Article

Iron and manganese co-limit growth of the Southern Ocean diatom Chaetoceros debilis

Abstract and Figures

In some parts of the Southern Ocean (SO), even though low surface concentrations of iron (Fe) and manganese (Mn) indicate FeMn co-limitation, we still lack an understanding on how Mn and Fe availability influences SO phytoplankton ecophysiology. Therefore, this study investigated the effects of Fe and Mn limitation alone as well as their combination on growth, photophysiology and particulate organic carbon production of the bloom-forming Antarctic diatom Chaetoceros debilis. Our results clearly show that growth, photochemical efficiency and carbon production of C. debilis were co-limited by Fe and Mn as highest values were only reached when both nutrients were provided. Even though Mn-deficient cells had higher photochemical efficiencies than Fe-limited ones, they, however, displayed similar low growth and POC production rates, indicating that Mn limitation alone drastically impeded the cell's performance. These results demonstrate that similar to low Fe concentrations, low Mn availability inhibits growth and carbon production of C. debilis. As a result from different species-specific trace metal requirements, SO phytoplankton species distribution and productivity may therefore not solely depend on the input of Fe alone, but also critically on Mn acting together as important drivers of SO phytoplankton ecology and biogeochemistry.
Content may be subject to copyright.
RESEARCH ARTICLE
Iron and manganese co-limit growth of the
Southern Ocean diatom Chaetoceros debilis
Franziska PauschID
1,2
*, Kai Bischof
2
, Scarlett Trimborn
1,2
1EcoTrace, Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven,
Germany, 2Marine Botany, University of Bremen, Bremen, Germany
*Franziska.Pausch@awi.de
Abstract
In some parts of the Southern Ocean (SO), even though low surface concentrations of
iron (Fe) and manganese (Mn) indicate FeMn co-limitation, we still lack an understanding
on how Mn and Fe availability influences SO phytoplankton ecophysiology. Therefore,
this study investigated the effects of Fe and Mn limitation alone as well as their combina-
tion on growth, photophysiology and particulate organic carbon production of the bloom-
forming Antarctic diatom Chaetoceros debilis. Our results clearly show that growth, pho-
tochemical efficiency and carbon production of C.debilis were co-limited by Fe and Mn as
highest values were only reached when both nutrients were provided. Even though Mn-
deficient cells had higher photochemical efficiencies than Fe-limited ones, they, however,
displayed similar low growth and POC production rates, indicating that Mn limitation
alone drastically impeded the cell’s performance. These results demonstrate that similar
to low Fe concentrations, low Mn availability inhibits growth and carbon production of C.
debilis. As a result from different species-specific trace metal requirements, SO phyto-
plankton species distribution and productivity may therefore not solely depend on the
input of Fe alone, but also critically on Mn acting together as important drivers of SO phy-
toplankton ecology and biogeochemistry.
Introduction
Large parts of the Southern Ocean (SO) are classified as high-nutrient, low-chlorophyll regions
due to the observed low phytoplankton productivity despite high concentrations of macronu-
trients. The low biomass in these areas results from the very low concentrations of the trace
metal iron (Fe) [1], which is required for the optimal growth and cellular function of phyto-
plankton [26]. Fe is integrated in photosystem I and II (PSI and II) and is used in redox reac-
tions in many pathways of the cell, including the electron transport chains of photosynthesis
and respiration [4,6,7]. Much less is known whether manganese (Mn) limits or co-limits phy-
toplankton growth alongside with Fe in the SO [5]. Fe and Mn enter the SO from several
sources including atmospheric dust [8,9], upwelling [10], sediments [11,12] and melting of gla-
ciers [13] and sea ice[14]. The flux of mineral-aerosol Fe and Mn into SO waters is probably
lower than anywhere else on Earth [1,15]. In fact, concentrations of dissolved Mn in waters of
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 1 / 16
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Pausch F, Bischof K, Trimborn S (2019)
Iron and manganese co-limit growth of the
Southern Ocean diatom Chaetoceros debilis. PLoS
ONE 14(9): e0221959. https://doi.org/10.1371/
journal.pone.0221959
Editor: Bruno Jesus, University of Nantes, FRANCE
Received: March 13, 2019
Accepted: August 19, 2019
Published: September 16, 2019
Copyright: ©2019 Pausch et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
available from Pangaea at: Pausch, Franziska;
Bischof, Kai; Trimborn, Scarlett (2019): The
Southern Ocean diatom Chaetoceros debilis is
limited in growth by iron and manganese together.
PANGAEA, https://doi.org/10.1594/PANGAEA.
905473.
Funding: ST and FP were funded by the Helmholtz
Impulse Fond (HGF Young Investigators Group
EcoTrace, VH-NG-901). ST was funded by
Deutsche Forschungsgemeinschaft (TR 899/4-1).
The funders had no role in study design, data
the Drake Passage [16,17] and the Weddell Sea [18] were found to be as low as dissolved Fe con-
centrations, being generally below 0.4 nmol L
-1
and potentially (co-)limiting with Fe. Indeed,
growth and species composition of phytoplankton assemblages from the Weddell-Scotia Con-
fluence were found to be influenced by enrichment with Fe or Mn alone [19]. Following ash
additions to natural phytoplankton assemblages of the Drake Passage, a more significant stimu-
lation of photosynthetic activity and biomass was observed relative to those amended only with
Fe [17]. The ash released significantly more Mn than Fe, therefore the strong phytoplankton
growth response to ash potentially resulted from the relief of manganese (co-)limitation.
According to the three different definitions of co-limitation by [20], the study by [17] hints
towards a Type I co-limitation, where two different nutrients do not have the same biochemical
function, but are both required for optimal growth. Hence, based on results of the ash-addition
experiments Mn can act as a limiting nutrient with Fe in certain parts of the SO. The spatial pat-
terns of Fe-Mn co-limitation across the SO, however, remain largely untested [5].
Manganese is the second most abundant trace metal in thylakoids after Fe [3]. It is essential
for phytoplankton growth [21] as it is required in the water-splitting complex of PSII where
four Mn ions are involved in the oxidation of water [2]. It is further needed for the antioxidant
enzyme superoxide dismutase (SOD), which detoxifies reactive oxygen species (ROS) and pre-
vents cell damage [6,2224]. To date, the influence of Mn-deficiency on phytoplankton physi-
ology was investigated only in the temperate diatoms Thalassiosira pseudonana,Thalassiosira
weissflogii and Thalassiosira oceanica [22,25,26]. These studies revealed that low concentra-
tions of either Fe or Mn alone reduced growth of T.pseudonana and T.oceanica and that only
with the addition of both metals together highest growth rates were reached, hence demon-
strating that both diatoms were co-limited by Fe and Mn [22]. Considering the lack of knowl-
edge on the effects of Fe-Mn co-limitation in particular on Antarctic phytoplankton
physiology, the purpose of this study was to elucidate whether Fe and Mn can co-limit the eco-
logically relevant Antarctic diatom Chaetoceros debilis. The latter forms large phytoplankton
blooms during spring time [27] and therefore contributes strongly to carbon export in the SO
[28]. The strain used for this study was isolated during the European iron fertilization experi-
ment (EIFEX) where it was one of the two species with a continuous exponential growth stim-
ulated by Fe fertilization [29]. The same species also dominated the phytoplankton community
after the Fe fertilization experiment SEEDS in the North East Pacific [30]. In this study, the
responses in growth rate, photophysiology, pigment composition and particulate organic car-
bon production of C.debilis were assessed under altered Fe and Mn availability.
Material and methods
Culture conditions
Fe-Mn manipulation experiments were performed with the Antarctic diatom Chaetoceros
debilis (Polarstern expedition ‘EIFEX’ ANT-XXI/3, In-Patch, 2004, 49˚ 36 S, 02˚ 05 E, isolated
by Philipp Assmy) which was grown in stock cultures with Fe- and Mn-enriched natural Ant-
arctic seawater (F/2
R
medium [31]). For the pre-acclimation phase and the main experiment,
dilute batch cultures of C.debilis were transferred into natural FeMn-poor Antarctic seawater
(sampled during Polarstern expedition ANT29-2 on September, 20, 2013, 60˚ 32’ S 26˚ 29’ W,
Table 1). This seawater was sterile filtered through acid-cleaned filter cartridges (0.2 μm, Sarto-
bran, Sartorius) and spiked with chelexed (Chelex 100, Sigma Aldrich, Merck) macronutrients
(100 μmol L
-1
Si, 100 μmol L
-1
NO
3-
, and 6.25 μmol L
-1
PO
43-
) and vitamins (30 nmol L
-1
B
1
,
23 nmol L
-1
B
7
, and 0.228 nmol L
-1
B
12
) according to F/2
R
medium [31]. To represent trace
metal concentrations typical for Antarctic high nutrient low chlorophyll waters, a mixture of
zinc (0.16 nmol L
-1
), copper (0.08 nmol L
-1
), cobalt (0.09 nmol L
-1
Co), molybdenum (0.05
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 2 / 16
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
nmol L
-1
) was added and adjusted to maintain the ratio of the original F/2 recipe. Our experi-
mental treatments consisted of four different combinations of Fe and Mn concentrations of
the culture medium (Table 1) without any Fe or Mn addition (-FeMn treatment) and to which
enrichments of Fe alone (-Mn treatment), Mn alone (-Fe treatment) or both trace metals
together (control treatment) were made. The trace metals Fe and Mn were added as FeCl
3
(4
nmol L
-1
, AAS standard, TraceCERT, Fluka) and MnCl
2
(2 nmol L
-1
, AAS standard, Trace-
CERT, Fluka). As suggested in [32], in an effort to minimize the alteration of the natural sea-
water trace metal chemistry and ligands, no ethylenediaminetetraacetic acid (EDTA) was
added. Due to the naturally present ligands (1.62 ±0.20 nmol L
-1
), it is expected that a part of
the added Fe was buffered rather than forming inorganic colloids. In order to prevent potential
contamination, the experiments were conducted under trace metal clean conditions and there-
fore all sampling and handling of the incubations was conducted under a laminar flow hood
(Class 100, Opta, Bensheim, Germany). Prior to use, the 2 L polycarbonate incubation bottles
and other equipment were cleaned for 7 days in a detergent bath containing 1% Citranox solu-
tion (Sigma-Aldrich, St. Louis, MO, USA) followed by rinsing 7 times with Milli-Q (Millipore,
Merck, Darmstadt, Germany). All bottles and other equipment were subsequently filled with 1
M hydrochloric acid (HCl) for 7 days. After 7 rinsing steps with Milli-Q, all equipment was
dried under the clean bench and stored triple-bagged in polyethylene bags until usage.
