pacific Ocean is thought
to be controlled by dissolved inorganic phosphate (DIP) availability because
of the presence of repleted trace metals concentrations compared to the
adjacent South Pacific central gyre
. The aim of the VAHINE
experiment was to (i) investigate the fate of the diazotroph-derived nitrogen (DDN) in oligotrophic
ecosystems by removing any potential DIP limitation for diazotrophs and
thereby potentially stimulate the growth of organisms (in particular
diazotrophs), (ii) enhance N2 fixation and DDN fluxes through the entire
ecosystem, and (iii) study the dynamics of biogeochemical C, N and P fluxes.
N2 fixation is expected to rapidly deliver new N to other organisms than
diazotrophs, thus reducing possible N growth limitation or co-limitation in
the ecosystem. Our goal was to monitor the dynamics of this new N toward the
food chain, the inorganic and organic N pools, as well as in the exported
particulate matter. The discussion will focus on expected and unexpected
results obtained in this study after the DIP enrichment, as well as the fate
of DDN in the ecosystem.
Patterns of change over time of the nitrogen fixation contribution
(%) to particulate matter export in SIME.
An expected enhancement of biogeochemical fluxes after the DIP enrichment
The mesocosms DIP enrichment performed at the end of day 4 associated with
the provision of new N by diazotrophy led to a strong increase in diazotrophs
(especially UCYN-C) abundances (Fig. a and b), biomass
(data not shown) and N2 fixation fluxes during P2, and a significant
development of UCYN-C occurred during that period. Whereas a strong increase
in N2 fixation was observed in SIME during P2 (consistent with the data
indicating a near 3-fold higher mean N2 fixation rate in P2 than P1; Fig. a), N2 fixation rates gradually decreased in
SIMC, indicating strong differences between the mesocosm conditions and
those encountered in lagoon waters. During the experiment, hydrological
parameters such as temperature and biogeochemical conditions were all similar
inside and outside the mesocosms, except the DIP conditions
, confirming that the DIP
enrichment stimulated N2 fixation in the mesocosms. Nevertheless, a slight
increase in N2 fixation rates was observed outside the mesocosms during P2
(+35 %), which could be explained by a provision of external DIP sources
to the lagoon, by growth on DOP sources
and/or by the increasing seawater temperature over the 25-day experiment as
mentioned in , which provided
favorable conditions for diazotroph growth . A
rapid decrease in TDIP was observed on day 5 after the DIP enrichment,
suggesting a rapid consumption of the DIP by the planktonic community both in observed data and in the model outputs. Diazotrophs were the first to respond
to the DIP enrichment in term of abundance, even if this response did not
lead to an immediate increase in the N2 fixation rate. The latter
significantly increased during P2 in relation to the development of UCYN-C
. In the model results, other
autotrophic organisms and heterotrophic bacteria declined until the middle of
P1 and started to grow 5–10 days after the DIP enrichment (except PHYL).
Despite this time lag between the DIP enrichment and the planktonic response,
the DIP enrichment resulted in an increase in the abundances of all
planktonic groups in the model outputs except PHYL and COP
(Fig. ). The DIP limitation at the beginning of the
experiment was represented in the model by setting the P cell contents of all
organisms at their minimum value, which led to an immediate uptake of DIP
after the enrichment at the end of day 4 (Fig. c and d).
On the cellular scale, this immediate DIP uptake resulted in a rapid increase
in intracellular P contents of autotrophs and heterotrophic bacteria up to
their maximum quota (Fig. c and d for diazotrophs). After
N2 fixation by diazotrophs, the DDN inputs were of benefit to
non-diazotrophic organisms. Autotrophic PP and heterotrophic BP increased in
the model after the DIP enrichment (+262 and +181 %, from day 5 to
day 23 for PP and BP, respectively). The enhanced PP (Fig. e) led to an increase in total suspended matter (Fig. 3e and f) and
finally in exported particulate material (Fig. d, f and h). The contribution of N2 fixation to PP (up to 10.0 % for SIME
and 6.0 % for SIMC) is in good agreement with corresponding measured
contributions, which were equal to 10.9 ± 5.0 % inside the mesocosms
and 5.7 ± 2.0 % in the lagoon waters .
