Iron and manganese co-limit growth of the
Southern Ocean diatom Chaetoceros debilis
*, Kai Bischof
, Scarlett Trimborn
1EcoTrace, Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven,
Germany, 2Marine Botany, University of Bremen, Bremen, Germany
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.
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) , which is required for the optimal growth and cellular function of phyto-
plankton [2–6]. 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 . Fe and Mn enter the SO from several
sources including atmospheric dust [8,9], upwelling , sediments [11,12] and melting of gla-
ciers  and sea ice. 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
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/
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.
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  were found to be as low as dissolved Fe con-
centrations, being generally below 0.4 nmol L
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 . 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 . 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 , the study by  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 .
Manganese is the second most abundant trace metal in thylakoids after Fe . It is essential
for phytoplankton growth  as it is required in the water-splitting complex of PSII where
four Mn ions are involved in the oxidation of water . It is further needed for the antioxidant
enzyme superoxide dismutase (SOD), which detoxifies reactive oxygen species (ROS) and pre-
vents cell damage [6,22–24]. 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 . 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  and therefore contributes strongly to carbon export in the SO
. 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 . The same species also dominated the phytoplankton community
after the Fe fertilization experiment SEEDS in the North East Pacific . 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
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
medium ). 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
Si, 100 μmol L
, and 6.25 μmol L
) and vitamins (30 nmol L
23 nmol L
, and 0.228 nmol L
) according to F/2
medium . To represent trace
metal concentrations typical for Antarctic high nutrient low chlorophyll waters, a mixture of
zinc (0.16 nmol L
), copper (0.08 nmol L
), cobalt (0.09 nmol L
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.
) 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
, AAS standard, TraceCERT, Fluka) and MnCl
(2 nmol L
, AAS standard, Trace-
CERT, Fluka). As suggested in , 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
), 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
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
) 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
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
in the -FeMn treatment, 93 603 ±10 805
in the -Mn treatment, 72 155 ±4158 cells mL
in the -Fe treatment and 114
819 ±26 661 cells mL
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.
Seawater 0.30–0.29 0.46–0.52
-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
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 . 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
(certified 495 ±46 ng L
) and 546 ±48 ng L
(certified 530 ±50 ng L
) for dFe and dMn, respectively.
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
in the -FeMn treatment, 93 603 ±10 805 cells mL
-Mn treatment, 72 155 ±4158 cells mL
in the -Fe treatment and 114 819 ±26 661 cells mL
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
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
) and maximum (F
) chlorophyll afluorescence of PSII was determined
6 times to calculate the maximum quantum yield of photochemistry in PSII (F
, 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
), the time constant for electron transport at
the acceptor side of PSII (τ
,μs) and the connectivity factor (p, dimensionless) were derived
according to , 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
with an accli-
mation phase of 5 min per light level. Each actual light intensity (E, μmol photons m
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
) according to the formula by [37,38]:
ETR ¼sPSII xððFq0=Fm0Þ=ðFv=FmÞÞ x E Eq 2
0denotes the effective PSII quantum yield under ambient light. According to ,
maximum ETR (ETR
), light utilization efficiency (α) and minimum saturating
,μmol photons m
) were calculated from the fitted irradiance-dependent
ETR using the SigmaPlot 13.0 software (SysStat Software Inc.).
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
m¼ ðln Nt2ln Nt1Þ=DtEq 3
represent the cell densities (cells mL
) at the sampling day t
tively, and Δtdenotes the time between the two measurements.
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.
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
) 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
, Nalge Nunc International, Rochester, NY, USA), concen-
trations of chlorophyll aand c
, fucoxanthin, diatoxanthin and diadinoxanthin were deter-
mined by reversed phase high performance liquid chromatography. The analysis was
performed on a LaChromElite
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-
ODS-2 column (25 cm x 4.6 mm, 5 μm particle size; Waters, Milford, MA, USA) with a
100-RP-18 guard cartridge was used for the separation of pigments, applying a
gradient according to . Peaks were detected at 440 nm, identified and quantified by co-
chromatography with standards for chlorophyll aand c
, 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.
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.
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
and 0.49 ±0.04 nmol dMn L
(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
Maximum quantum yield and functional absorption cross-sections of PSII
The maximum quantum yield of PSII (F
) 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
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
of C.debilis was highest (control:
The functional absorption cross section of PSII (σ
) was significantly affected by the avail-
ability of both trace metals (ANOVA: F = 18.33, p= 0.0006, Fig 1B). As for F
, the addition
of Mn alone (-Fe: 3.29 ±0.27 nm
) did not change σ
relative to the -FeMn treatment
(3.37 ±0.15 nm
). Only when Fe alone (-Mn: 2.56 ±0.16 nm
) or Fe and Mn together (control:
2.53 ±0.10 nm
) were given, σ
decreased significantly by 24% and 25%, respectively, rela-
tive to the -FeMn treatment. Irrespective of whether Fe alone or both trace metals were added
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
), -Fe (0.31 ±0.03 d
) and -Mn (0.26 ±0.01 d
) treatments, the addition
of Fe and Mn together yielded the highest growth rates (0.40 ±0.02 d
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
) and -Fe (24.91 ±2.21 pg cell
) treatments, they were,
Fig 1. The dark-acclimated maximum PSII quantum yield F
(A) and the dark-adapted functional absorption
cross section of PSII photochemistry σ
(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).
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
). POC quotas of
the control treatment (27.38 ±1.81 pg cell
) 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).
Table 2. Connectivity between adjacent photosystems (p), time constant for electron transfer at PSII (τ
), absolute maximum electron transport rates (ETR
light saturation point (I
) 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
-FeMn 0.32 ±0.01
-Fe 0.35 ±0.01
-Mn 0.42 ±0.01
Control 0.45 ±0.02
�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
) of the control
treatment could unfortunately not be determined due to a technical problem with the FRRf software FastPro8.
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
), -Fe (7.72 ±1.05 pg cell
) and -Mn (8.14 ±0.35
) 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
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
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
) was significantly altered in
response to the different Fe and Mn concentrations (ANOVA: F = 11.38, p= 0.0029, Table 2).
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
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
significantly affected by the different trace metal additions (ANOVA: F = 17.63, p= 0.0007,
Table 2). ETR
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
) 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
using the light-adapted σ
showed the same trend. The light saturation point of PSII electron transport (I
between 408 ±26 and 532 ±35 μmol photons m
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.
Cellular concentrations of light-harvesting pigments (LH = chlorophyll a+ chlorophyll c
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.
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).
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  as “Type I: Independent nutrient co-
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
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
values of the FeMn- and Fe-deficient
cells suggest reduced photochemical efficiency, a common feature of Fe-limited phytoplankton
[7,41–44]. To counterbalance the reduced amount of Fe-containing photosynthetic reaction
centers [41,43,45,46], both FeMn- and Fe-poor cells displayed larger σ
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 , 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  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 [45,51–54], 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 . 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
, 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).
-FeMn 89 ±2
-Fe 80 ±2
-Mn 118 ±5
Control 135 ±17
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
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  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 . Furthermore, minimum saturating irradiance (I
) 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 . 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
. 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 . 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  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
than to those in
which only Fe was added . 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 . 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  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
Christian Vo¨lkner, Tina Brenneis, Dorothee Wilhelms-Dick and Britta Meyer-Schlosser are
thanked for the support in the laboratory.
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.
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