All incubations were grown at 2˚C under a light:dark cycle of 16:8 h at a light intensity of
100 μmol photons m
2
s
1
using light-emitting diodes (LED) lamps (SolarStinger LED Sun
Strip Marine Daylight, Econlux, Cologne, Germany). Light intensities were adjusted using
with a light sensor (ULM-500 Universal Light Meter equipped with a Spherical Micro Quan-
tum Sensor US-SQS, Walz GmbH, Effeltrich, Germany). As differences in the maximum
quantum yield of photochemistry in PSII (F
v
/F
m
) were observed between treatments during
pre-acclimation after 15 days, the main experiment was started. Main experiments were con-
ducted in triplicates with a starting cell density of ~ 250 cells mL
-1
and were run in parallel.
During the experiments, the growth rates in all incubations were monitored, confirming expo-
nential growth. Depending on the experimental treatment, cells were harvested after 14 and 18
days with cell densities of 79 042 ±6 904 cells mL
-1
in the -FeMn treatment, 93 603 ±10 805
cells mL
-1
in the -Mn treatment, 72 155 ±4158 cells mL
-1
in the -Fe treatment and 114
819 ±26 661 cells mL
-1
in the control treatment. Samples for each treatment were taken at the
same time of day to allow a direct comparison of all measured parameters.
Determination of total dissolved Fe and Mn concentrations of the seawater
Samples to determine total dissolved Fe (dFe) and Mn (dMn) concentrations of the Antarctic
seawater and the different culture mediums were taken (Table 1). To this end, 100 mL of each
Table 1. Total dissolved Fe (dFe) and Mn (dMn) concentrations from the natural Antarctic seawater as well as
from the four different treatments (-FeMn, -Fe, -Mn, control) were determined in the culture medium (0.2 μm fil-
tered Antarctic seawater). Values represent the range of duplicate measurements of the different culture mediums.
dFe
(nmol L
-1
)
dMn
(nmol L
-1
)
Seawater 0.30–0.29 0.46–0.52
Culture medium:
-FeMn 0.46–0.48 0.51–0.58
-Fe 0.78–0.83 2.17–2.35
-Mn 3.19–3.70 0.57–0.58
Control 2.52–2.59 2.15–2.17
https://doi.org/10.1371/journal.pone.0221959.t001
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 3 / 16
sample were filtered through HCl-cleaned polycarbonate filters (0.2 μm pore size, 47-mm,
Nuclepore, Whatman, GE Healthcare, Chicago, IL, USA) using a trace metal clean filtration
system under a clean bench. The filtrate was then filled into a 125 mL PE bottle and stored tri-
ple-bagged at 2˚C until analysis. Between each filtration, the filtration system was cleaned in
an acid bath with 1 M HCl and rinsed with Milli-Q.
Concentrations of total dFe and dMn of each seawater sample and process blanks were ana-
lysed using a SeaFast system (Elemental Scientific, Omaha, NE, USA) [33,34] coupled to an
inductively coupled plasma mass spectrometer (ICP-MS, Element2, Thermo Fisher Scientific,
resolution of R = 4000). An iminodiacetate (IDA) chelation column (part number CF-N-0200,
Elemental Scientific) was used in the pre-concentration step. All labware used for analysis was
pre-cleaned according to the Geotraces cookbook [35]. Prior to the analysis of dFe and dMn,
0.2 μm pre-filtered seawater samples were acidified to pH 1.7 with double distilled HNO and
UV-oxidized using a 450 W photochemical UV power supply (ACE GLASS Inc., Vineland N.
J., USA). Two blanks were processed the same way during each UV digestion step. The
ICP-MS was optimized daily to achieve oxide forming rates below 0.3%. Each seawater sample
was analyzed in duplicates via standard addition to minimize any matrix effects, which might
influence the quality of the analysis. A pH of 1.7 was needed in order to minimize the forma-
tion of Mn and Fe hydroxides and was sufficiently high to minimize the loss of other trace
metals on the SeaFast column (pers. comm. Mr. Klemens, Elemental Scientific). To assess the
accuracy and precision of the method, a NASS-6 (National Research Council of Canada) refer-
ence standard was analyzed in a 1:10 dilution (corresponding to environmentally representa-
tive concentrations) at the beginning, during and at the end of a run (two batch runs; n = 6),
yielding 487 ±23 ng L
-1
(certified 495 ±46 ng L
-1
) and 546 ±48 ng L
-1
(certified 530 ±50 ng L
-
1
) for dFe and dMn, respectively.
Chlorophyll afluorescence
At the end of the experiments, chlorophyll afluorescence measurements were conducted for
each replicate at 2˚C using a Fast Repetition Rate fluorometer (FRRf, FastOcean PTX sensor,
Chelsea Technologies Group (CTG) Ltd, West Molesey, UK) connected with a FastAct Labo-
ratory system (CTG Ltd). Chlorophyll afluorescence measurements were conducted with cell
densities of 79 042 ±6 904 cells mL
-1
in the -FeMn treatment, 93 603 ±10 805 cells mL
-1
in the
-Mn treatment, 72 155 ±4158 cells mL
-1
in the -Fe treatment and 114 819 ±26 661 cells mL
-1
in the control treatment. Excitation wavelengths of the fluorometer’s LEDs were 450 nm, 530
nm and 624 nm with an automated adjustment of the light intensity (between 0.66–1.2 x 10
22
).
The single turnover mode was set with a saturation phase consisting of 100 flashlets on a 2 μs
pitch followed by a relaxing phase of 40 flashlets on a 50 μs pitch. After 10 min of dark acclima-
tion, the minimum (F
0
) and maximum (F
m
) chlorophyll afluorescence of PSII was determined
6 times to calculate the maximum quantum yield of photochemistry in PSII (F
v
/F
m
, rel. unit)
using the equation (Eq.):
Fv=Fm¼ ðFmF0Þ=FmEq 1
From the single turnover measurements of dark-acclimated cells, also the functional
absorption cross section of PSII (σ
PSII,
nm
2
PSII
-1
), the time constant for electron transport at
the acceptor side of PSII (τ
Qa
,μs) and the connectivity factor (p, dimensionless) were derived
according to [36], using FastPro8 Software (Version 1.0.55, Kevin Oxborough, CTG Ltd.).
Electron transport rates (ETR)-irradiance curves with sequential increasing irradiances
were performed, with 9 irradiances ranging from 0 to 1,700 μmol photons m
-2
s
-1
with an accli-
mation phase of 5 min per light level. Each actual light intensity (E, μmol photons m
-2
s
-1
)
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 4 / 16
emitted from the FastAct Laboratory system (CTG Ltd.) was measured with a light sensor
(ULM-500 Universal Light Meter equipped with a Spherical Micro Quantum Sensor US-SQS,
Walz GmbH, Effeltrich, Germany) and used to calculate absolute electron transport rates
(ETR, e
-
PSII
-1
s
-1
) according to the formula by [37,38]:
ETR ¼sPSII xððFq0=Fm0Þ=ðFv=FmÞÞ x E Eq 2
where F
q
0/F
m
0denotes the effective PSII quantum yield under ambient light. According to [39],
maximum ETR (ETR
max
, e
-
PSII
-1
s
-1
), light utilization efficiency (α) and minimum saturating
irradiance (I
k
,μmol photons m
-2
s
-1
) were calculated from the fitted irradiance-dependent
ETR using the SigmaPlot 13.0 software (SysStat Software Inc.).
Growth
Cell count samples of C.debilis for each treatment were taken on a daily basis at the same time
of the day. All samples were fixed with 10% acid Lugol’s solution and stored at 2˚C in the dark
until enumeration. At least 400 C.debilis cells were counted in stripes under a magnification of
400x in combination with a 1.6x optovar using Utermo¨hl chambers (Hydrobios, Altenholz,
Germany) on an inverted microscope (Axio Observer D1, Carl Zeiss AG, Oberkochen, Ger-
many). Cell-specific growth rate (μ) was calculated per day (d
-1
) as
m¼ ðln Nt2ln Nt1Þ=DtEq 3
where N
t1
and N
t2
represent the cell densities (cells mL
-1
) at the sampling day t
1
and t
2
, respec-
tively, and Δtdenotes the time between the two measurements.
Elemental composition
For the analyses of the content of particulate organic carbon (POC) and particulate organic
nitrogen (PON), 250 mL of the C.debilis cultures was gently filtered (<20 mmHg) onto pre-
combusted glass-fibre filters (15h, 500˚C, GF/F, ~0.6 μm, 25 mm, Whatman, Wisconsin,
USA). Filters were stored in pre-combusted glass petri dishes at -20˚C until sample prepara-
tion. Prior the analysis, filters were dried at 50˚C for >12 h before they were acidified with
200 μL of 0.2 M HCl to remove inorganic carbon. After being dried at 50˚C overnight, filters
were coated in tin foil and compressed into small pellets and analysed on an automated carbon
nitrogen elemental analyser (Euro EA—CN Elemental Analyzer, HEKAtech GmbH, Wegberg,
Germany). Cellular contents of POC and PON were corrected for blank measurements and
normalized to cell density and filtered volume. To calculate cellular daily production rates of
POC and PON, cellular quotas were multiplied by the corresponding growth rate of the
respective treatment. Molar ratios of carbon to nitrogen (C:N) were also calculated.
Pigment analysis
For the analysis of the photosynthetic pigments, 250 mL of the C.debilis cultures were gently
filtered (<20 mmHg) onto 25-mm GF/F filters (~0.6 μm, 25 mm, Whatman, Wisconsin,
USA), which were then immediately frozen in liquid nitrogen (N
2
) and stored at -80˚C until
analysis. Pigments samples were homogenized and extracted in 90% acetone for 24 h at 4˚C in
the dark. After centrifugation (5 min, 4˚C, 13000 rpm) and filtration through a 0.45 μm pore
size nylon syringe filter (Nalgene
1
, Nalge Nunc International, Rochester, NY, USA), concen-
trations of chlorophyll aand c
2
, fucoxanthin, diatoxanthin and diadinoxanthin were deter-
mined by reversed phase high performance liquid chromatography. The analysis was
performed on a LaChromElite
1
system equipped with a chilled autosampler L-2200 and a
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 5 / 16
DAD detector L-2450 (VWR-Hitachi International GmbH, Darmstadt, Germany). A Spheri-
sorb
1
ODS-2 column (25 cm x 4.6 mm, 5 μm particle size; Waters, Milford, MA, USA) with a
LiChropher
1
100-RP-18 guard cartridge was used for the separation of pigments, applying a
gradient according to [40]. Peaks were detected at 440 nm, identified and quantified by co-
chromatography with standards for chlorophyll aand c
2
, fucoxanthin, diatoxanthin and diadi-
noxanthin (DHI Lab Products, Hørsholm, Denmark) using the software EZChrom Elite ver.
3.1.3. (Agilent Technologies, Santa Clara, CA, USA). Pigment contents were normalized to fil-
tered volume and cell densities to yield cellular quotas.
