Influence of irradiance and iron on the growth of colonial Phaeocystis antarctica: implications for

18 Pages·2010·0.5 MB·English

Vol. 57: 203–220, 2009 AQUATIC MICROBIAL ECOLOGY Printed November 2009
doi: 10.3354/ame01334 Aquat Microb Ecol Published online October 14, 2009
Influence of irradiance and iron on the growth
of colonial Phaeocystis antarctica: implications for
seasonal bloom dynamics in the Ross Sea, Antarctica
Nathan S. Garcia1,3,*, Peter N. Sedwick2, Giacomo R. DiTullio1
1College of Charleston, 331 Fort Johnson Road, Charleston, South Carolina 29412, USA
2Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, 4600 Elkhorn Avenue, Norfolk,
Virginia 23529, USA
3Present address: University of Southern California, 3616 Trousdale Parkway, Los Angeles, California 90089, USA
ABSTRACT: Laboratory culture experiments were used to investigate the growth rate of colonial
Phaeocystis antarcticaas a function of irradiance and dissolved iron concentration. The experiments
were conducted with a P. antarcticastrain isolated from the southern Ross Sea, Antarctica, and made
use of natural, low-iron (<0.2 nM dissolved Fe) filtered seawater as a growth medium, thereby avoid-
ing the addition of synthetic organic ligands to regulate dissolved iron concentrations. Under iron-
and nutrient-replete conditions, colonial P. antarctica attained an average maximum cell-specific
growth rate of 0.37 d–1 at an irradiance of 68 µE m–2 s–1, above which growth rates decreased to
0.27d–1at an irradiance of 314 µE m–2s–1. The dependence of growth rate on ambient dissolved iron
concentration was examined in dose–response type bioassay experiments using realistic sub-
nanomolar additions of dissolved iron. The experimental results indicate significant changes in the
iron requirements for growth of colonial P. antarcticaas a function of irradiance, with our estimates
of the half-saturation constant for growth with respect to dissolved iron (Kμ) ranging from 0.26 nM at
~20 µE m–2s–1, to 0.045 nM at ~40 µE m–2s–1and to 0.19 nM at ~90 µE m–2s–1. We interpret these vari-
ations in Kμas reflecting an increase in the cellular iron requirements of colonial P. antarcticaat sub-
optimal and supraoptimal irradiance, such that the cells require higher ambient dissolved iron con-
centrations to attain maximum growth rates under such irradiance conditions. The experiments also
provide evidence of a relationship between iron availability and the relative proportion of colonial
versus solitary P. antarctica cells, whereby the colonial form appears to be favored by higher dis-
solved iron concentrations. Our experimental results suggest that the initiation and termination of
colonial P. antarcticablooms in the Ross Sea are determined by the combined effects of irradiance-
driven changes in cellular iron requirements and a seasonal decrease in dissolved iron availability.
KEY WORDS: Phaeocystis antarctica· Growth· Iron· Light· Ross Sea· Bloom dynamics
Resale or republication not permitted without written consent of the publisher
INTRODUCTION early summer (October to December), followed by
diatom blooms in mid- to late summer (Bunt & Wood
The largest and most predictable phytoplankton 1963, El-Sayed et al. 1983, Comiso et al. 1993, DiTullio
blooms in the Southern Ocean are observed over the & Smith 1996, Smith & Gordon 1997, Arrigo et al.
Antarctic continental shelves, particularly in the south- 1998b, Goffart et al. 2000, Smith et al. 2000, Arrigo &
ern Ross Sea (Smith & Gordon 1997, Arrigo et al. van Dijken 2004). P. antarctica is considered a key-
1998a, Arrigo & van Dijken 2003). Here the phyto- stone species in this region, because of its major role in
plankton community is typically dominated by blooms the biogeochemical cycling of carbon, sulfur and nutri-
of colonial Phaeocystis antarcticain austral spring and ent elements (Gibson et al. 1990, Smith et al. 1991,
*Email: [email protected] © Inter-Research 2009 · www.int-res.com
204 Aquat Microb Ecol 57: 203–220, 2009
DiTullio & Smith 1995, Arrigo et al. 1999, DiTullio et al. and irradiance exert controls on the distribution and
2000, Sweeney et al. 2000, Schoemann et al. 2005). The species composition of phytoplankton blooms in the
factors that control the spatial and temporal distribu- Ross Sea (Sedwick et al. 2000, 2007, Boyd 2002, Arrigo
tion of P. antarctica blooms in the southern Ross Sea et al. 2003). Indeed, recent numerical models of the
are not well understood. Bottom-up controls by irradi- Ross Sea ecosystem (Arrigo et al. 2003, Arrigo & Tagli-
ance, iron and vitamin B have been suggested abue 2005, Tagliabue & Arrigo 2005) have successfully
12
(Arrigo et al. 1998b, Fitzwater et al. 2000, Sedwick et simulated the broad, regional-scale features of P.
al. 2000, Smith et al. 2003a, Bertrand et al. 2007), antarcticaand diatom blooms by incorporating explicit
whereas top-down control by grazing pressure is not limitation of growth rates by iron availability and verti-
thought to be significant (Caron et al. 2000, Tagliabue cal mixing (i.e. irradiance). These models prescribe an
& Arrigo 2003). Field observations document a general elevated growth rate for P. antarcticaat low irradiance,
decrease in the depth of the surface mixed layer, which and for diatoms at high irradiance, with an additional
allows an increase in mean irradiance during the constraint of photoinhibition for P. antarctica at high
growing season (Arrigo et al. 1998b, Gordon et al. light (Arrigo et al. 2003). The models also assume a sig-
2000, Smith et al. 2003a, Sweeney 2003, Worthen & nificantly higher iron requirement for the growth of
Arrigo 2003). Over the same period, the concentrations diatoms relative to colonial P. antarctica, by prescrib-
of dissolved iron in the surface mixed layer apparently ing a higher Fe/C uptake ratio and higher half-satura-
decrease from ~0.2–0.5 nM or more to <0.2 nM, while tion constant for growth with respect to iron (Kμ) for
dissolved macronutrient concentrations remain rela- diatoms (Tagliabue & Arrigo 2005). The resulting
tively high (Fitzwater et al. 2000, Sedwick et al. 2000, model simulations imply that iron availability controls
Smith et al. 2003a, Coale et al. 2005). annual primary production, whereas the differential
Studies of the effects of light on the growth of Phaeo- responses of P. antarcticaand diatoms to variable light
cystis antarcticasuggest that this species is inherently regimes control the distribution of these algal groups
well adapted to low and/or highly variable irradiance in space and time.