Hence, the DIP enrichment not only stimulated N2 fixation and PP but also
the percentage of PP sustained by N2 fixation. The newly synthesized
biomass had two possible fates, namely remineralization or export.
Throughout the experiment, has shown that DDAs were the most abundant diazotrophs in the mesocosms during P1.
However, it has been shown by
that they did not represent a significant biomass, and the associated export flux was low compared to the export flux measured during P2.
Moreover, due to their rapid settling , DDN produced by DDAs were not of benefit to the system. For these
reasons, we decided not to include DDAs in the model.
During P1, the export in SIME is unexpectedly in good agreement with data, probably due to the overestimation of UCYN-C by the model
(Fig. b). The export during P1 was however lower than
during P2, during which we observed a higher increase in suspended
particulate matter (Fig. d, f and h) enhanced by the more
significant UCYN-C growth, not clearly noticeable with the logarithmic scale
in Fig. b. Moreover, the presence of large (100–500 µm) UCYN-C aggregates in the mesocosms facilitated their export
into the traps where UCYN-C accounted for up to 22.4 ± 4.0 % of total
C export at the height of their extensive development
. This indicates that UCYN-C cannot only contribute to direct export but promote indirect export. The high
content of TEP measured in traps by on days 15 and 16 in correlation with the increase in UCYN-C
abundances observed by led to
the assumption that the presence of TEP in the field would facilitate export
flux and especially the sinking of UCYN-C during P2. This phenomenon was taken
into account in the model by allowing, each day from day 10, the settling of
10 % of all the model compartments (living and non-living, particulate
and dissolved) in addition to the detrital particulate matter. C, N and P export
in SIME closely follows the mesocosm trap measurements
(Fig. d, f and h). SIME shows higher C, N and P
exports (+28, +35 and +158 %, respectively) compared to
SIMC. Large-size N2-fixing organisms are known to directly contribute
to C export in coastal and oceanic environments
, but small-size UCYN-C,
although very few studies have focused on them, were considered less
efficient at promoting export due to their small size (typically
1–6 µm) associated with low individual sinking rates and the tight
grazing control that leads to high recycling rates in the euphotic zone. In
the present study, both our experimental and model results indicated that
UCYN-C also significantly contributed to export under DIP repleted
conditions, both directly by the sinking of UCYN-C cells and indirectly
after the transfer of DDN to non-diazotrophic plankton, which was
subsequently exported.
An unexpected delay for UCYN-C development and biogeochemical fluxes enhancement
The new N provided by N2 fixation after the DIP enrichment resulted in
high PP and BP rates, as well as in an increase in export and planktonic
abundances. However, these responses were not observed immediately after the
DIP enrichment on day 4 but 5–10 days later (Figs. and ). The massive UCYN-C development occurred during P2,
with a maximum population growth on day 21 in the model, consistent with the
observation of the maximum in the UCYN-C abundances reported in
, on days 20, 15 and 19 in M1,
M2 and M3, respectively (Fig. b).
What factor may explain the 5–10-day delay between the DIP enrichment and the large UCYN-C development?
On the cellular scale (Fig. a), the DIP enrichment had an
immediate influence on cell-specific growth rate of UCYN-C, with a 4-fold
increase in a few hours. However, this immediate response was not observed on the population scale (Fig. a). At the beginning of the
simulation, the P cell quota of UCYN-C was at a minimum, and UCYN-C cell-specific
growth rate was therefore equal to zero. Though DIP and DOP were very low at
the beginning of the simulation, UCYN-C could however take up part of this
available P, thereby increasing their P quota and their growth rate. UCYN-C
reached their maximum cellular P quota the day after the DIP enrichment
(Fig. c) and DIP did not further limit the UCYN-C growth
until day 17. The peak in cell-specific growth rate at day 5
(Fig. c) corresponds to the temporary absence of
significant nutrient limitation, while oscillations during the following days
correspond to the day–night rhythm in UCYN-C C quota associated with C
starvation during the night (the specific growth rate is modulated by the
lowest intracellular quota). When C is the most limiting nutrient, the
night–day oscillations are passed on to the growth rate. The strong increase