Statistics
Kolmogorov-Smirnov tests with Liliefors correction were applied to test for normal distribu-
tion of the data and Brown-Forsythe tests were applied to test for equal variances. To test for
significant differences between treatments one-way analyses of variance (ANOVA) with addi-
tional Bonferroni’s multiple comparison post tests were applied using the program GraphPad
Prism (Version 5.00 for Windows, Graph Pad Software, San Diego California, USA). The sig-
nificance testing was done at the p<0.05 level.
Results
Total dissolved Fe (dFe) and Mn (dMn) concentrations
The naturally FeMn-poor Antarctic seawater, used for the experiment, contained 0.30 ±0.01
nmol dFe L
-1
and 0.49 ±0.04 nmol dMn L
-1
(Table 1). Culture media was prepared using this
seawater, to which either no Fe and Mn (-FeMn), only Mn (-Fe) or Fe (-Mn) alone or both
trace metals together (control) were added. Hence, concentrations of dFe and dMn of the cul-
ture medium were altered depending on the treatment (Table 1). While dFe concentrations of
the culture medium of the -FeMn and the -Fe treatments were similar, they were reduced rela-
tive to ones of the -Mn and control treatments. The culture medium of the -FeMn and -Mn
treatments exhibited similar dMn concentrations. In comparison to the latter treatments, dMn
concentrations of the culture medium of the -Fe and control treatments were enhanced after
Mn enrichment.
Maximum quantum yield and functional absorption cross-sections of PSII
The maximum quantum yield of PSII (F
v
/F
m
) of C.debilis was significantly influenced by the
availability of both trace metals (ANOVA: F = 92, p<0.0001, Fig 1A). While Mn enrichment
(-Fe: 0.33 ±0.01) did not alter the F
v
/F
m
in comparison with cells grown in FeMn-poor water
(-FeMn: 0.31 ±0.01), Fe addition increased the yield, reaching a value of 0.42 ±0.01. Only
after enrichment with both trace metals the F
v
/F
m
of C.debilis was highest (control:
0.47 ±0.02).
The functional absorption cross section of PSII (σ
PSII
) was significantly affected by the avail-
ability of both trace metals (ANOVA: F = 18.33, p= 0.0006, Fig 1B). As for F
v
/F
m
, the addition
of Mn alone (-Fe: 3.29 ±0.27 nm
2
) did not change σ
PSII
relative to the -FeMn treatment
(3.37 ±0.15 nm
2
). Only when Fe alone (-Mn: 2.56 ±0.16 nm
2
) or Fe and Mn together (control:
2.53 ±0.10 nm
2
) were given, σ
PSII
decreased significantly by 24% and 25%, respectively, rela-
tive to the -FeMn treatment. Irrespective of whether Fe alone or both trace metals were added
σ
PSII
remained unchanged.
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 6 / 16
Growth, elemental composition and stoichiometry
Growth rates of C.debilis were significantly altered in response to the different Fe and Mn con-
centrations (ANOVA: F = 35.67, p<0.0001, Fig 2A). While growth rates were similar between
-FeMn (0.26 ±0.01 d
-1
), -Fe (0.31 ±0.03 d
-1
) and -Mn (0.26 ±0.01 d
-1
) treatments, the addition
of Fe and Mn together yielded the highest growth rates (0.40 ±0.02 d
-1
).
Cellular contents of particulate organic carbon (POC) were significantly influenced by dif-
ferent Fe and Mn concentrations (ANOVA: F = 6.508, p= 0.0154, Fig 2B). POC quotas were
similar for -FeMn (25.11 ±2.21 pg cell
-1
) and -Fe (24.91 ±2.21 pg cell
-1
) treatments, they were,
Fig 1. The dark-acclimated maximum PSII quantum yield F
v
/F
m
(A) and the dark-adapted functional absorption
cross section of PSII photochemistry σ
PSII
(B) of C.debilis grown in naturally FeMn-poor Antarctic seawater (-FeMn)
and to which additions of either Mn alone (-Fe), Fe alone (-Mn) or both (control) were given. Values represent the
means ±SD (n = 3). Different letters indicate significant differences between treatments (p<0.05).
https://doi.org/10.1371/journal.pone.0221959.g001
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 7 / 16
however, lower than compared to the -Mn treatment (31.40 ±1.93 pg cell
-1
). POC quotas of
the control treatment (27.38 ±1.81 pg cell
-1
) were similarly high as all other treatments. POC
production rates were significantly affected by Fe and Mn availability (ANOVA: F = 18.38,
Fig 2. Growth rates (A), cellular production rate of particulate organic carbon (POC,B) and molar ratios of carbon to nitrogen (C:N, C) of C.debilis
grown in naturally FeMn-poor Antarctic seawater (-FeMn) and to which additions of either Mn alone (-Fe), Fe alone (-Mn) or both (control) were
given. Values represent the means ±SD (n = 3). Different letters indicate significant differences between treatments (p<0.05).
https://doi.org/10.1371/journal.pone.0221959.g002
Table 2. Connectivity between adjacent photosystems (p), time constant for electron transfer at PSII (τ
Qa
), absolute maximum electron transport rates (ETR
max
),
light saturation point (I
k
) and light use efficiency (α) were measured for C.debilis grown in naturally FeMn-poor Antarctic seawater (-FeMn) and to which additions
of either Mn alone (-Fe), Fe alone (-Mn) or both (control) were given. Values represent the means ±SD (n = 3). Different letters indicate significant differences between
treatments (p<0.05).
Treatment P
(rel. unit)
τ
Qa
(μs)
ETR
max
(e
-
PSII
-1
s
-1
)
I
k
(μmol
photons m
-2
s
-1
)
α
(rel. unit)
-FeMn 0.32 ±0.01
a
426 ±29
a
869 ±150
a
495 ±90
a
1.81 ±0.08
a
-Fe 0.35 ±0.01
a
461 ±39
ab
945 ±72
a
532 ±35
a
1.78 ±0.16
a
-Mn 0.42 ±0.01
b
510 ±27
bc
531 ±64
b
408 ±26
a
1.30 ±0.12
b
Control 0.45 ±0.02
b
551 ±12
c
528 ±50
b
427 ±89
a
1.21 ±0.07
b
Please note that the marked values need to be treated with caution as ETRs values for the two highest irradiances (1152 and 1504 μmol photons m
2
s
1
) of the control
treatment could unfortunately not be determined due to a technical problem with the FRRf software FastPro8.
https://doi.org/10.1371/journal.pone.0221959.t002
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 8 / 16
p= 0.0006, Fig 2C). The same trend as for growth was observed for the POC production rates,
with the -FeMn (6.51 ±0.40 pg cell
-1
d
-1
), -Fe (7.72 ±1.05 pg cell
-1
d
-1
) and -Mn (8.14 ±0.35
pg cell
-1
d
-1
) treatments showing similar high rates. Again, only when Fe and Mn were added
together did POC production reached the highest values of 10.66 ±0.77 pg cell
-1
d
-1
. Irrespec-
tive of changes in Fe and Mn availabilities, carbon to nitrogen ratios (C:N) remained
unchanged among all treatments and ranged between 6.97 ±0.18 and 7.37 ±0.08 mol mol
-1
(Fig 2D).
Chlorophyll afluorescence
The connectivity of adjacent PSIIs (p) was significantly affected in response to the different Fe
and Mn concentrations (ANOVA: F = 62.29, p<0.0001, Table 2). Similar pvalues were
observed for -FeMn and -Fe treatments. These values were, however, lower compared to the
-Mn and control treatments. Between -Mn and control treatments, no difference in pwas
found. The time constant for electron transfer at PSII (τ
Qa
) was significantly altered in
response to the different Fe and Mn concentrations (ANOVA: F = 11.38, p= 0.0029, Table 2).
While τ
Qa
values did not change when C.debilis was grown under -FeMn or -Fe conditions,
these values were, however, significantly higher when Fe was added alone (-Mn) or in combi-
nation with Mn (control). The only exception was the -Fe treatment, for which τ
Qa
did not dif-
fer from the value determined for the -Mn treatment.
ETR-irradiance curves showed differences in both shape and amplitude in response to
changes in Fe and Mn availability (Fig 3). Maximum electron transport rates (ETR
max
) were
significantly affected by the different trace metal additions (ANOVA: F = 17.63, p= 0.0007,
Table 2). ETR
max
values were similar for the -FeMn and -Fe treatments, but were higher than
those measured of the -Mn and control treatments. Please note that ETR values for the two
highest irradiances (1152 and 1504 μmol photons m
2
s
1
) of the control treatment could
unfortunately not be determined due to a technical problem with the FRRf software FastPro8.
For unknown reasons the software did not follow the protocol during the measurements for
these two light intensities as no dark acclimation of 5 minutes was performed. Due to this,
these data were excluded from the analysis. Calculating ETR
max
using the light-adapted σ
PSII
showed the same trend. The light saturation point of PSII electron transport (I
k
) ranged
between 408 ±26 and 532 ±35 μmol photons m
-2
s
-1
among treatments and were neither
influenced by the addition of Fe or Mn alone nor the combination of both (Table 2). The light
use efficiency (α) was significantly influenced by the availability of Fe and Mn (ANOVA:
F = 23.09, p= 0.0003, Table 2), with αvalues of the -FeMn and -Fe treatments being higher
than those of the -Mn and control treatments. In comparison, αremained unaltered between
the -FeMn and -Fe as well as between the -Mn and control treatments.
Pigments
Cellular concentrations of light-harvesting pigments (LH = chlorophyll a+ chlorophyll c
2
+
fucoxanthin) were significantly altered by the availability of Fe and Mn (ANOVA: F = 24.21,
p= 0.0002, Table 3). LH quotas remained unchanged when C.debilis was grown under -FeMn
or -Fe conditions, these values were, however, increased when Fe was added alone (-Mn) or in
combination with Mn (control). Between -Mn and control treatments, there was no difference
in LH quotas. The availability of Fe and Mn influenced cellular concentrations of light-protec-
tive pigments (LP = diadinoxanthin + diatoxanthin) (ANOVA: F = 7.317, p= 0.0111, Table 3).
While LP quotas of the -Fe and control treatments differed, LH quotas were similar among the
other treatments. The availability of Fe and Mn had a strong influence on LH:LP ratios
(ANOVA: F = 7.273, p<0.0133, Table 3). Only after enrichment with both trace metals the
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 9 / 16
LH:LP ratio become the smallest relative to all other treatments. In comparison, LH:LP ratios
were similar between -FeMn, -Fe and -Mn treatments.
Discussion
Previous studies reported that growth and species composition of phytoplankton assemblages
of different SO regions was influenced solely by the addition of Mn [1,17,19]. As these studies
were undertaken with natural phytoplankton assemblages, it has not yet been investigated how
Mn and Fe availability influences SO phytoplankton physiology and which species are actually
Fig 3. Absolute electron transport rates (ETR) were measured in response to increasing irradiance in C.debilis grown in naturally FeMn-poor Antarctic seawater
(-FeMn, A) and to which additions of either Mn alone (-Fe, B), Fe alone (-Mn, C) or both (control, D) were given. Values represent the means ±SD (n = 3).
https://doi.org/10.1371/journal.pone.0221959.g003
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 10 / 16
susceptible to the limitation of these two trace metals. This study shows that growth of the Ant-
arctic bloom-forming diatom C.debilis was co-limited by Fe and Mn and that Mn deficiency
alone inhibited growth and carbon production to the same degree as Fe limitation. Therefore,
we define Fe-Mn co-limitation according to [20] as “Type I: Independent nutrient co-
limitation”.