(Palmisano et al. 1986, Arrigo et al. 1999, van Leeuwe However, the broad-scale agreement of model simu-
& Stefels 2007). On this basis, it has been argued that lations with field observations does not necessarily
the ability of P. antarctica to maintain high rates of imply that such models are mechanistically accurate.
photosynthesis at low or variable irradiance allows this At present, there is considerable uncertainty regarding
species to dominate the poorly stratified waters of the the phytoplankton iron parameters used in such mod-
southern Ross Sea during spring (Leventer & Dunbar els, and the use of fixed values for these parameters
1996, Arrigo et al. 1998b, 1999, Goffart et al. 2000). may be inappropriate (Tagliabue & Arrigo 2005).
This hypothesis can explain the observed spatial In this context, there is a pressing need for quantita-
and temporal separation of P. antarctica and diatom tive information concerning the iron requirements for
blooms in the Ross Sea, if it is assumed that diatoms are growth of colonial Phaeocystis antarctica; that is, the
better adapted to the stable higher irradiance of more growth rate of colonial P. antarctica as a function of
stratified waters (Arrigo et al. 1998b, 1999, 2003, ambient dissolved iron concentration. Quantitatively,
Sedwick et al. 2000). However, this idea is challenged this information is provided by Kμ, which represents
by the results of field and laboratory studies that indi- the ambient dissolved iron concentration at which
cate little difference between the photosynthesis– phytoplankton achieve a growth rate (μ) that is 50% of
irradiance characteristics of P. antarctica and diatoms the maximum iron-saturated growth rate (μ ) for a
max
in the southern Ross Sea (van Hilst & Smith 2002, given irradiance under nutrient replete conditions (e.g.
Smith & van Hilst 2004). see Timmermans et al. 2001, 2004). Coale et al. (2003)
Availability of iron is also likely to influence the tim- performed shipboard dose-response iron-addition
ing and location of Phaeocystis antarctica and diatom experiments in the Ross Sea and observed an increase
blooms in the Ross Sea. Certainly there is good evi- in the abundance of haptophytes (presumably P.
dence for the role of iron availability in limiting phyto- antarctica) in response to iron addition, with their
plankton growth rates in this region, as shown in the results suggesting relatively low Kμvalues in the range
results of shipboard bioassay experiments (Martin et of 0.005 to 0.043 nM dissolved iron. However, these
al. 1990, Sedwick & DiTullio 1997, Sedwick et al. 2000, experiments were conducted under full sea-surface
Coale et al. 2003). This and the antagonistic relation- irradiance during late summer when there is an
ship between the iron requirements of phytoplankton increased abundance of solitary P. antarctica cells
and irradiance, whereby phytoplankton require more (Smith et al. 2003b); hence, the experimental results
iron per cell under sub-optimal irradiance (Raven might not be representative of the colonial P. antarctica
1990, Sunda & Huntsman 1997, van Leeuwe & Stefels blooms that dominate the southern Ross Sea in spring
2007), lead to the hypothesis that both iron availability and early summer.
Garcia et al.: Light and iron effects on Phaeocystis antarcticagrowth 205
In this paper, we report the results of laboratory
experiments that examined the growth of predomi-
nantly colonial Phaeocystis antarctica cultures as a
function of both irradiance and dissolved iron concen-
tration. For these experiments we used a recent isolate
of P. antarctica and filtered low-iron seawater, both
collected from the southern Ross Sea, in an effort to
simulate natural field conditions. Our results indicate
that the iron requirements for growth of colonial P.
antarctica (as expressed by Kμ) vary significantly as a
function of irradiance and suggest a new conceptual
model for the role of iron availability and irradiance in
regulating the growth of P. antarcticain the Ross Sea.
MATERIALS AND METHODS
Fig. 1. The Ross Sea indicating collection locations for low-
To glean meaningful data from laboratory studies of iron filtered seawater (Stn B) and phytoplankton (Stn 25)
marine phytoplankton, the experimental conditions used in this study
should approach natural field conditions as closely as
possible. This includes not only the physical and chem-
ical conditions of the experiments, but also the culture cultures, and the absence of other phytoplankton
history of the phytoplankton that are used. Ideally, the species was verified by microscopy. One of these P.
algae used should be recent field isolates, rather than antarctica cultures, NBP03-05A-25, was the primary
laboratory clones that have been cultured over ex- source culture for all experiments described in this
tended periods of time under temperature, irradiance report.