in the cell-specific growth rate on day 5 led to an increase in UCYN-C
abundances (Fig. b). After day 5, photosynthesis and N
uptake were then not rapid enough to sustain the increased C and N needs, and
N, and mostly C at night, became limiting (Fig. c). As a
consequence, UCYN-C cell-specific growth rate decreased slightly after day 5 and more rapidly after day 18 when DIP once again became limiting
(Fig. a). Figure illustrates the time lag
between the variations at the cellular level for specific growth rate and
growth at the population level. The growth rate of the UCYN-C population also
increased from the beginning of the simulation since the specific growth rate
and the abundance of UCYN-C increased, but this was almost imperceptible
until the exponential increase started around day 11. From day 18, when the
specific growth rate began to strongly decrease, the population growth rate
still increased but more slowly and finally decreased after the maximum of
5.107 cell L-1 s-1 reached on day 22 (Fig. a). TRI abundance was less influenced by the DIP enrichment than UCYN-C
abundance. However, the DIP enrichment led to an increase in TRI growth rate
on day 5 on both the population and trichome scale (Fig. c). Since a trichome includes 100 cells of Trichodesmium, the time
lag between the responses at the trichome and population levels was therefore
far less than that evidenced for UCYN-C. Furthermore, although TRI growth was
not nutrient-limited from day 5 to day 15 as the three cellular quotas (C, N
and P) were at their maximum value (Fig. d), the TRI
population did not increase significantly because of its low maximum division
rate as compared to the timescale of the experiment (3 weeks; consistent with in situ data).
Discussion on the time duration of enrichment experiments
The aforementioned time lag between cellular and population responses is also
useful for understanding what may be viewed as a contradiction: on one hand,
we observed a clear and net increase in PP, BP and export productions after
the DIP enrichment, both in the mesocosms and in the SIME, but on the
other hand, oligotrophic waters are generally known to be more DIN- than
DIP-limited. After reviewing the main studies conducted on nutrient limitation, and especially on N and P limitation in oligotrophic waters,
concluded that N was the first limiting nutrient
for phytoplankton in nutrient-depleted areas as nutrient-addition experiments
did not lead to a significant increase in autotrophic activity after P
additions, whereas it did after N additions
.
Similar results were obtained in the South Pacific gyre for autotrophs
and heterotrophs
.
also observed a proximal N limitation of BP at the beginning of
the present mesocosm experiment (before the DIP enrichment) on short timescales (days). This apparent contradiction regarding DIP limitation may therefore be
explained by the time duration of the aforementioned DIP enrichment
experiments that was not long enough to evidence the response of the
planktonic ecosystem. The enrichment mesocosm experiment conducted during the
VAHINE project made it possible to monitor the ecosystem and the associated
biogeochemical fluxes over a longer period of time (23 days) compared to the
nutrient-addition experiments cited above. Since we observed a significant
increase in PP and BP about 5–10 days after the DIP enrichment in both
experimental (M2 and M3, 5 days for M1) and simulation results, we may
conclude that around 5–10 days are necessary for the newly fixed N by
diazotrophs to sustain the observed high production rates and to see an
effective change in the planktonic populations (in term of abundances,
structure and function). In the light of the foregoing, two conclusions may
therefore be drawn. First, 5 days may be the lowest time limit to
characterize the real nutrient-limiting primary, bacterial and export
productions, at least in marine areas where N2 fixation is a significant
process. Therefore, short-term (∼ 2 days) nutrient-addition experiments
may not be well-suited to studying nutrient limitation in marine ecosystems.
Second, the initial DIP limitation considered in the model clearly indicates
that DIP limitation observed at the cellular level does not reflect the
response on the population scale (in terms of primary, bacterial and export
productions), which may be delayed. Therefore, in order to correctly assess
the nutrient limitation during short-term nutrient-addition experiments,
nutrient limitation diagnostics operating at a cellular level (such as
enzymatic responses) need to be applied rather than classical measurements of
PP or BP increase after the enrichment.