FeMn- and Fe-deficient cells showed similar physiological responses
The determination of dFe and dMn concentrations in the culture medium of all treatments
revealed that the four experimental treatments were successfully achieved (Table 1). In line
with this, at the time of sampling the F
v
/F
m
of FeMn-deficient cells (0.31 ±0.01) was identical
with the one of Fe-deficient cells (0.34 ±0.01), but strongly reduced compared to FeMn-replete
cells (0.47 ±0.02; Fig 1A). Hence, the similar low F
v
/F
m
values of the FeMn- and Fe-deficient
cells suggest reduced photochemical efficiency, a common feature of Fe-limited phytoplankton
[7,4144]. To counterbalance the reduced amount of Fe-containing photosynthetic reaction
centers [41,43,45,46], both FeMn- and Fe-poor cells displayed larger σ
PSII
relative to the
FeMn-replete cells (Fig 1B). A Fe-dependent disconnection of antennae from PSII reaction
centres [42,45,47,48] was also apparent for FeMn- and Fe-deficient cells (Table 2). Hence, the
photophysiological response of the FeMn-deficient incubations was largely driven by low Fe
availability. Along with these photophysiological changes, FeMn- and Fe-deficient cells also
displayed significantly lower growth, and POC production rates relative to the FeMn-replete
cells (Fig 2A, 2B and 2D). Similar low growth and carbon fixation rates were previously esti-
mated under Fe limitation alone in the same species [49,47], with other Chaetoceros species
[42,50] and with various other Antarctic diatoms [45,51]. In a previous study by [47], a smaller
cell volume of the same C.debilis strain was observed under Fe-limitation alone. However,
POC quotas were reduced as a consequence of Fe-limitation after normalization to either cell
number or cell volume. Since the cell size was not measured for this experiment and therefore
no cell volume-normalized POC quotas could be derived, we assume based on [47] that not
only cell volume decreased under Fe-limitation, but also the cell volume-normalized POC con-
tent per cell of C.debilis.
Low Fe availability usually causes chlorosis [45,5154], as seen by the reduced light harvest-
ing pigment quotas of both FeMn- and Fe-deplete cells (Table 3). Moreover, Fe is required in
various components of the electron transport chain: as a consequence of Fe-limitation, fewer
Fe-containing proteins are synthesized, interrupting the transfer of electrons in the photosyn-
thetic electron transport chain [55]. Electron transfer was largely hindered under Fe-limiting
conditions [4,42,51]. Hence, POC production rates were significantly lower in both FeMn-
and Fe-deplete relative to the FeMn enriched cells (Fig 2C). This generally resulted in reduced
rates of photosynthesis [42,45,51,54,56], which affects cellular growth rates (Fig 2A).
Table 3. Cellular concentrations of light-harvesting (LH: sum of chlorophyll a, chlorophyll c
2
, and fucoxanthin) and light-protective pigments (LP: sum of diadi-
noxanthin and diatoxanthin) as well as the ratio of light-protective to light-harvesting pigments (LP:LH) were determined in C.debilis grown in naturally FeMn-
poor Antarctic seawater (-FeMn) and to which additions of either Mn alone (-Fe), Fe alone (-Mn) or both (control) were given. Values represent the means ±SD
(n = 3). Different letters indicate significant differences between treatments (p<0.05).
Treatment LH
(fg cell
-1
)
LP
(fg cell
-1
)
LP:LH
(%)
-FeMn 89 ±2
a
58 ±0
ab
0.72 ±0.07
a
-Fe 80 ±2
a
56 ±6
a
0.74 ±0.06
a
-Mn 118 ±5
b
70 ±4
ab
0.73 ±0.08
a
Control 135 ±17
b
71 ±7
b
0.56 ±0.01
b
https://doi.org/10.1371/journal.pone.0221959.t003
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 11 / 16
Unexpectedly, re-oxidation time of Q
a
was shorter (Table 2) while electron transfer of both
FeMn- and Fe-deficient cells was significantly enhanced relative to FeMn-replete cells (Fig 3,
Table 2). The higher ETRs combined with reduced carbon fixation suggest that electrons
entered alternative pathways like the Mehler reaction [57] and the cyclic electron transport
flow around PSI. Considering, however, that the latter pathways are rather Fe-expensive, less
Fe-expensive processes such as the putative plastid plastoquinol terminal oxidase (PTOX)
pathway were of importance [58]. Furthermore, minimum saturating irradiance (I
k
) as well as
light utilization efficiency (α) were both enhanced in the FeMn- and Fe-deplete relative to the
control treatment, indicating enhanced acclimation to short-term high light stress [4]. The
potential to counteract short-term high light stress potentially resulted from the elevated ratios
of LP:LH (Table 3), indicating improved capacity of FeMn- and Fe-deficient cells to cope with
high light exposure than the FeMn-enriched cells.
Mn deficiency inhibited growth of C.debilis
As the water-splitting complex of PSII contains four Mn ions, a lack of Mn can affect the pho-
tochemical activity of the overall pool of PSII as previously observed in temperate diatoms
[22]. Accordingly, the photochemical efficiency of Mn-deplete cells was reduced relative to
FeMn-sufficient cells, but still much higher than FeMn- and Fe-deficient cells. Apparently, the
addition of Fe alone to Mn-deplete cells led to restoration of photophysiological adjustments,
with better connected adjacent PSIIs, higher PSII photochemical efficiency as well as smaller
PSII functional absorption cross sections (Fig 1A and 1B,Table 2). As a consequence, Mn-defi-
cient cells exhibited efficient linear electron transport, which resulted in enhanced POC build-
up relative to the FeMn- and Fe-poor incubations (Figs 2B and 3,Table 2). These findings sug-
gest that amendment of Fe alone led to the reorganization of the thylakoid membrane enabling
Mn-deficient cells undisturbed linear electron flow, which provided ATP and NADPH for
subsequent carbon fixation. Even though photosynthetic activity was restored, the Mn-defi-
cient cells grew as slowly as the FeMn- and Fe-deplete incubations (Fig 2B). Reduced growth
of Mn-limited temperate diatoms was previously reported [22,25] and was explained by their
impacted capacity of the antioxidant enzyme superoxide dismutase (SOD). The latter detoxi-
fies reactive oxygen species (ROS) and prevents cell damage, but requires Mn. As a conse-
quence, Mn-deficient cells were probably compromised by reduced activity of the Mn-
containing SOD, increasing oxidative stress and the requirements for energy and resources to
repair damaged cellular constituents [22]. In support for this, to counteract light stress Mn-
deficient cells displayed a higher LP:LH ratio than cells grown under FeMn-replete conditions
(Table 3). The distinct response between Mn- and Fe-deficient cells further supports the find-
ing of [22] that Fe and Mn do not substitute one another.
Only the supply of both Fe and Mn yielded highest growth
Only when Fe and Mn were provided together, was maximum photosynthetic efficiency
reached, which allowed unimpeded entry of electrons into the photosynthetic electron trans-
port chain (Fig 1A). Similarly, the additions of Mn-containing volcanic ashes to natural com-
munities from the Drake passage resulted in a stronger increase of the F
v
/F
m
than to those in
which only Fe was added [17]. Here, only the addition of both metals allowed C.debilis to
achieve maximum POC production and to reach highest growth rates (Fig 2A and 2C), indi-
cating relief from FeMn co-limitation. Similarly, growth rates reached maximum values only
when cultures of the two diatoms T.pseudonana and T.oceanica were enriched with both
trace metals together [22]. Sufficient quantities of Mn-containing SOD and therefore less
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 12 / 16
oxidative stress [22] were indicated by the low LP:LH ratio of FeMn-replete C.debilis cells
compared to all other treatments (Table 3).
The results of this study hint towards the finding that low phytoplankton growth and pro-
ductivity in SO open ocean waters may not be explained solely by the limitation of Fe, but also
with Mn together. In fact, C.debilis was found to be co-limited by Fe and Mn at similarly low
concentrations as previously reported in the Drake Passage [17,18]. As FeMn- and Fe-deficient
C.debilis cells showed similar physiological responses, we hypothesize that the occurrence of
FeMn-limited phytoplankton species may be masked in the natural environment. Fe-Mn-
enrichment experiments using different SO phytoplankton communities will be helpful to
identify Mn and Fe co-limited species, but also such species that have very low requirements of
both metals and therefore are better adapted to thrive in FeMn-poor waters. Because of the
potential ecological and biogeochemical implications, a mechanistic understanding of photo-
synthesis and trace metal requirements of SO phytoplankton is needed. Variability in stoichi-
ometries of trace metal supply and biological demand are key determinants of trace metal
limitation and as such for phytoplankton productivity. Deciphering the mechanisms that
underpin this variability and the consequences for SO phytoplankton species is crucial for
accurately predicting the consequences of ongoing anthropogenic perturbations to SO
biogeochemistry.
Acknowledgments
Christian Vo¨lkner, Tina Brenneis, Dorothee Wilhelms-Dick and Britta Meyer-Schlosser are
thanked for the support in the laboratory.
Author Contributions
Conceptualization: Franziska Pausch, Scarlett Trimborn.
Data curation: Franziska Pausch, Scarlett Trimborn.
Formal analysis: Franziska Pausch.
Investigation: Franziska Pausch.
Project administration: Scarlett Trimborn.
Resources: Scarlett Trimborn.
Supervision: Scarlett Trimborn.
Visualization: Franziska Pausch.
Writing – original draft: Franziska Pausch, Kai Bischof, Scarlett Trimborn.
Writing – review & editing: Franziska Pausch, Kai Bischof, Scarlett Trimborn.
References
1. Martin JH, Gordon RM, Fitzwater SE. Iron in Antarctic waters. Nature. 1990; 345: 156–158.
2. Raven JA. Predictions of Mn and Fe use efficiencies of phototrophic growth as a function of light avail-
ability for growth and of C assimilation pathway. New Phytol. 1990; 116: 1–18.
3. Raven JA, Evans MCW, Korb RE. The role of trace metals in photosynthetic electron transport in O
2
-
evolving organisms. Photosyn. Res. 1999. 60; 111–149.
4. Behrenfeld MJ, Milligan AJ. Photophysiological expressions of iron stress in phytoplankton. Annu Rev
Mar Sci. 2013; 5: 217–246.
5. Moore CM, Mills MM, Arrigo KR, Berman-Frank I, Bopp L, Boyd PW, et al. Processes and patterns of
oceanic nutrient limitation. Nature Geoscience. 2013; 6: 701–710.