and nutrient conditions that are different from the nat- All growth media used for maintaining cultures and
ural environment. Over a period of years, such condi- conducting laboratory experiments were prepared
tions might be expected to result in significant gene with low-iron filtered seawater collected from the
mutations and/or deletions in the cultured algal strains southern Ross Sea near 76°02’S, 169°53’E (Fig. 1) in
relative to phytoplankton in the field. In the present December 2003. Approximately 4000 l of this low-iron
study, we conducted experiments with a field isolate of seawater was collected from ~10 m water depth
Phaeocystis antarcticathat was cultured in our labora- through acid-cleaned polyethylene tubing, which was
tory over a period of approximately 2 yr, and used nat- deployed on a non-metal line from the research vessel
ural, low-iron, surface seawater from the Ross Sea as a ‘Nathaniel B. Palmer’ while slowly underway. The sea-
basic growth medium. water was pumped aboard using an electrically oper-
Field collections and culture isolation. A unialgal ated polypropylene-Santoprene double-diaphragm
strain of Phaeocystis antarctica was isolated from pump (Jabsco), filtered through in-line, acid-rinsed
mixed algal assemblages collected in the southern 1-µm/0.2-µm pleated-polypropylene cartridge filters
Ross Sea during December 2003. These assemblages with a 1 µm prefilter (Cole-Parmer), and collected in
were collected near 76°05’S, 170°08’E (Fig. 1) in acid-cleaned 125 l opaque polyethylene barrels with
Niskin bottles mounted on a CTD rosette deployed air-tight polyethylene screw closures. Subsequent
from the research vessel ‘Nathaniel B. Palmer’ (cruise measurements of iron in sub-samples of this filtered
NBP03-05A). The algal assemblages were maintained seawater (Sedwick et al. 2007) revealed relatively low
in L1 nutrient medium (Guillard & Hargraves 1993) dissolved iron (dFe) concentrations of 0.07 ± 0.02 nM
diluted 20-fold with filtered surface seawater (here- (mean ± 1 SD, n = 4). This seawater is hereafter
after L1/20 medium), at 0°C in a shipboard Percival referred to as filtered low-iron seawater. For use in the
incubator. After the cruise, the algal cultures were experiments described in this paper, the seawater was
transported to the Hollings Marine Laboratory in removed from the polyethylene barrels through acid-
Charleston, South Carolina, where P. antarctica cleaned silicone tubing using a peristaltic pump, with
colonies were isolated using a capillary pipette, then in-line filtration through a pre-rinsed 0.2 µm Supor
transferred through a series of 5 or more glass wells CritiCap filter capsule (Pall), following a stringent
containing L1/20 medium. Several unialgal cultures of trace-metal clean transfer protocol.
P. antarctica were grown from these isolated colonies Laboratory irradiance experiments. Batch-culture
in L1/20 medium at 0°C. The colonial form dominated incubation experiments were performed to examine
206 Aquat Microb Ecol 57: 203–220, 2009
Table 1. Summary of data from light experiments with Phaeocystis antarctica. See details in ‘Materials and methods’ and
‘Results’. Data in boldtext are plotted in Fig. 3
Light experiment Total cell density (cells ml–1) Growth period (t )
d
Irradiance treatment at sampling time points from 0 to 39 d Growth rate (d–1)
Expt 1 0 8 13 16 20.5 t–t t–t t –t t –t t–t
0 8 8 13 13 16 16 20.5 8 16
28 µE m–2s–1 130 1141 7587 23822 0.272 0.254 0.237
68 µE m–2s–1 130 3599 20974 36072 0.416 0.353 0.181 0.288
Expt 2 0 16 22 t–t t –t
0 16 16 22
68 µE m–2s–1 250 1733 15575 0.121 0.366
Expt 3 0 3 8 11 t–t t–t t–t
0 3 3 8 8 11
150 µE m–2s–1 3209 4947 20161 28396 0.144 0.281 0.114
Expt 4 0 7 16 24 29 t–t t–t t –t t –t
0 7 7 16 16 24 24 29
20 µE m–2s–1 600 634 2807 14204 27824 0.008 0.165 0.203 0.134
0 7 15 20 24 t–t t–t t –t t –t
0 7 7 15 15 20 15 24
50 µE m–2s–1 600 556 5764 36247 –0.01 0.292 0.204
68 µE m–2s–1 600 638 9176 28175 0.009 0.333 0.224
Expt 5 0 27 30 34 39 t–t t –t t –t t –t t –t
0 27 27 30 30 34 34 39 27 39
16abµE m–2s–1 496 2876 4797 10600 22090 0.065 0.171 0.198 0.147 0.170
27 28 30 34 t –t t –t t –t
27 28 28 30 30 34
*85bµE m–2s–1 2876 3191 5372 15303 0.104 0.260 0.262
*314bµE m–2s–1 2876 3426 10268 13206 0.175 0.549 0.063
Expt 6 0 8 11 15 t–t t–t t –t t–t
0 8 8 11 11 15 8 15
85abµE m–2s–1 728 5871 6884 25388 0.261 0.053 0.326 0.209
8 9 11 12 15 t–t t–t t–t t –t t –t
8 9 9 11 9 12 11 15 12 15
*16bµE m–2s–1 5871 4103 7415 17338 –0.358 0.197 0.283
*314bµE m–2s–1 5871 3654 6902 14750 –0.474 0.318 0.190
Expt 7 0 3 7 10 14 t–t t–t t –t t –t t–t
0 3 3 7 7 10 10 14 7 14
314abµE m–2s–1 1555 1795 5301 12016 32092 0.048 0.271 0.273 0.246 0.257
7 8 10 14 t–t t–t t –t t–t
7 8 8 10 10 14 8 14
*85bµE m–2s–1 5301 8873 14076 36449 0.515 0.231 0.238 0.235
*16bµE m–2s–1 5301 9175 14159 24604 0.549 0.217 0.138 0.164
*Initial irradianceawas different from final irradianceband growth rates may not represent light-acclimated conditions
the growth rate of colonial Phaeocystis antarctica in ent and iron concentrations of approximately 15 µM ni-
iron- and nutrient-replete media at irradiance levels trate + nitrite, 1.3 µM phosphate and 25 nM dFe. Given
ranging from 16 to 314 µE m–2s–1(Table 1). We carried these relatively high initial concentrations of macronu-
out these irradiance experiments in 1.2 l polycarbonate trients and iron, we assume that observed exponential-
bottles at 0°C under continuous fixed irradiance gener- phase growth rates represent growth under iron- and
ated by 61 cm (24 in) T8 or T12 cool white flourescent nutrient-replete conditions.
