Atmospheric CO2 availability induces varying responses in net photosynthesis, toxin production and N2 fixation rates in heterocystous filamentous Cyanobacteria (Nostoc and Nodularia)

Abstract

Heterocystous Cyanobacteria of the genus Nodularia form major blooms in brackish waters, while terrestrial Nostoc species occur worldwide, often associated in biological soil crusts. Both genera, by virtue of their ability to fix N 2 and conduct oxygenic photosynthesis, contribute significantly to global primary productivity. Select Nostoc and Nodularia species produce the hepatotoxin nodularin and whether its production will change under climate change conditions needs to be assessed. In light of this, the effects of elevated atmospheric CO 2 availability on growth, carbon and N 2 fixation as well as nodularin production were investigated in toxin and non-toxin producing species of both genera. Results highlighted the following: Biomass and volume specific biological nitrogen fixation (BNF) rates were respectively almost six and 17 fold higher in the aquatic Nodularia species compared to the terrestrial Nostoc species tested, under elevated CO 2 conditions.
There was a direct correlation between elevated CO 2 and decreased dry weight specific cellular nodularin content in a diazotrophically grown terrestrial Nostoc species, and the aquatic Nodularia species, regardless of nitrogen availability.
Elevated atmospheric CO 2 levels were correlated to a reduction in biomass specific BNF rates in non-toxic Nodularia species.
Nodularin producers exhibited stronger stimulation of net photosynthesis rates (NP) and growth (more positive Cohen’s d) and less stimulation of dark respiration and BNF per volume compared to non-nodularin producers under elevated CO 2 levels.

This study is the first to provide information on NP and nodularin production under elevated atmospheric CO 2 levels for Nodularia and Nostoc species under nitrogen replete and diazotrophic conditions.

Full text

Vol.:(0123456789)
1 3
Aquatic Sciences (2021) 83:33
https://doi.org/10.1007/s00027-021-00788-6
RESEARCH ARTICLE
Atmospheric CO2 availability induces varying responses
innet photosynthesis, toxin production and N2 fixation rates
inheterocystous filamentous Cyanobacteria (Nostoc andNodularia)
NicolaWannicke1 · AchimHerrmann2 · MichelleM.Gehringer2
Received: 21 April 2020 / Accepted: 3 February 2021
© The Author(s) 2021
Abstract
Heterocystous Cyanobacteria of the genus Nodularia form major blooms in brackish waters, while terrestrial Nostoc species
occur worldwide, often associated in biological soil crusts. Both genera, by virtue of their ability to fix N2 and conduct oxy-
genic photosynthesis, contribute significantly to global primary productivity. Select Nostoc and Nodularia species produce
the hepatotoxin nodularin and whether its production will change under climate change conditions needs to be assessed.
In light of this, the effects of elevated atmospheric CO2 availability on growth, carbon and N2 fixation as well as nodularin
production were investigated in toxin and non-toxin producing species of both genera. Results highlighted the following:
• Biomass and volume specific biological nitrogen fixa-
tion (BNF) rates were respectively almost six and 17 fold
higher in the aquatic Nodularia species compared to the
terrestrial Nostoc species tested, under elevated CO2 con-
ditions.
• There was a direct correlation between elevated CO2 and
decreased dry weight specific cellular nodularin content
in a diazotrophically grown terrestrial Nostoc species,
and the aquatic Nodularia species, regardless of nitrogen
availability.
• Elevated atmospheric CO2 levels were correlated to a
reduction in biomass specific BNF rates in non-toxic
Nodularia species.
• Nodularin producers exhibited stronger stimulation of
net photosynthesis rates (NP) and growth (more positive
Cohen’s d) and less stimulation of dark respiration and
BNF per volume compared to non-nodularin producers
under elevated CO2 levels.
This study is the first to provide information on NP and nodularin production under elevated atmospheric CO2 levels for
Nodularia and Nostoc species under nitrogen replete and diazotrophic conditions.
Keywords Climate change· Nitrogen fixation· Nodularia· Nodularin· Nostoc· Photosynthesis
Abbreviations
BNF Biological nitrogen fixation
HC High CO2 (2000ppm)
LC Low CO2 (~ 440ppm)
POC Particulate organic carbon
PON Particulate organic nitrogen
NP Nett photosynthesis
NPP Net primary production
CCM Carbon concentrating mechanism
Introduction
Cyanobacteria, in their role as primary producers, form an
essential part of the global C and N cycles, both in terrestrial
and aquatic environments (Visser etal. 2016; Elbert etal.
2012). The process of oxygenic photosynthesis, whereby
energy from the sun is used to reduce inorganic carbon
with the accompanying oxidation of water, is thought to
Aquatic Sciences
* Michelle M. Gehringer
mgehring@rhrk.uni-kl.de; mmgehringer@yahoo.com
1 Research Group Plasma-Agriculture, Leibniz Institute
forPlasma Science andTechnology, Felix‐Hausdorff‐Str. 2,
17489Greifswald, Germany
2 Department ofMicrobiology, Technical University
ofKaiserslautern, Paul-Ehrlich Straße, 67653Kaiserslautern,
Germany