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 13 / 16
6. Twining BS, Baines SB. The trace metal composition of marine phytoplankton. Annu Rev Mar Sci.
2013; 5: 191–215.
7. Greene RM, Geider RJ, Kolber Z, Falkowski PG. Iron-induced changes in light harvesting and photo-
chemical energy conversion processes in eukaryotic marine algae. Plant Physiol 1992; 100: 565–575.
https://doi.org/10.1104/pp.100.2.565 PMID: 16653030
8. Moore JK, Braucher O. Sedimentary and mineral dust sources of dissolved iron to the world ocean. Bio-
geosciences. 2008; 5: 631–656.
9. Boyd PW, Ellwood MJ. The biogeochemical cycle of iron in the ocean. Nat Geosci. Nature Publishing
Group; 2010; 3: 675–682. https://doi.org/10.1038/ngeo964
10. de Baar HJW, de Jong JTM, Bakker DCE, Lo
¨scher BM, Veth C, Bathmann U V., et al. Importance of
iron for plankton blooms and carbon dioxide drawdown in the Southern Ocean. Nature. 1995; 373:
412–415. https://doi.org/10.1038/373412a0
11. De Jong J, Schoemann V, Lannuzel D, Croot P, De Baar H, Tison JL. Natural iron fertilization of the
Atlantic sector of the Southern Ocean by continental shelf sources of the Antarctic Peninsula. J Geo-
phys Res Biogeosciences. 2012; 117. https://doi.org/10.1029/2011JG001679
12. Middag R, de Baar HJW, Klunder MB, Laan P. Fluxes of dissolved aluminum and manganese to the
Weddell Sea and indications for manganese co-limitation. Limnol Oceanogr. 2013; 58: 287–300.
https://doi.org/10.4319/lo.2013.58.1.0287
13. Raiswell R, Tranter M, Benning LG, Siegert M, De’ath R, Huybrechts P, et al. Contributions from gla-
cially derived sediment to the global iron (oxyhydr)oxide cycle: Implications for iron delivery to the
oceans. Geochim Cosmochim Acta. 2006; 70: 2765–2780. https://doi.org/10.1016/j.gca.2005.12.027
14. Sedwick PN, Ditullio GR. Regulation of algal blooms in Antarctic shelf waters by the release of iron from
melting sea ice. Geophys Res Lett. 1997; 24: 2515–2518.
15. Duce RA, Tindale NW. Atmospheric transport of iron and its deposition in the ocean. Limnol Oceanogr.
1991: 36; 1715–1726.
16. Middag R, de Baar HJW, Klunder MB, Laan P. Fluxes of dissolved aluminum and manganese to the
Weddell Sea and indications for manganese co-limitation. Limnol Oceanogr. 2013; 8: 287–300.
17. Browning TJ, Bouman HA, Henderson GM, Mather TA, Pyle DM, Schlosser C, et al. Strong responses
of Southern Ocean phytoplankton communities to volcanic ash. Geophys Res Lett. 2014; 41: 2851–
2857.
18. Middag R, de Baar HJW, Laan P, Cai PH, Van Ooijen DJV. Dissolved manganese in the Atlantic sector
of the Southern Ocean. Deep-Sea Res. II. 2011; 58: 2661–2677.
19. Buma AGJ, de Baar HJW, Nolting RF, Van Bennekom AJ. Metal enrichment experiments in the Wed-
dell-Scotia Seas: Effects of iron and manganese on various plankton communities. Limnol Oceanogr.
1991; 36: 1865–1878.
20. Saito MA, Goepfert TJ, Ritt JT. Some thoughts on the concept of colimitation: Three definitions and the
importance of bioavailability. Limnol Oceanogr. 2008; 53: 276–290. https://doi.org/10.4319/lo.2008.53.
1.0276
21. Morel FMM, Price NM. The biogeochemical cycles of trace metals in the oceans. Science. 2003; 300:
944–947. https://doi.org/10.1126/science.1083545 PMID: 12738853
22. Peers G, Price NM. A role for manganese in superoxide dismutases and growth of iron-deficient dia-
toms. Limnol Oceanogr. 2004; 49: 1774–1783.
23. Wolfe-Simon F, Grzebyk D, Schofield O, Falkowski PG. The role and evolution of superoxide dismu-
tases in algae. J Phycol. 2005; 41: 453–465.
24. Allen MD, Kropat J, Tottey S, DelCampo JA, Merchant SS. Manganese deficiency in Chlamydomonas
results in loss of photosystem II and MnSOD function, sensitivity to peroxides, and secondary phospho-
rus and iron deficiency. Plant Physiol. 2006; 143: 263–277. https://doi.org/10.1104/pp.106.088609
PMID: 17085511
25. Sunda WG, Huntsman SA. Effect of competitive interactions between manganese and copper on cellu-
lar manganese and growth in estuarine and oceanic species of the diatom Thalassiosira. Limnol Ocea-
nogr. 1983; 28: 924–934.
26. Sunda WG, Huntsman SA. Antagonisms between cadmium and zinc toxicity and manganese limitation
in a coastal diatom. Limnol Oceanogr. 1996; 41: 373–387.
27. Thomson PG, McMinn A, Kiessling I, Watson M, Goldsworthy PM. Composition and succession of dino-
flagellates and chrysophytes in the upper fast ice of Davis Station, East Antarctica. PolarBiol. 2006;
29: 337–345. https://doi.org/10.1007/s00300-005-0060-y
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 14 / 16
28. Smetacek V, Assmy P, Henjes J. The role of grazing in structuring Southern Ocean pelagic ecosystems
and biogeochemical cycles. Antarct Sci. 2004; 16: 541–558. https://doi.org/10.1017/
S0954102004002317
29. Assmy P, Henjes J, Klaas C, Smetacek V. Mechanisms determining species dominance in a phyto-
plankton bloom induced by the iron fertilization experiment EisenEx in the Southern Ocean. Deep Res
Part I Oceanogr Res Pap. 2007; 54: 340–362. https://doi.org/10.1016/j.dsr.2006.12.005
30. Tsuda A, Kiyosawa H, Kuwata A, Mochizuki M, Shiga N, Saito H, et al. Responses of diatoms to iron-
enrichment (SEEDS) in the western subarctic Pacific, temporal and spatial comparisons. Prog Ocea-
nogr. 2005; 64: 189–205. https://doi.org/10.1016/j.pocean.2005.02.008
31. Guillard RRL, Ryther JH. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Deto-
nula confervacea (Cleve) Gran. Can J Microbiol. 1962; 8: 229–239. https://doi.org/10.1139/m62-029
PMID: 13902807
32. Gerringa LJA, de Baar HJW, Timmermans KR. A comparison of iron limitation of phytoplankton in natu-
ral oceanic waters and laboratory media conditioned with EDTA. Mar Chem 68, 335–346 (2000).
33. Hathorne EC, Haley B, Stichel T, Grasse P, Zieringer M, Frank M. Online preconcentration ICP-MS
analysis of rare earth elements in seawater. Geochem Geophys Geosyst. 2012; 13(1): Q01020.
34. Rapp I, Schlosser C, Rusiecka D, Gledhill M, Achterberg EP. Automated preconcentration of Fe, Zn,
Cu, Ni, Cd, Pb, Co, and Mn in seawater with analysis using high-resolution sector field inductively-cou-
pled plasma mass spectrometry. Anal Chim Acta. 2017; 976: 1–13. https://doi.org/10.1016/j.aca.2017.
05.008 PMID: 28576313
35. Cutter G, Casciotti K, Croot P, Geibert W, Heimbu¨rger L-E, Lohan M, et al. Sampling and Sample-han-
dling Protocols for GEOTRACES Cruises. Version 3, Toulouse, France, GEOTRACES International
Project Office; 2017. pp. 139 & Appendices.
36. Oxborough K, Moore CM, Suggett DJ, Lawson T, Chan HG, Geider RJ. Direct estimation of functional
PSII reaction center concentration and PSII electron flux on a volume basis: a new approach to the anal-
ysis of Fast Repetition Rate fluorometry (FRRf) data. Limnol Oceanogr Methods. 2012; 10: 142–154.
37. Suggett DJ, MacIntyre HL, Geider RJ. Evaluation of biophysical and optical determinations of light
absorption by photosystem II in phytoplankton. Limnol Oceanogr Methods. 2004; 2: 316–332.
38. Suggett DJ, Moore CM, Hickman AE, Geider RJ. Interpretation of fast repetition rate (FRR) fluores-
cence: Signatures of phytoplankton community structure versus physiological state. Mar Ecol Prog Ser.
2009; 376: 1–19.
39. Ralph PJ, Gademann R. Rapid light curves: A powerful tool to assess photosynthetic activity. Aquatic
Botany. 2005; 82: 222–237.
40. Wright SW, Jeffrey SW, Manoura RFC, Llewellyn CA, Bjornland T, Repeta D, et al. Improved HPLC
method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser.
1991; 77: 183–196.
41. Hopkinson BM, Mitchell BG, Reynolds RA, Wang H, Selph KE, Measures I, et al. Iron limitation across
chlorophyll gradients in the southern Drake Passage: Phytoplankton responses to iron addition and
photosynthetic indicators of iron stress. Limnol Oceanogr. 2007; 52: 2540–2554.
42. Petrou K, Trimborn S, Rost B, Ralph P, Hassler CS. The impact of iron limitation on the physiology of
the Antarctic diatom Chaetoceros simplex. Mar Biol. 2014; 161: 925–937. https://doi.org/10.1007/
s00227-014-2392-z PMID: 24719494
43. Trimborn S, Hoppe CJM, Taylor BB, Bracher A, Hassler CS. Physiological characteristics of open
ocean and coastal phytoplankton communities of Western Antarctic Peninsula and Drake Passage
waters. Deep Sea Res. Part I: Oceanogr Res Papers. 2015; 98: 115–124.
44. Trimborn S, Brenneis T, Hoppe CJM, Laglera LM, Norman L, Santos-Echeandı
`a J, et al. Iron sources
alter the response of Southern Ocean phytoplankton to ocean acidification. Mar Ecol Prog Ser. 2017;
578: 35–50.
45. Strzepek RF, Hunter KA, Frew RD, Harrison PJ, Boyd PW. Iron-light interactions differ in Southern
Ocean phytoplankton. Limnol Oceanogr. 2012; 57: 1182–1200.
46. Schuback N, Schallenberg C, Duckham C, Maldonado MT, Tortell PD. Interacting effects of light and
iron availability on the coupling of photosynthetic electron transport and CO
2
assimilation in marine phy-
toplankton. PloS One. 2015; 10(7): e0133235. https://doi.org/10.1371/journal.pone.0133235 PMID:
26171963
47. Trimborn S, Thoms S, Bischof K, Beszteri S. Susceptibility of two Southern Ocean phytoplankton key
species to iron limitation and high light. Front Mar Sci. 2019; 6: 167. https://doi.org/10.3389/FMARS.