lights (Philips). Marked changes in the diel light cycle The protocols used for our irradiance experiments
are expected in the southern Ross Sea during the grow- are summarized in Fig. 2a. The experiments were initi-
ing season, owing to seasonal changes in the solar an- ated by diluting approximately 1 l of colonial Phaeo-
gle as well as short-term changes in the depth of the cystis antarctica culture in exponential growth phase
surface mixed layer (Arrigo et al. 1998b, Gordon et al. to a volume in the range of 18 to 43 l, using L1/457
2000, Smith & van Hilst 2004). We did not attempt to medium chilled to 0°C. This produced experimental
mimic these in situtemporal variations in irradiance in ‘inoculum cultures’ with initial cell densities in the
our experiments; instead, we chose to conduct the ex- range of 200 to 750 cells ml–1, with the exception of
periments under continuous irradiance using a range of irradiance Expts 3 (~3000 cells ml–1) and 7 (~1500 cells
values typical of the mean mixed-layer irradiance in the ml–1). These inoculum cultures were then gently mixed
southern Ross Sea from early spring through early sum- and used to fill multiple 1.2 l polycarbonate incubation
mer (Smith et al. 2000, Hiscock 2004, Smith & van Hilst bottles, leaving 200 ml of headspace in each bottle. In
2004). The growth medium used for these experiments addition, subsamples were taken from the inoculum
was L1 nutrient medium (Guillard & Hargraves 1993) cultures for measurements of chlorophyll a(chl a) con-
diluted 457-fold with filtered low-iron seawater (here- centration and cell density (in cells ml–1). The 1.2 l bot-
after L1/457 medium), which yielded initial macronutri- tles were immediately transferred to a refrigerated
Garcia et al.: Light and iron effects on Phaeocystis antarcticagrowth 207
a
Algal assemblages collected 125 l barrels
from Ross Sea maintained in
Ross Sea water amended with
nutrients from L1 recipe
diluted 20-fold (L1/20)
Ross seawater
collected filtered and Irradiance experiments
stored in barrels #1 #2 #3 #4
Colonies isolated with
~42 l filtered seawater Low
capillary pipette and
Light
maintained in L1/20 Irradiance experiments
50 l carboys
High
Light
~43 l ~43 l
h
Culture oug Culture Irradiance experiments
Expt #1 hr Expt #7
t Low
#5
Light
Cultures diluted to L/457 Cultures diluted with
#6
nutrient concentration Ross Sea water amended
High
(~25 nM dFe) several with L1/457 nutrients #7
Light
months prior to experiment for light experiments
b
Algal assemblages collected 125 l barrels Iron experiments
~42 l
from Ross Sea maintained in 50 l carboys
filtered
Ross Sea water amended with
seawater
nutrients from L1 recipe
~43 l ~43 l ~43 l
diluted 20-fold (L1/20)
Ross seawater Culture Culture Culture
collected filtered and Expt #1 Expt #2 Expt #3
stored in barrels 750 ml
culture Cultures diluted with Ross Sea
Colonies isolated with
water for iron experiments
capillary pipette and
maintained in L1/20 Expt Expt Expt
#1 #2 #3
Iron experiments
Cultures diluted #1 #2 #3
20-fold yielding
High
~1.25 nM dFe
dFe
and acclimated
to 20, 40, and
90 µE m–2 s–1
Cultures diluted to L/457
nutrient concentration
Low
(~25 nM dFe) several
dFe
months prior to experiment
Fig. 2. Culturing techniques and protocols for (a) irradiance experiments and (b) iron-addition experiments. In Irradiance Expts 5
to 7, light intensity was changed in the middle of the experiments (see ‘Materials and methods’). Irradiance was 20, 40 and
90 µE m–2s–1for iron Expts 1, 2 and 3, respectively; dFe: dissolved iron
208 Aquat Microb Ecol 57: 203–220, 2009
incubator (Powers Scientific model FMS33SDF), or to trace-metal clean techniques, we were able to produce
an ice bath maintained in a cold room and incubated at experimental unialgal inoculum cultures that were
0°C over periods of 11 to 39 d at the fixed irradiance predominantly colonial cells of P. antarctica in a sea-
levels listed in Table 1. During the incubation period, water medium containing low concentrations of dis-
duplicate 1.2 l bottles were terminally subsampled (i.e. solved iron (0.05 to 0.22 nM) typical of Ross Sea surface
the entire incubation bottle was used in the subsam- waters (Martin et al. 1990, Fitzwater et al. 2000, Sed-
pling procedure) at 3 to 4 time points for measure- wick et al. 2000, Coale et al. 2005).
ments of chl a concentration and total cell density. Using this approach, we conducted iron-addition
Because of the heat generated when producing high experiments to examine the effect of ambient dissolved
irradiance, treatments greater than 100 µE m–2 s–1 iron concentrations on the growth of colonial Phaeo-
were incubated in an ice-water bath inside a 4°C cold cystis antarcticaat irradiances of approximately 20, 40
room, rather than in the refrigerated incubator. To min- and 90 µE m–2 s–1 (hereafter referred to as iron addi-
imize biases due to spatial variations of the irradiance tion Expts 1, 2 and 3, respectively), as summarized in
field in the incubators or ice bath, bottle positions were Table2and Fig. 2b. In these experiments, P. antarctica
alternated on a daily basis. Given initial cell densities isolates that had been maintained as semi-continuous
of around 500 cells ml–1 in the inoculum cultures cultures in L1/20 medium were diluted with low-iron
(except irradiance Expts 3 and 7), we assume that the filtered seawater to produce primary starting cultures
P. antarcticacells were acclimated to the experimental that were predominantly colonial P. antarctica in
irradiance levels for the periods when exponential L1/457 medium (~25 nM dFe) approximately 3 mo
growth rates were calculated, during which total cell before the start of each experiment. These primary
densities were in the range of 5000 to 10000 cells ml–1 starting cultures were maintained in an incubator at
(see Table 1for details). For Expts 3 and 7, cells were 0°C at a continuous irradiance of ~50 µE m–2s–1. At 1 to
acclimated to experimental light levels for approxi- 2 wk before the start of each iron-addition experiment,
mately 5 generations before the beginning of the a secondary starting culture of P. antarctica was pre-
experiments. To examine the effects of rapid changes pared by diluting the primary starting culture 20-fold
in irradiance on cellular physiology and biochemistry with 0.2 µm filtered, low-iron seawater in an acid-
of P. antarctica, a subset of incubation bottles from cleaned 1.2 l polycarbonate bottle, using a trace-metal
irradiance Expts 5 to 7 were exposed to a range of irra- clean protocol. The resulting growth medium for these
diance levels (see Table 1 [final irradiance], Fig. 2a) secondary starting cultures contained approximately
following acclimation to the initial experimental irradi- 1.25 nM dFe. These secondary starting cultures were
ance for periods of 7 to 27 d. acclimated to continuous irradiances of 20, 40 or 90 µE
Laboratory iron dose-response experiments. Re- m–2s–1(for iron addition Expts 1, 2 or 3, respectively) at
sults of preliminary laboratory and shipboard experi- 0°C in a refrigerated incubator, as described for the
ments indicated that colonial Phaeocystis antarcticais irradiance experiments, for 1 to 2 wk before commenc-
able to access dissolved iron that is complexed by the ing the iron-addition experiments.