The fate of DDN in the planktonic ecosystem and exported matter
At the start of the simulation, DDN was almost exclusively in TRI since the
flux of N2 fixation by UCYN-C was negligible compared to that of TRI, and
the situation was reversed at the end of the simulation when UCYN-C abundance
became predominant. Due to DON exudation and NH4+ release by TRI, the
proportion of DDN first increased in the DON and NH4+ pools and then in
the NO3- pool due to nitrification. Before day 10, planktonic organisms
did not significantly benefit from the DDN, as its proportion decreased in
BAC and PHYS between days 4 and 8 and in HNF between days 6 and 10. For BAC
and PHYS, this was mainly due to the decrease (which is overestimated by the
model) in abundance of these two groups between days 5 and 8 due to grazing
by HNF and CIL. After day 10, the DDN proportion increased in all the
non-diazotrophic plankton groups, while it decreased in the non-living
pools, though somewhat later (i.e., from day 13) in DON. This decrease in DDN
proportion in the non-living pools is both due to the assimilation of mineral
and organic nutrients by phyto- and bacterioplankton and to the sinking of
the produced organic matter through aggregation processes. Since mineral N is
first taken up, the uptake of DON occurs later, namely during P2, as shown in
. As a consequence, the
decrease in DDN–DON percentage was also delayed as compared to that of
NO3- and NH4+. DDN–DET increased quite regularly until day 10 as long
as the sinking rate was constant and then decreased with the increase in
this sinking rate. As a result, DDN in the particulate matter collected in
traps increased from day 10 to the end, consistent with the
δ15N budget performed by , thereby indicating a 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 up to 60 %). mDON
appeared to be the pool which mainly benefitted from the DDN. This is due to
the DON release by diazotrophs, especially TRI, which was at its maximum N
quota throughout the simulation (Fig. c and d). Since the TRI maximum cell division rate was low, their N2 fixation rate is indeed
high enough to allow Trichodesmium to fulfill their N reserves and
reach their maximum N quota (Fig. d). The same is not
true for UCYN-C, for which the division rate (boosted by the P enrichment) was
too high, as compared to their N fixation rate, to reach their N maximum
quota. However, in the model, DDN exudation by diazotrophs released equal
amounts of NH4+ and DON. During P1, DDN accumulated in DON (up to almost
40 % on day 13; Fig. c), whereas DDN in NH4+
decreased rapidly from day 5 as it was immediately used by heterotrophic
bacteria and phytoplankton (Fig. d). DDN in DON decreased
later (i.e., during P2, when the DON pool began to be used) as the inorganic N
pool was depleted. Finally, though DDN transited in the same proportions in
NH4+ and DON, it mostly accumulated in DON since DDN–NH4+ was taken
up more rapidly, these results substantiating those found by
. Among the living compartments, PHYS, BAC,
HNF and CIL were the main beneficiaries of DDN. PHYS and BAC were indeed the
main consumers of NH4+ and labile DON (while PHYL was not allowed by the
model to uptake DON), and HNF and CIL, respectively, feed on BAC and PHYS and
on PHYS and HNF. DDN therefore mainly transited through the actors of the
microbial loop, which is consistent with nanoSIMS measurements performed
after 24 h of incubation with 15N2 on water sampled on day 17, showing
that 18 ± 4 % of the DDN was found in picophytoplankton against 3 ± 2 % in diatoms .
According to the model, only 5 % of the total DDN were recovered in the
traps at the end of the simulation. This proportion is likely underestimated
by the fact that UCYN-C sinking is probably underestimated in the model. The
contribution of UCYN-C to POC export on day 17 during P2 was indeed
0.25 % in the model simulation, against up to 22.4 ± 4.0 % in the
data during the same period as reported in . In the same way, the ratio DDN / total N in traps was equal to 1 %
at the end of the simulation, which is dramatically lower than the measured
value, which is equal to 80 % .
This discrepancy is partially due to the different methodologies used to make
these estimations. In the post-processing treatment, we considered that the
initial DDN was zero in every compartment, which is obviously not true, but
this hypothesis was constrained by the fact that the initial DDN in all the
model compartments was unknown, and arbitrary allocations of DDN in
compartments would have added additional uncertainty to the model results. As
a consequence, our results are necessarily underestimated as compared to the
measured values since the latter include the history of previous N2
fixation in the field (i.e., before the beginning of the mesocosm experiment).
If we consider that an initial content of DDN in the traps equals 30 % as
measured by , the final modeled DDN
content would be 31 %, which is still underestimated but more realistic.