2019.00167
48. Behrenfeld MJ, Kolber ZS. Widespread Iron Limitation of Phytoplankton in the South Pacific Ocean. Sci-
ence. 1999; 283: 840–843. https://doi.org/10.1126/science.283.5403.840 PMID: 9933166
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 15 / 16
49. Hoffmann LJ, Peeken I, Lochte K. Iron, silicate, and light co-limitation of three Southern Ocean diatom
species. Polar Biol. 2008; 31: 1067–1080.
50. Timmermans K, Davey M, van der Wagt B, Snoek J, Geider R, Veldhuis M, et al. Co-limitation by iron
and light of Chaetoceros brevis,C.dichaeta and C.calcitrans (Bacillariophyceae). Mar Ecol Prog Ser.
2001; 217: 287–297.
51. Alderkamp A-C, Kulk G, Buma AGJ, Visser RJW, Van Dijken GL, Mills MM, et al. The effect of iron limi-
tation on the photophysiology of Phaeocystis antarctica (Prymnesiophyceae) and Fragilariopsis cylin-
drus (Bacillariophyceae) under dynamic irradiance. J Phycol. 2012; 48: 45–59. https://doi.org/10.1111/
j.1529-8817.2011.01098.x PMID: 27009649
52. Reinbothe C, Bartsch S, Eggink LL, Hoober JK, Brusslan J, Andrade-Paz R, et al. A role for chlorophyl-
lide a oxygenase in the regulated import and stabilization of light-harvesting chlorophyll a/b proteins.
Proc Natl Acad Sci. 2006; 103: 4777–4782. https://doi.org/10.1073/pnas.0511066103 PMID:
16537436
53. Van Leeuwe MA, Stefels J. Effects of iron and light stress on the biochemical composition of Antarctic
Phaeocystis sp. (Prymnesiophyceae). II. Pigment composition. J Phycol. 1998; 34: 496–503.
54. Koch F, Beszteri S, Harms L, Trimborn S. The impacts of iron limitation and ocean acidification on the
cellular stoichiometry, photophysiology and transcriptome of Phaeocystis antarctica. Limnol Oceanogr.
2019; 64: 357–375.
55. Geider RJ, La Roche J. The role of iron in phytoplankton photosynthesis, and the potential for iron-limi-
tation of primary productivity in the sea. Photosyn Res. 1994; 39: 275–301. https://doi.org/10.1007/
BF00014588 PMID: 24311126
56. Greene RM, Geider RJ, Falkowski PG. Effect of iron limitation on photosynthesis in a marine diatom.
Limnol Oceanogr. 1991; 36: 1772–1782.
57. Mehler AH. Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen
and other Hill reagents. Arch Biochem Biophys. 1957; 33: 65–77.
58. Mackey KRM, Paytan A, Grossman AR, Bailey S. A photosynthetic strategy for coping in a high-light,
low-nutrient environment. Limnol Oceanogr. 2008; 53: 900–913.
Fe and Mn co-limit diatom growth
PLOS ONE | https://doi.org/10.1371/journal.pone.0221959 September 16, 2019 16 / 16
... Hence, clear evidence of how Mn availability influences SO phytoplankton growth at the species level and thereby shapes SO phytoplankton composition is still lacking in the field. A laboratory study using the bloom-forming SO diatom Chaetoceros debilis showed that only the supply of Fe and Mn together led to optimal growth, photochemical efficiency and carbon production 28 . ...
... Fe-limited phytoplankton is also known to suffer from higher oxidative stress requiring also more Mn to produce the antioxidant enzyme superoxide dismutase and thereby preventing cell damage 48 . Some studies suggested that low F v /F m values may also be associated with low Mn concentrations [21][22][23]28 . In our study, only the addition of Fe and Mn together led to the highest F v /F m values of both final communities (Table 3) indicating that besides Fe, Mn availability also influenced the F v /F m signal. ...
... Hence, the addition of Mn next to Fe enabled the cells to prevent oxidative stress and to reach maximum photosynthetic efficiency. Based on this and previous studies [21][22][23]28 , one needs to be careful when interpreting low F v / F m values detected in the field as they could be the result of multiple TM limitations, in our specific case Fe and Mn. ...
Article
Full-text available
While it has been recently demonstrated that both iron (Fe) and manganese (Mn) control Southern Ocean (SO) plankton biomass, how in particular Mn governs phytoplankton species composition remains yet unclear. This study, for the first time, highlights the importance of Mn next to Fe for growth of two key SO phytoplankton groups at two locations in the Drake Passage (West and East). Even though the bulk parameter chlorophyll a indicated Fe availability as main driver of both phytoplankton assemblages, the flow cytometric and microscopic analysis revealed FeMn co-limitation of a key phytoplankton group at each location: at West the dominant diatom Fragilariopsis and one subgroup of picoeukaryotes, which numerically dominated the East community. Hence, the limitation by both Fe and Mn and their divergent requirements among phytoplankton species and groups can be a key factor for shaping SO phytoplankton community structure.
... The influence of Mn on diatom physiology has only been examined in four diatom species to date (Sunda andHuntsman 1983, 1996;Peers and Price 2004;Pausch et al. 2019). These studies reveal a decrease in F v /F m when cells were Mn limited and further reductions when they were Fe-Mn co-limited (Pausch et al. 2019). ...
... The influence of Mn on diatom physiology has only been examined in four diatom species to date (Sunda andHuntsman 1983, 1996;Peers and Price 2004;Pausch et al. 2019). These studies reveal a decrease in F v /F m when cells were Mn limited and further reductions when they were Fe-Mn co-limited (Pausch et al. 2019). Diatom intracellular quotas for Mn range from 2 to 200 mmol:mol P and are second only to Fe in rank order of trace metal minimum cellular quotas Ho et al. 2003). ...
... Adjust pH to 6.6.Dissolve Fe-citrate and citric acid in warm H 2 O separately and add 1 mL HCl L −1 after mixing both 56,57 . Measuring antenna size in addition to energy flow between PSII complexes will allow more precise measurements of algal response 10 . ...
Preprint
Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross section (σPSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm′), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, we describe how to measure PSII photophysiology of an epizoic alga Colacium sp.Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton) using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. Müller 1776, Cladocera, Daphniidae) on background fluorescence and σPSII, Fv/Fm, Fq′/Fm′, and NPQNSV of planktonic Colacium sp. To validate our methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol can determine effects of Ca and Mn on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.
... Polynyas are often highly biologically active since they are the first open water areas to be exposed to the increasing solar irradiance during springtime and are often located near trace metal sources (Arrigo and Van Dijken, 2003;Arrigo and van Dijken, 2015). There should be sufficient nutrients and light available to sustain these larger phytoplankton blooms in the ASP (Alderkamp et al., 2013;Kwon et al., 2021;Oliver et al., 2019;Park et al., 2017) (Wu et al., 2019) (Pausch et al., 2019). The trace metal iron (Fe) and possibly manganese (Mn) are known to limit primary productivity in the Southern Ocean (Brand et al., 1983;Browning et al., 2021;Peers and Price, 2004). ...
Article
Coastal areas around Antarctica such as the Amundsen Sea are important sources of trace metals and biological hotspots, but are also experiencing the effects of climate change, including the rapid thinning of ice sheets. In the central Amundsen Sea Polynya (ASP), both bio-essential dissolved Fe (DFe) and dissolved Mn (DMn) were found to be depleted at the surface, indicating substantial biological uptake and/or precipitation. Close to the Dotson Ice Shelf (DIS) there were elevated surface concentrations of DMn (>3 nM) but surprisingly not for DFe (<0.3 nM). While Fe-binding ligand data suggests that ligands were abundant near the DIS, these were most likely not strong enough to outcompete scavenging and thus increase DFe substantially in the outflow. In contrast to the dissolved phase, particulate Fe (PFe) and Mn (PMn) concentrations (both labile and refractory fractions) were elevated over the entire water column close to the DIS and partly in the central ASP. We hypothesize that DFe was released from the DIS and immediately established an equilibrium with the labile particulate Fe (L-PFe)pool, via (reversible) scavenging, as indicated by a positive correlation between L-PFe and DFe in the outflow. This scavenging results in relatively low DFe concentrations, but the pool of labile PFe likely buffers the DFe pool when DFe is decreasing, e.g. due to uptake by phytoplankton. The DFe distribution also shows that inflowing modified circumpolar deep water (mCDW) and benthic sediments are clear and important sources for both DFe and DMn in the ASP. Refractory Fe and Mn likely have a lithogenic source, whereas the labile fractions are mostly biogenic in surface waters, and authigenic in deep waters (>100 m depth). We compared different uptake ratios, underlining that uptake ratio estimates do not necessarily capture natural variability and it is likely better to use a range of values. In the future, climate change may increase the heat flux of mCDW and thereby the melting of the DIS. This will most likely cause an increased input of Fe and Mn into the ASP, which may fuel increased levels of primary productivity in the ASP.
... This study used Aquil medium due to its wide application in trace metal experiments (Price et al., 1989). The medium is composed of artificial seawater in which Milli-Q 18.2 M cm −1 grade water is mixed with ultra-pure salts to reproduce the major ion composition of seawater (Pausch et al., 2019). The medium was filtered through a 0.2 µm pore size filter and sterilized in a microwave for a total of 11 min in acid-cleaned polycarbonate bottles (2 L) (Price et al., 1989). ...
Article
Full-text available
Ocean alkalinity enhancement (OAE) is a proposed method for removing carbon dioxide (CO2) from the atmosphere by the accelerated weathering of (ultra-)basic minerals to increase alkalinity – the chemical capacity of seawater to store CO2. During the weathering of OAE-relevant minerals relatively large amounts of trace metals will be released and may perturb pelagic ecosystems. Nickel (Ni) is of particular concern as it is abundant in olivine, one of the most widely considered minerals for OAE. However, so far there is limited knowledge about the impact of Ni on marine biota including phytoplankton. To fill this knowledge gap, this study tested the growth and photo-physiological response of 11 marine phytoplankton species to a wide range of dissolved Ni concentrations (from 0.07 to 50 000 nmol L−1). We found that the phytoplankton species were not very sensitive to Ni concentrations under the culturing conditions established in our experiments, but the responses were species-specific. The growth rates of 6 of the 11 tested species showed generally limited but still significant responses to changing Ni concentrations (36 % maximum change). Photosynthetic performance, assessed by measuring the maximum quantum yield (Fv/Fm) and the functional absorption cross-section (σPSII) of photosystem II (PSII), was sensitive to changing Ni in 3 out of 11 species (35 % maximum change) and 4 out of 11 species (16 % maximum change), respectively. The limited effect of Ni may be partly due to the provision of nitrate as the nitrogen source for growth as previous studies suggest higher sensitivities when urea is the nitrogen source. Furthermore, the limited influence may be due to the relatively high concentrations of synthetic organic ligands added to the growth media in our experiments. These ligands are commonly added to control trace metal bioavailability and therefore for example “free Ni2+” concentrations by binding the majority of the dissolved Ni. Our data suggest that dissolved Ni does not have a strong effect on phytoplankton under our experimental conditions, but we emphasize that a deeper understanding of nitrogen sources, ligand concentrations, and phytoplankton composition is needed when assessing the influence of Ni release associated with OAE.