siderophore desferrioxamine-B (DFOB), even when The iron addition experiments were initiated by
DFOB is added to cultures in more than a 10-fold molar removing ~250 ml of a light-acclimated secondary
excess of total dissolved iron (Garcia 2006, Sedwick et starting culture for initial measurements of chl a and
al. 2007). These and the results of other laboratory accessory pigments (see DiTullio et al. 2007). The
studies (e.g. see Boyé & van den Berg 2000, van remaining ~750 ml of the secondary starting culture
Leeuwe & Stefels 2007) suggest that haptophytes are was then added to 40 to 45 l of filtered low-iron sea-
able to produce specific Fe(III)-binding organic ligands water that had been pre-chilled to ~0°C in an acid-
and/or reduce ligand-bound Fe(III) at the cell surface cleaned 50 l polyethylene carboy. Our measurements
(Shaked et al. 2005). With the assumption that organi- verified that the filtered low-iron seawater used to
cally complexed iron may be biologically available to dilute the Phaeocystis antarctica cultures contained
P. antarctica, we chose to conduct our iron-addition relatively low concentrations of dissolved iron: 0.17 ±
experiments without adding synthetic organic ligands 0.10 (SD) nM(n = 4) for Expt 1, 0.041 ±0.006 (SD) nM
such as DFOB or EDTA to control dissolved inorganic (n = 4) for Expt 2 and 0.071 ±0.013 (SD) nM (n = 4) for
Fe(III) concentrations. This was done to mimic chemi- Expt 3. Thus, we produced large-volume inoculum cul-
cal conditions in the marine environment. To this end, tures of P. antarcticain seawater media with initial dFe
we performed experiments in which cultures of colo- concentrations measured in sub-samples of 0.22 ±
nial P. antarctica were successively diluted with fil- 0.00nM (n = 2) for Expt 1, 0.053 ±0.011 nM (n = 2) for
tered, low-iron, Ross Sea seawater, similar to the meth- Expt 2 and 0.076 ±0.014 nM (n = 2) for Expt 3 (Table 2),
ods described by Boyé & van den Berg (2000) and whereas the concentration of EDTA (remaining from
Timmermans et al. (2001, 2004). By applying stringent dilution of the L1 medium) was negligible (<0.02 nM).
Garcia et al.: Light and iron effects on Phaeocystis antarcticagrowth 209
Table 2. Summary of data from iron addition experiments. Nitrate + nitrite concentration was used to calculate growth rates in
Expt 1, whereas total cell density was used to calculate growth rates in Expts 2 and 3. See ‘Materials and methods’ for details.
Data in boldtext were used to calculate Kμ, μmaxand r2. Error associated with Kμand μmaxis the SE of the slope and y-intercept,
respectively, of the Eadie-Hofstee regression
Added Initial Number [NO3–+ NO2–] (µM) or Growth period (t, d) Kμ (nM dFe)
dFe (nM) total dFe of 1 l total cell density (cells ml–1) at Growth rate (d–1) μ (d–1)
max
(nM) bottles sampling time points from 0 to 31 d r2
Expt 1: 20 µE m–2s–1 0 16 25 31 t –t t –t t –t 0.26 ±0.0014
16 25 25 31 16 31
0.0 0.22 7 13.38 13.20 12.78 12.21 0.135 0.111 0.125 0.29 ±0.00062
0.2 0.42 7 13.38 12.96 11.22 8.16 0.181 0.147 0.167 0.99
0.6 0.82 7 13.38 13.02 11.66 6.83 0.173 0.222 0.193
1.8a 2.02 7 13.38 13.30 11.44 10.56 a0.345 0.062 0.232
Expt 2: 40 µE m–2s–1 0 7 13 16 t–t t –t t–t 0.045 ±0.0041
7 13 13 16 7 16
0.0 0.05 7 1102 1290 4344 5326 0.202 0.068 0.158 0.38 ±0.011
0.09 0.14 7 1102 1958 11490 12186 0.295 0.020 0.203 0.96
0.18 0.23 7 1102 2000 12195 20419 0.301 0.172 0.258
0.36 0.41 7 1102 2073 15107 24477 0.331 0.161 0.274
0.72 0.77 8 1102 2181 19758 43132 0.367 0.260 0.332
Expt 3: 90 µE m–2s–1 0 20 26 31 t –t t –t t –t 0.19 ± 0.040
20 26 26 31 20 31
0.0 0.08 7 271 296 524 786 0.095 0.081 0.089 0.33 ±0.042
0.05 0.13 7 271 307 700 1255 0.137 0.117 0.128 0.84
0.10 0.18 7 271 511 1115 1399 0.130 0.019 0.092
0.30 0.38 7 271 482 1871 5236 0.226 0.206 0.217
0.90 0.98 8 271 747 1853 8018 0.151 0.293 0.216
aIndicates treatment was omitted from calculations of Kμ, μmax, and r2(see ‘Results’ and ‘Discussion’)
The resulting low-iron inoculum culture was gently approximately 20, 40 or 90µEm–2s–1, for iron addition
mixed before removing ~3 l for initial measurements of Expts 1, 2 or 3, respectively. As in the irradiance
dFe, chl a, macronutrients (dissolved inorganic nitrate experiments, bottle positions inside the incubator
+ nitrite, phosphate, silicate), and cell density. This were alternated on a daily basis to minimize biases
inoculum culture was used to fill multiple acid-washed due to spatial variations in the irradiance field. At
1.2 l polycarbonate bottles that had been pre-rinsed each sampling time point, duplicate bottles for each
with filtered low-iron seawater, leaving 200 ml of iron treatment were terminally sampled for analysis of
headspace in each bottle. For each experiment, suffi- chl a, dissolved macronutrients and cell density (cell
cient 1.2 l bottles were filled to provide duplicate bot- density data from Expt 1 are considered semi-quanti-
tles for 4 to 5 different iron treatments (see Table 2,Fig. tative only; see ‘Analytical methods’ section below).