This approximation of the initial zero DDN content in organisms is therefore
not sufficient to explain the huge difference to observations concerning
the DDN proportion in traps. Another source of error lies in the implicit
representation of the aggregation process given in this study. It has been considered that from day 10, 10 % of all the model variables are
allowed to sink in addition to the detrital particulate compartment. However,
it seems that this leads to an underestimation of UCYN-C sinking. As already
mentioned, showed that the UCYN-C
contribution to the particulate C collected in traps on day 17 was up to 22.4 ± 4.0 % as against 0.25 % for the model. The in situ value has
been estimated using a value of the intracellular C content per cell of 22 pgC cell-1, determined according to the measured UCYN-C cell size in the
mesocosms and the equations of . However,
the modeled C intracellular content of UCYN-C at day 17 is about 150 times
lower (0.13 pgC cell-1). This difference in UCYN-C C contents is due to
the straightforward hypothesis we made in the model which was to consider the
UCYN-C diazotrophs as PHYS. Our aim was to use the same model developed for
the oligotrophic ocean and particularly the Mediterranean Sea (Eco3M-MED) in
every oligotrophic region of the ocean. Moreover, we considered that it was
potentially informative to consider that the diazotrophs added in the model
were similar in all points to PHYS and PHYL except that they were able to fix
N2. In the model, PHYS represents picophytoplankton and the small
nanophytoplankton, and its C intracellular content ranged between 0.08 and
0.25 pgC cell-1, which seems to be an underestimated value for UCYN-C.
During the VAHINE experiment,
have shown
that large cells of UCYN-C (size about 5.7 µm) were present with a
C content estimated at 22 pgC cell-1. With the latter C content, we
established that the mUCYN-C contribution to export would reach 28 %, a
result consistent with the 22.4 ± 4.0 % estimated by
. Finally, the overestimation of
UCYN-C abundance by the model also supports the idea that UCYN-C sinking is
underestimated by the model. The aggregation process induced by TEP
or by specific molecules
such as extracellular polysaccharides (EPSs; ),
which is not explicitly represented in the model, might explain the
preferential export of UCYN-C in the mesocosms. Hence, though aggregation was
probably overestimated in the mesocosms as compared to natural situations,
the contribution of this process seems to be significant in C export. The
overestimation of UCYN-C in the model during P2 might also be explained by an
underestimation of the grazing by HNF and CIL. Nevertheless, we did not go
further in this assumption since few data regarding grazing rates by
zooplankton were available in this study. Overall, despite the clear
underestimation by the model of the UCYN-C sinking and DDN export, the main
conclusions delivered by the model concerning the fate of DDN through the
planktonic food web remain unchanged.
Conclusions
The DIP enrichment conducted during the VAHINE mesocosms experiment in the
oligotrophic water of the New Caledonia lagoon (southwest Pacific Ocean) led
to a clear increase in primary, bacterial and export productions. Two
simulations, with and without considering the DIP enrichment, were run. Their
comparison enabled the quantification of the increase in the main
biogeochemical fluxes due to the DIP enrichment. This modeling work was also
intended to investigate the fate of the N provided by N2 fixation (i.e., DDN) throughout the planktonic food web. The dynamics of the functional
groups provided by the simulation with the DIP enrichment were generally
consistent with the measured values, especially the development of UCYN-C 10
days after the DIP enrichment. The time lag of 5–10 days (concomitant with
the increase in primary, bacterial and export productions) raises the
question of the suitability of the classical methods used to quantify primary
and bacterial nutrient limitation, at least in areas where N2 fixation may
sustain a large proportion of new PP. This modeling study also enabled us to
monitor the fate of the new N input by N2 fixation (DDN) in the ecosystem.
According to the model, DDN is mainly found in the dissolved pool (NH4+
and DON) before benefiting the whole planktonic community. At the end of the
simulation, 43, 33 and 15 % were found in non-diazotroph
organisms, UCYN-C and DON, respectively. The exported matter collected in the
traps at 15 m depth showed that export is essentially due to the sinking of
small organisms. Although the measured and simulated C, N and P export was consistent in magnitude, the simulated percentage of DDN in traps was
significantly lower than that of experimental measurements. During the
experiment, UCYN-C export was high, probably due to their aggregation in
larger particles because of the secretion of TEP or EPS, which increased their
own sinking velocity rather than the sinking velocity of the whole suspended
matter as considered in the present model. Directly or indirectly, small
diazotrophs significantly contribute to the particulate export through the
aggregation process, which needs to be further investigated in future work.