... Manganese (Mn) is an essential micro-nutrient for all known photosynthetic organisms (Morel and Price, 2003) because it is integral to water oxidation in photosystem II and can serve as a co-factor in key enzymes, including Mn superoxide dismutase (Peers and Price, 2004;Hansel, 2017). Due to its importance for phytoplankton metabolism, Mn can limit or co-limit marine phytoplankton growth (Pausch et al., 2019). For example, Mn concentrations are depleted in parts of the offshore Southern Ocean (Latour et al., 2021) and recent work suggests Mn-or Fe/Mn co-limitation is prevalent in Drake Passage . ...
Article
Full-text available
Manganese (Mn) is an essential micro-nutrient that can limit or, along with iron (Fe), co-limit phytoplankton growth in the ocean. Glacier meltwater is thought to be a key source of trace metals to high latitude coastal systems, but little is known about the nature of Mn delivered to glacially-influenced fjords and adjacent coastal waters. In this work, we combine in-situ dissolved Mn (dMn) measurements of surface waters with Mn K-edge X-ray absorption spectroscopy (XAS) data of suspended particles in four fjords of West Greenland. Data were collected from transects of up to 100 km in fjords with different underlying bedrock geology from 64 to 70°N. We found that dMn concentrations generally decreased conservatively with increasing salinity (from 80-120 nM at salinity <8 to <40 nM at salinities >25). Dissolved Fe (dFe) trends in these fjords similarly declined with increasing distance from glacier outflows (declining from >20 nM to <8 nM). However, the dMn/dFe ratio increased rapidly likely due to the greater stability of dMn at intermediate salinities (i.e. 10 – 20) compared to rapid precipitation of dFe across the salinity gradient. The XAS data indicated a widespread presence of Mn(II)-rich suspended particles near fjord surfaces, with structures akin to Mn(II)-bearing phyllosilicates. However, a distinct increase in Mn oxidation state with depth and the predominance of birnessite-like Mn(IV) oxides was observed for suspended particles in a fjord with tertiary basalt geology. The similar dMn behaviour in fjords with different suspended particle Mn speciation (i.e., Mn(II)-bearing phyllosilicates and Mn(IV)-rich birnessite) is consistent with the decoupling of dissolved and particulate Mn and suggests that dMn concentrations on the scale of these fjords are controlled primarily by dilution of a freshwater dMn source rather than exchange between dissolved and particle phases. This work provides new insights into the Mn cycle in high latitude coastal waters, where small changes in the relative availabilities of dMn, dFe and macronutrients may affect the identity of the nutrient(s) proximally limiting primary production.
... which is considered typical of naturally iron-fertilised regions (Korb et al., , 2012Lasbleiz et al., 2016) and has been experimentally shown to be limited by iron (e.g. Pausch et al., 2019;Petrou et al., 2014). However, within Group D stations, both total abundance and diatom abundance was significantly lower, and, relative to diatoms, there were greater proportions of both flagellates and Phaeocystis antarctica, which can be suggestive of lower productivity and lower export waters. ...
Article
Full-text available
The South Sandwich Islands (SSI) are a biologically productive archipelago situated in the eastern Scotia Sea to the south of the eastward flowing Antarctic Circumpolar Current (ACC). The islands support important populations of higher predators, including several penguin species, seals and humpback whales. Despite this, the plankton ecology of the region has been little studied and information on mesoscale structure and environmental forcing of plankton ecology is particularly limited. We conducted a comprehensive oceanographic and net sampling campaign during the CCAMLR Area 48 Survey (January and February 2019), incorporating phytoplankton, mesozooplankton and macrozooplankton/nekton. Satellite chlorophyll-a (chl-a) data showed the development of a large bloom that was initiated two months prior to our study period at the south-eastern edge of the archipelago and propagated northwards along the eastern side, limited to the east by mesoscale features associated with the southern boundary of the ACC (SB). Multivariate cluster analysis revealed distinct mesoscale structure within the plankton community, with four spatially defined groups of phytoplankton and macrozooplankton/nekton, and three cluster groups of mesozooplankton. North of the SB, we found some spatial congruence between the three plankton assemblages, with a distinct, spatially coherent, cluster in each, corresponding to a warmer water community. Here, biomass was dominated by mesozooplankton, particularly calanoid copepods Rhincalanus gigas, Calanus propinquus, C. simillimus and Euchaetidae. The corresponding phytoplankton community was dominated by small diatoms, particularly Thalassionema spp., Pseudo-nitzschia spp., Fragilariopsis spp. and Chaetoceros spp., whilst Themisto gaudichaudii, Euphausia triacantha and myctophids were the major contributors to the macrozooplankton/nekton community. South of the SB, there was some spatial congruence between phytoplankton and macrozooplankton/nekton community structure on the western side of the archipelago, as well as on the eastern side that corresponded to the location of the bloom, but less association with mesozooplankton structure. Macrozooplankton/nekton structure was strongly driven by environmental conditions 1–2 months prior to the survey, including sea-ice distribution, surface phytoplankton concentration and productivity, whilst mesozooplankton was more tightly coupled to in-situ prevailing conditions such as surface temperature and integrated chl-a. Top-down pressure between trophic levels may have also had an influence on spatial patterns although direct evidence is lacking. Antarctic krill (Euphausia superba) was found with relatively low biomass at our net sampling sites (median biomass of 0.04 mg m⁻³ or <0.01 g m⁻²) while myctophids and the euphausiid Thysanoessa spp. predominated. We suggest that the highly productive and species rich pelagic community of the SSI supports multiple trophic pathways, and that off-shelf these may operate independently of Antarctic krill.
Article
The California Current System is a diatom‐dominated region characterized by seasonal coastal upwelling and additional elevated mesoscale activity. Cyclonic mesoscale eddies in the region trap productive coastal waters with their planktonic communities and transport them offshore with limited interaction with surrounding waters, effectively acting as natural mesocosms, where phytoplankton populations undergo ecological succession as eddies age. This study examines diatom community composition within two mesoscale cyclonic eddies that formed in the same region of the California Current System 2 months apart and in the California Current waters surrounding them. The diatom communities were analyzed in the context of shifting environmental gradients and through a lens of community succession to expand our understanding of biophysical interactions in California Current System cyclonic eddies. Diatom communities within each eddy were different from non‐eddy communities and varied in concert with salinity and dissolved iron (Fe) concentrations. The younger, nearshore eddy displayed higher macronutrient and dissolved Fe concentrations, had higher values for diatom Shannon diversity and evenness, and had nutrient ratios indicative of either eventual silicic acid (Si) or Fe limitation or possibly co‐limitation. The older, offshore eddy displayed low macronutrient and dissolved Fe concentrations, was likely nitrate‐limited, and had lower diatom Shannon diversity and evenness indices. Sequences from the genus Rhizosolenia, some of which form vertically migrating mats to bypass nitrate limitation, dominated in the older eddy. This is of potential significance as the prevalence of Rhizosolenia mats could impact estimates of carbon cycling and export in the wider California coastal area.
Article
Contrasting models predict two different climate change scenarios for the Southern Ocean (SO), forecasting either less or stronger vertical mixing of the water column. To investigate the responses of SO phytoplankton to these future conditions, we sampled a natural diatom dominated (63%) community from today’s relatively moderately mixed Drake Passage waters with both low availabilities of iron (Fe) and light. The phytoplankton community was then incubated at these ambient open ocean conditions (low Fe and low light, moderate mixing treatment), representing a control treatment. In addition, the phytoplankton was grown under two future mixing scenarios based on current climate model predictions. Mixing was simulated by changes in light and Fe availabilities. The two future scenarios consisted of a low mixing scenario (low Fe and higher light) and a strong mixing scenario (high Fe and low light). In addition, communities of each mixing scenario were exposed to ambient and low pH, the latter simulating ocean acidification (OA). The effects of the scenarios on particulate organic carbon (POC) production, trace metal to carbon ratios, photophysiology and the relative numerical contribution of diatoms and nanoflagellates were assessed. During the first growth phase, at ambient pH both future mixing scenarios promoted the numerical abundance of diatoms (∼75%) relative to nanoflagellates. This positive effect, however, vanished in response to OA in the communities of both future mixing scenarios (∼65%), with different effects for their productivity. At the end of the experiment, diatoms remained numerically the most abundant phytoplankton group across all treatments (∼80%). In addition, POC production was increased in the two future mixing scenarios under OA. Overall, this study suggests a continued numerical dominance of diatoms as well as higher carbon fixation in response to both future mixing scenarios under OA, irrespective of different changes in light and Fe availability.
Article
Full-text available
The production and removal of ammonium (NH4+) are essential upper-ocean nitrogen cycle pathways, yet in the Southern Ocean where NH4+ has been observed to accumulate in surface waters, its mixed-layer cycling remains poorly understood. For surface seawater samples collected between Cape Town and the Marginal Ice Zone in winter 2017, we found that NH4+ concentrations were 5-fold higher than is typical for summer and lower north than south of the Subantarctic Front (0.01–0.26 µM versus 0.19–0.70 µM). Our observations confirm that NH4+ accumulates in the Southern Ocean's winter mixed layer, particularly in polar waters. NH4+ assimilation rates were highest near the Polar Front (12.9 ± 0.4 nM d−1) and in the Subantarctic Zone (10.0 ± 1.5 nM d−1), decreasing towards the Marginal Ice Zone (3.0 ± 0.8 nM d−1) despite the high ambient NH4+ concentrations in these southernmost waters, likely due to the low temperatures and limited light availability. By contrast, rates of NH4+ oxidation were higher south than north of the Polar Front (16.0 ± 0.8 versus 11.1 ± 0.5 nM d−1), perhaps due to the lower-light and higher-iron conditions characteristic of polar waters. NH4+ concentrations were also measured along five transects of the Southern Ocean (Subtropical Zone to Marginal Ice Zone) spanning the 2018/19 annual cycle. These measurements reveal that mixed-layer NH4+ accumulation south of the Subantarctic Front derives from sustained heterotrophic NH4+ production in late summer through winter that, in net, outpaces NH4+ removal by temperature-, light-, and iron-limited microorganisms. Our observations thus imply that the Southern Ocean becomes a biological source of CO2 to the atmosphere in autumn and winter not only because nitrate drawdown is weak but also because the ambient conditions favour net heterotrophy and NH4+ accumulation.