2b). For each iron treatment, an extra 1.2 l bottle was To avoid contamination with iron, the preparation of
filled and incubated for the analysis of accessory pig- the low-iron inoculum cultures and the filling of the
ments at the final sampling time point (see DiTullio et 1.2 l incubation bottles were carried out using strin-
al. 2007). In addition, for the highest iron treatment in gent trace-metal clean techniques inside a plastic
each experiment, an extra 1.2 l bottle was filled, incu- ‘bubble’ enclosure under positive-pressure Class-100
bated and successively sampled during the course of filtered air. In addition, all materials contacting the
the experiment to monitor changes in algal biomass low-iron filtered seawater and experimental inocula
and thereby select appropriate sampling time points. were rigorously acid cleaned using methods similar to
During the bottle-filling process, the inoculum cul- those described by Sedwick et al. (2000). Strict axenic
ture was gently mixed to minimize biases associated conditions were not maintained; however, to minimize
with settling or floating of the Phaeocystis antarctica bacterial contamination of the experimental cultures
colonies. Iron was added to each 1.2 l bottle (except the low-iron seawater used to prepare and dilute the
the control treatments) as a 17.9 µM ferric nitrate experimental inocula was filtered through a 0.2 µm
solution prepared in 0.1% by volume HCl (Fisher Supor CritiCap capsule filter (Pall) immediately
trace metal grade); the nitrate and HCl added in this before use. In addition, the 50 l inoculum carboy and
iron solution had negligible effects on the nitrate con- 1.2 l incubation bottles were thoroughly rinsed with
centration and pH of the inoculum cultures. Once this 0.2 µm filtered seawater under Class-100 filtered
filled, the 1.2 l incubation bottles were transferred air immediately before filling.
into a refrigerated incubator, where they were main- Analytical methods. Colonial and total Phaeocystis
tained at 0°C under a continuous irradiance of antarctica cells were enumerated using the methods
210 Aquat Microb Ecol 57: 203–220, 2009
described by Garcia (2006). Briefly, solitary Phaeocys- are therefore assumed to be representative of the colo-
tis antarcticacell density (n, cells ml–1) was estimated nial form of P. antarctica.
s
by loading 20 ml of a culture sample into a Multisizer 3 We infer net growth rates (μ) in the experimental
particle counter (Beckman-Coulter) and counting par- incubations from the accumulation of total Phaeocystis
ticles in the 3 to 9 µm size range. Total P. antarcticacell antarctica cells, except in the case of iron addition
density (n, cells ml–1) was similarly estimated using a Expt 1, for which we use net decrease (drawdown) in
t
230 ml culture sample that was acidified to pH 3.1 for dissolved nitrate + nitrite concentration as a proxy for
20 min by addition of 10% (by volume) HCl solution. net increase in cellular biomass, assuming balanced
Colonial P. antarctica cell density (n , cells ml–1) was growth. These growth rates were calculated from
c
then calculated by difference as n = n – n. Because increases in total cell number (or nitrate + nitrite draw-
c t s
this cell enumeration method was developed during down) between sampling time points that yielded the
the course of our study, the cell density data reported maximum calculated growth rate for a given experi-
for iron addition Expt 1 should be regarded as semi- mental treatment. As a result, the growth rates and
quantitative only. Selected sub-samples of the experi- associated Kμ and μmax values we report for iron
mental cultures were preserved for microscopic obser- addition Expt 1 differ from those previously reported
vations and cell enumeration by adding 1% (by from this experiment by Sedwick et al. (2007), who cal-
volume) of modified Lugol’s solution (30 g l–1 potas- culated growth rates using nitrate + nitrite concentra-
sium iodide, 20 g l–1iodine, 315 ml l–1ethanol, 35 ml l–1 tions at time points of 25 and 31 d for all experimental
gluteraldehyde, 15 ml l–1 glacial acetic acid), which treatments. In detail, the cell-specific net growth rates
preserves both whole colonies and solitary cells of P. for the incubation experiments were calculated from
antarcticafor microscopy (V. Schoemann pers. comm.). the mean total cell density (N) of replicate bottles at
Dissolved inorganic nutrients (nitrate + nitrite, phos- successive time points t and t , using the equation:
1 2
phate and silicic acid) were determined in samples fil-
μ = [ln(N ) – ln(N )]/(t – t ) (1)
tered through 0.45 µm Supor Acrodisc filters (Pall) by t2 t1 2 1
the Marine Science Institute Analytical Laboratory, For iron addition Expt 1, in the absence of reliable
University of California, Santa Barbara, using standard cell density data, we used this same equation to calcu-
flow analysis methods. Duplicate sub-samples for mea- late nitrate-specific growth rates, where N and N
t1 t2
surement of particulate chl a were collected by filter- instead represent the net drawdown in nitrate + nitrite
ing cultures through GF/F glass fiber filters (Whatman) from the initiation of the incubations to sampling times
under a gentle vacuum of 1 to 2 pounds per square t and t , respectively.
1 2
inch; filters were then extracted in 90% acetone for Some deviations from balanced growth are likely to
24h, and chl adetermined in the extracts using a 10AU have occurred during our batch-type incubation
fluorometer (Turner Designs) following standard Joint experiments. For this reason, we chose not to use mea-
Global Ocean Flux Study (JGOFS) protocols (Knap et sured changes in chl a to estimate net growth rates,
al. 1996). Dissolved iron was determined in filtered because changes in irradiance and iron availability are
seawater and in culture samples that were filtered known to cause strong variations in the cellular chl a
through 0.4 µm polycarbonate membranes (Poretics) content of Phaeocystis antarctica. Here net growth
using flow injection analysis, as described by Sedwick rates estimated from nitrate + nitrite drawdown, as are
et al. (2005). The incident irradiance for the experi- presented for iron addition Expt 1, are expected to be
mental cultures was estimated by submerging a QSL- more reliable, because cellular C:N ratios are thought
100 light meter sensor (Biospherical Instruments) to be less plastic than C:chl a ratios in the case of
inside a 1.2 l polycarbonate bottle filled with water, unbalanced growth (Stefels & van Leeuwe 1998, van
then recording the irradiance after ~30 min, which Leeuwe & Stefels 1998, Schoemann et al. 2005). How-
allowed the fluorescent lights to equilibrate with the ever, the potential for deviations from balanced growth
incubator set temperature (0°C). mean that the nitrate-specific growth rate estimates
Interpretation of experimental data. Microscope ob- presented for iron addition Expt 1 are less robust than
servations and particle-counter measurements indi- the cell-specific growth rates presented for iron addi-
cate that all of the experimental incubations contained tion Expts 2 and 3.
unialgal cultures of Phaeocystis antarctica that were Experimental uncertainties on calculated μ values
dominated (≥50%) by cells in colonies, and all growth were estimated by propagating the relative standard
rates were calculated between experimental time deviations on the mean total cell densities (or nitrate +
points when incubation cultures were numerically nitrite drawdown), N and N , through Eq. (1). To
t1 t2
dominated (>55 to 70%) by colonial P. antarcticacells. assess the statistical significance of differences in mea-
Our experimentally derived data for growth rates as a sured variables between experimental treatments, we
function of irradiance and dissolved iron concentration used a 1-way ANOVA at the 95% confidence level (p<
Garcia et al.: Light and iron effects on Phaeocystis antarcticagrowth 211
0.05); where experiments included multiple treat- 0.5
c
mtheen sttsa, twistei cuasle sdig onritfhicoagnocnea lo fc odmifpfearreisnocness tboe dtweteeernm tihnee pecifi )–1 0.4 *
exFpoerr itmhee nirtaoln t-raedadtmitieonnt se.xperiments, we fitted Monod ell-s te (d 0.3
c a
(or Michaelis-Menten) saturation functions to the m h r 0.2
growth rate versus dissolved iron data, as has previ- mu wt
o
ously been done by Coale et al. (1996, 2003), Timmer- axi gr 0.1
mans et al. (2001, 2004) and Blain et al. (2002). These M
saturation functions have the general form: 0.0
0 100 200 300
μ = μmax[dFe/(Kμ+ dFe)] (2) Irradiance (µE m–2 s–1)
where μis net growth rate, dFe is the initial dissolved
iron concentration in the growth medium, μmax is the Fig. 3. Phaeocystis antarctica. Maximum cell-specific net
theoretical maximum growth rate under iron-replete growth rate (μ) of colonial P. antarcticaas a function of irradi-
conditions, and Kμ is the half-saturation constant for ance: data are from specific irradiance experiments (see
Table 1). Error bars are SE and estimated as described in
growth with respect to dissolved iron. The saturation
‘Materials and methods’, except the data point indicated by
functions were fitted to our experimental μversus dFe an asterisk (#) for which the error bars represent the SE on
data using a least-squares best fit to the Eadie-Hofstee growth rates calculated from 3 separate experiments
linear transformation of the Monod function, in which μ
is plotted against μ/dFe. The Monod function requires
that μversus μ/dFe yields a straight line with slope Kμ Growth rate as a function of dissolved iron
and y-intercept μ . Given that there are significant
max
uncertainties associated with our experimental esti- Results of our 3 iron-addition experiments are pre-
mates of μ, the Eadie-Hofstee transformation should sented in Table 2and Fig. 4. The upper panels of Fig.4
provide the most robust estimates of Kμ and μmax be- show mean chl a concentrations (for iron addition
cause this method assigns equal weight to all data Expt 1, Fig. 4a) or mean total cell densities (for iron
points (Zivin & Waud 1982). The standard error (SE) on addition Expts 2 and 3, Fig. 4b,c), and the middle pan-
the slope (Kμ) and the y-intercept (μmax) of the Eadie- els show average dissolved nitrate + nitrite concentra-
Hofstee regression plots was calculated with R sta- tions (NO –+ NO –, Fig. 4d,f), all plotted as a function
3 2
tistical software. of incubation time. The lower panels show maximum
net growth rates, as calculated from either NO –+ NO –
3 2
drawdown (iron addition Expt 1, Fig. 4g) or increase in
RESULTS total cell density (iron addition Expts 2 and 3, Fig. 4h,i),
all plotted as a function of initial dissolved iron concen-
Growth rate as a function of irradiance tration. These iron-addition experiments are princi-
pally diagnostic for iron deficiency: where net growth
The data from our laboratory irradiance experiments rates are significantly enhanced in experimental treat-
yield estimates of maximum net cell-specific growth ments relative to the control (or other) treatments as a
rates for colonial Phaeocystis antarcticaas a function of result of iron amendment, we infer that growth rates in
irradiance under iron- and nutrient-replete conditions the control (or other) treatments were limited by iron
and continuous light (Table 1, Fig. 3). These results deficiency.