Article
Full-text available
For photoheterotrophic growth, a Chlamydomonas reinhardtii cell requires at least 1.7 × 10⁷ manganese ions in the medium. At lower manganese ion concentrations (typically <0.5 μ m), cells divide more slowly, accumulate less chlorophyll, and the culture reaches stationary phase at lower cell density. Below 0.1 μ m supplemental manganese ion in the medium, the cells are photosynthetically defective. This is accompanied by decreased abundance of D1, which binds the Mn4Ca cluster, and release of the OEE proteins from the membrane. Assay of Mn superoxide dismutase (MnSOD) indicates loss of activity of two isozymes in proportion to the Mn deficiency. The expression of MSD3 through MSD5, encoding various isoforms of the MnSODs, is up-regulated severalfold in Mn-deficient cells, but neither expression nor activity of the plastid Fe-containing superoxide dismutase is changed, which contrasts with the dramatically increased MSD3 expression and plastid MnSOD activity in Fe-deficient cells. Mn-deficient cells are selectively sensitive to peroxide but not methyl viologen or Rose Bengal, and GPXs, APX, and MSRA2 genes (encoding glutathione peroxidase, ascorbate peroxidase, and methionine sulfoxide reductase 2) are slightly up-regulated. Elemental analysis indicates that the Mn, Fe, and P contents of cells in the Mn-deficient cultures were reduced in proportion to the deficiency. A natural resistance-associated macrophage protein homolog and one of five metal tolerance proteins were induced in Mn-deficient cells but not in Fe-deficient cells, suggesting that the corresponding gene products may be components of a Mn²⁺-selective assimilation pathway.
Article
Full-text available
Although iron (Fe) availability primarily sets the rate of phytoplankton growth and primary and export production in the Southern Ocean, other environmental factors, most significantly light, also affect productivity. As light availability strongly influences phytoplankton species distribution in low Fe-waters, we investigated the combined effects of increasing light (20, 200, and 500 μmol photons m-2 s-1) in conjunction with different Fe (0.4 and 2 nM) availability on the physiology of two ecologically relevant phytoplankton species in the Southern Ocean, Chaetoceros debilis (Bacillariophyceae) and Phaeocystis antarctica (Haptophyceae). Fe-deficient cells of P. antarctica displayed similar high growth rates at all irradiances. In comparison, Fe-deplete C. debilis cells grew much slower under low and medium irradiance and were unable to grow at the highest irradiance. Interestingly, Fe-deficient C. debilis cells were better protected against short-term excessive irradiances than P. antarctica. This tolerance was apparently counteracted by strongly lowered growth and particulate organic carbon production rates of the diatom relative to the prymnesiophyte. Overall, our results show that P. antarctica was the more tolerant species to changes in the availability of Fe and light, providing it a competitive advantage under a high light regime in Fe-deficient waters as projected for the future.
Article
Full-text available
Phaeocystis antarctica is an integral player of the phytoplankton community of the Southern Ocean (SO), the world’s largest high nutrient low chlorophyll (HNLC) region, and faces chronic iron (Fe) limitation. As the SO is responsible for 40% of anthropogenic CO2 uptake, P. antarctica must also deal with ocean acidification (OA). However, mechanistic studies investigating the effects of Fe limitation and OA on trace metal (TM) stoichiometry, transcriptomic and photophysiological responses of this species, as well as on the Fe chemistry, are lacking. This study reveals that P. antarctica responded strongly to Fe limitation by reducing its growth rate and particular organic carbon (POC) production. Cellular concentrations of all TMs, not just Fe, were greatly reduced, suggesting that Fe limitation may drive cells into secondary limitation by another TM. P. antarctica was able to adjust its photophysiology in response to Fe limitation, resulting in similar absolute electron transport rates across PSII. Even though OA stimulated growth in Fe-limited and -replete treatments, the slight reduction in cellular POC resulted in no net effect on POC production. In addition, relatively few genes were differentially expressed due to OA. Finally, this study demonstrates that, under our culture conditions, OA did not affect inorganic Fe or humic-acid like substances in seawater, but triggered the production of humic-acid like substances by P. antarctica. This species is well adapted to OA under all Fe conditions, giving it a competitive advantage over more sensitive species in a future ocean.
Article
Full-text available
The projected rise in anthropogenic CO2 and associated ocean acidification (OA) will change trace metal solubility and speciation, potentially altering Southern Ocean (SO) phytoplankton productivity and species composition. As iron (Fe) sources are important determinants of Fe bioavailability, we assessed the effect of Fe-laden dust versus inorganic Fe (FeCl3) enrichment under ambient and high pCO² levels (390 and 900 μatm) in a naturally Fe-limited SO phytoplankton community. Despite similar Fe chemical speciation and net particulate organic carbon (POC) production rates, CO²-dependent species shifts were controlled by Fe sources. Final phytoplankton communities of both control and dust treatments were dominated by the same species, with an OA-dependent shift from the diatom Pseudo-nitzschia prolongatoides towards the prymnesiophyte Phaeocystis antarctica. Addition of FeCl3 resulted in high abundances of Nitzschia lecointei and Chaetoceros neogracilis under ambient and high pCO2, respectively. These findings reveal that both the characterization of the phytoplankton community at the species level and the use of natural Fe sources are essential for a realistic projection of the biological carbon pump in the Felimited pelagic SO under OA. As dust deposition represents a more realistic scenario for the Felimited pelagic SO under OA, unaffected net POC production and dominance of P. antarctica can potentially weaken the export of carbon and silica in the future.
Article
Full-text available
Iron availability directly affects photosynthesis and limits phytoplankton growth over vast oceanic regions. For this reason, the availability of iron is a crucial variable to consider in the development of active chlorophyll a fluorescence based estimates of phytoplankton primary productivity. These bio-optical approaches require a conversion factor to derive ecologically-relevant rates of CO2-assimilation from estimates of electron transport in photosystem II. The required conversion factor varies significantly across phytoplankton taxa and environmental conditions, but little information is available on its response to iron limitation. In this study, we examine the role of iron limitation, and the interacting effects of iron and light availability, on the coupling of photosynthetic electron transport and CO2-assimilation in marine phytoplankton. Our results show that excess irradiance causes increased decoupling of carbon fixation and electron transport, particularly under iron limiting conditions. We observed that reaction center II specific rates of electron transport (ETRRCII, mol e- mol RCII-1 s-1) increased under iron limitation, and we propose a simple conceptual model for this observation. We also observed a strong correlation between the derived conversion factor and the expression of non-photochemical quenching. Utilizing a dataset from in situ phytoplankton assemblages across a coastal - oceanic transect in the Northeast subarctic Pacific, this relationship was used to predict ETRRCII: CO2-assimilation conversion factors and carbon-based primary productivity from FRRF data, without the need for any additional measurements.
Article
A rapid, automated, high-throughput analytical method capable of simultaneous analysis of multiple elements at trace and ultratrace levels is required to investigate the biogeochemical cycle of trace metals in the ocean. Here we present an analytical approach which uses a commercially available automated preconcentration device (SeaFAST) with accurate volume loading and in-line pH buffering of the sample prior to loading onto a chelating resin (WAKO) and subsequent simultaneous analysis of iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), cadmium (Cd), lead (Pb), cobalt (Co) and manganese (Mn) by high-resolution inductively-coupled plasma mass spectrometry (HR-ICP-MS). Quantification of sample concentration was undertaken using isotope dilution for Fe, Zn, Cu, Ni, Cd and Pb, and standard addition for Co and Mn. The chelating resin is shown to have a high affinity for all analyzed elements, with recoveries between 83 and 100% for all elements, except Mn (60%) and Ni (48%), and showed higher recoveries for Ni, Cd, Pb, Co and Mn in direct comparison to an alternative resin (NOBIAS Chelate-PA1). The reduced recoveries for Ni and Mn using the WAKO resin did not affect the quantification accuracy. A relatively constant retention efficiency on the resin over a broad pH range (pH 5–8) was observed for the trace metals, except for Mn. Mn quantification using standard addition required accurate sample pH adjustment with optimal recoveries at pH 7.5 ± 0.3. UV digestion was necessary to increase recovery of Co and Cu in seawater by 15.6% and 11.4%, respectively, and achieved full break-down of spiked Co-containing vitamin B12 complexes. Low blank levels and detection limits could be achieved (e.g., 0.029 nmol L⁻¹ for Fe and 0.028 nmol L⁻¹ for Zn) with the use of high purity reagents. Precision and accuracy were assessed using SAFe S, D1, and D2 reference seawaters, and results were in good agreement with available consensus values. The presented method is ideal for high throughput simultaneous analysis of trace elements in coastal and oceanic seawaters. We present a successful application of the analytical method to samples collected in June 2014 in the Northeast Atlantic Ocean.
Article
During summer 1995-96, we measured iron in the water column and conducted iron-enrichment bottle-incubation experiments at a station in the central Ross Sea (76°30′S, 170°40′W), first, in the presence of melting sea ice, and 17 days later, in ice-free conditions. We observed a striking temporal change in mixed-layer dissolved iron concentrations at this station, from 0.72-2.3 nM with sea ice present, to 0.16-0.17 nM in ice-free conditions. These changes were accompanied by a significant drawdown in macronutrients and an approximate doubling of algal (diatom) biomass. Our incubation experiments suggest that conditions were ironreplete in the presence of sea ice, and iron-deficient in the absence of sea ice. We surmise that bioavailable iron was released into seawater from the melting sea ice, stimulating phytoplankton production and the biological removal of dissolved iron from the mixed layer, until iron-limited conditions developed. These observations suggest that the episodic release of bioavailable iron from melting sea ice is an important factor regulating phytoplankton production, particularly ice-edge blooms, in seasonally ice-covered Antarctic waters.
Article
The trace metals aluminum (Al) and manganese (Mn) were studied in the Weddell Sea in March 2008. Concentrations of dissolved Al ([Al]) were slightly elevated (0.23-0.35 nmol L-1) in the surface layer compared to the subsurface minimum (0.07-0.21 nmol L-1) observed in the winter water. Atmospheric deposition is the main source of Al to the central Weddell Sea (22 mu mol m(-2) yr(-1)), and the residence time of dissolved Al in the upper mixed layer is similar to 0.8 yr. The flux from the shelf and slope regions equals about 50% of the atmospheric input of Al to the western Weddell Sea. The highest [Al] in the Weddell Sea bottom water (WSBW) is related to the formation of deep water, and the associated downward flux is in the range of 3-10 mu mol Al m(-2) yr(-1). The concentrations of dissolved Mn ([Mn]) were depleted in the surface layer, likely as a result of biological uptake, as indicated by the correlation among Mn, major nutrients, and fluorescence. The significant negative relation between the Delta Mn:Delta P ratio and the ambient concentration of dissolved iron indicates iron-Mn co-limitation. The flux of Mn from the continental margin is about 2.2 times greater than atmospheric input (1.1 mu mol m(-2) yr(-1)). The flux of both Al and Mn from the continental margin indicates melting of continental ice (icebergs) or direct continental runoff. The slightly elevated [Mn] in the WSBW is due to a relatively small flux of 1 mu mol Mn m(-2) yr(-1) associated with WSBW formation.