suggest that the growth rate of colonial P. antarctica The results of iron addition Expt 1 have been dis-
increases with irradiance up to an irradiance of cussed by Sedwick et al. (2007). In this experiment,
approximately 68 µE m–2 s–1, where maximum cell- iron amendment resulted in significant increases in
specific growth rates (μ ) were in the range of 0.33 to chl a (Fig. 4a) and decreases in NO – + NO – (Fig. 4d)
max 3 2
0.42 d–1 (average μ = 0.37 d–1). For irradiances relative to the control treatments after 31 d incubation.
max
greater than 68 µE m–2s–1, our data suggest a decrease The +0.6 nM Fe treatments achieved the greatest
in net growth rates, which decline to 0.27 d–1at an irra- increase in chl a (~5 µg l–1) and drawdown of NO – +
3
diance of 314 µE m–2s–1(Fig. 3). This observed decline NO – (~6.5 µM), relative to the starting inoculum.
2
presumably reflected a reduction in photosynthetic Assuming that these changes reflect a net accumula-
rates as a result of photoinhibition, although in the case tion of cellular biomass in the incubation bottles, the
of colonial P. antarctica, there is also the possibility that results indicate that the growth rate of colonial Phaeo-
these changes reflected unbalanced growth (see cystis antarctica in the starting inoculum was limited
‘Discussion’). by iron deficiency. Further, these data suggest that the
212 Aquat Microb Ecol 57: 203–220, 2009
20 µE m–2 s–1 40 µE m–2 s–1 90 µE m–2 s–1
7 50
–1Chl a (µgl) 0123456 a +++C100o...n862t rnnnoMMMl dddFFFeee* Total cell density –1xcells ml) 1000123400000 b ++++C0000o....n7310t2689ro nnnnlMMMM ddddFFFFeeee 100 c ++++C0000o....n9310t0005ro nnnnlMMMM ddddFFFFeeee
(
0 5 10 15 20 25 30 0 5 10 15 0 5 10 15 20 25 30
d e f
M)
12 12 12
µ
(2
–O 8 +1.8 nM dFe* 8 +0.72 nM dFe 8 +0.90 nM dFe
N +0.6 nM dFe +0.36 nM dFe +0.30 nM dFe
+ +0.2 nM dFe +0.18 nM dFe +0.10 nM dFe
– 3 4 Control 4 +0.09 nM dFe 4 +0.05 nM dFe
O
Control Control
N
0 0 0
0 5 10 15 20 25 30 0 5 10 15 0 5 10 15 20 25 30
Incubation time (d)
owth 0.5 g wth 0.5 h µ = 0.38 d–1 0.5 i
pecific gr–1)ate (d000...234 µmaxM = o0n.2o9d dfi–t1 ecific gro–1ate (d)000...234 Mro2 n=o d0m. 9faixt6 000...234 µmMax o=n 0o.d3 3fi td–1
e-sr0.1 r2 = 0.99 -spr0.1 0.1 r2 = 0.84
Nitrat 0.0 Kµ = 0.26 nM dFe Cell 0.0 Kµ = 0.045 nM dFe 0.0 Kµ = 0.19 nM dFe
0.0 0.4 0.8 1.2 1.6 2.0 0.0 0.4 0.8 1.2 1.6 2.0 0.0 0.4 0.8 1.2 1.6 2.0
Dissolved iron (nM)
Fig. 4. Results of iron addition Expts 1, 2 and 3 (left, center and right panels, respectively). Phaeocystis antarctica(a) chlorophyll
a(chl a) or (b) cell density versus incubation time for the different iron treatments. (d–f) Dissolved nitrate + nitrite versus incuba-
tion time for the different iron treatments. (g) Nitrate- or (h,i) cell-specific maximum net growth rates of P. antarcticaversus ini-
tial dissolved iron concentrations; also shown are Monod curves fitted using Kμand μmaxvalues estimated from Eadie-Hofstee
transformation of the data (see ‘Materials and methods’); dFe: dissolved iron. Asterisk indicates that treatment was omitted from
calculations of Kμ, μmaxand r2(see ‘Results’ and ‘Discussion’)
cellular biomass, hence, net growth rate of P. antarc- decreases in NO –+ NO –(Fig. 4e,f) relative to the con-
3 2
tica, increased in relative proportion with the dissolved trol treatments. These data indicate that the cellular
iron addition, with the notable exception of the biomass, total cell density and net cell-specific growth
+1.8 nM Fe treatments, which exhibited a lesser bio- rate (Fig. 4h,i) of Phaeocystis antarctica increased
logical response than the +0.2 nM and +0.6 nM Fe according to the added concentration of dissolved iron.
treatments (Fig. 4g). The anomalous results obtained Clearly, a deficiency in dissolved iron limited the
for the +1.8 nM Fe treatments is thought to reflect the growth rate of P. antarcticain all treatments relative to
loss of biologically available iron from the growth the +0.72 nM dFe (iron addition Expt 2) and +0.90 nM
medium owing to precipitation or adsorption (see dFe (iron addition Expt 3) treatments. The shorter
‘Discussion’). duration of iron addition Expt 2 (16 versus 31 d for iron
There were no such anomalous results for iron addi- addition Expts 1 and 3), in which there was a more
tion Expts 2 and 3, in which the highest initial dis- rapid biological response to the added iron, is readily
solved iron concentrations were 0.77 nM and 0.98nM, explained by the higher biomass in the starting inocu-
respectively (Table 2). In both of these experiments, lum (1100 cells ml–1for iron addtion Expt 2 compared
after incubation with added iron, we observed with 175 and 270 cells ml–1 for iron addition Expts 1
increases in total cell density (Fig. 4b,c) that mirrored and 3, respectively).