N.Wannicke et al.
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33 Page 2 of 17
have evolved during the Archean era when there was no
free oxygen in the Earth’s atmosphere (Lyons etal. 2014).
The enzyme catalysing CO2 fixation in Cyanobacteria and
modern-day C3 plants is ribulose-1,5-bisphosphate carbox-
ylase/oxygenase (Rubisco), thought to be the most abun-
dant enzyme on Earth. Rubisco binds CO2 and generates 2
molecules of 3-phosphoglycerate (3PGA) which is further
processed in the Calvin-Benson-Bassham (CBB) cycle to
produce ribulose-1,5-biphosphate and glutamate. In order
to reduce undesirable oxygenase activity, Cyanobacteria
have evolved the carbon concentrating mechanism (CCM)
to increase the effective concentration of CO2 around the
Rubisco active site by up to 1000-fold (Price 2011). CO2 dif-
fuses freely into the cell and is converted to bicarbonate in an
NADPH-dependent reaction. Most Cyanobacteria sequenced
to date carry the high flux, low affinity CO2 converting
enzyme, NDH-I4, as well as the low flux, high affinity NDH-
I3 variant. Uptake of bicarbonate from the surrounding liquid
requires an investment in energy and the synthesis of spe-
cific transporters. Two sodium –dependent symporters, BicA
(high flux, low affinity) and SbtA (low flux, high affinity)
bicarbonate transporters occur occasionally together with
the BCT1 high affinity low flux transporter, found in almost
all Cyanobacteria investigated to date on the cell membrane
(Burnap etal. 2015; Visser etal. 2016). The presence of
BicA provides aquatic Cyanobacterial species a growth
advantage under elevated levels of HCO3− availability (San-
drini etal. 2014). Oxygenic photosynthetic organisms that
rely on the construction of a carbon concentrating mecha-
nism (CCM) are thought to be sensitive to changes in pCO2
(e.g. Raven etal. 1991; Rost etal. 2003; Price 2011; Shi
etal. 2012; Raven etal. 2017). The plasticity of the CCM to
elevated levels of atmospheric CO2 was found to be high in
Cyanobacteria when compared to the more recently evolved
haplophytes and diatoms (Van de Waal etal. 2019). This
phenotypic plasticity in carbon fixation was demonstrated on
Microcystis grown under conditions of elevated CO2 (Ji etal.
2020). The maximum CO2 uptake rate of Microcystis grown
at 1000ppm CO2 was increased 1.5–1.8 times compared to
the low CO2 control cultures, suggesting that elevated CO2
conditions may stimulate Cyanobacterial bloom growth (Ji
etal. 2020). Furthermore, by reducing the levels of dissolved
CO2 and increasing the pH in dense blooms, Cyanobacterial
species succession is thought to be driven towards strains
with a more efficient carbon concentrating mechanisms
(Lines and Beardall 2018).
Globally, an increase in phytoplankton blooms, includ-
ing Cyanobacterial harmful algal blooms, has been recorded
since the 1980’s (Ho etal. 2019). While the reasons for the
observed increase is unclear, temperature, elevated atmos-
pheric CO2 levels and eutrophication especially of the
freshwater lakes are potential drivers of this phenomenon.
Approximately a third of all anthropogenic CO2 released
dissolves in the oceans, reducing the pH by increasing the
partial pressure of CO2, accompanied by a smaller rela-
tive increase in HCO3− and a decrease in CO32− (Sabine
etal. 2004; Raven etal. 2017). Although the speciation of
dissolved inorganic carbon is directly linked to pH, how
changes in their balance affects Cyanobacterial bloom
occurrence and toxicity is unclear (Raven etal. 2020).
The increase in growth rate observed for the marine, non-
heterocystous, filamentous diazotrophic Cyanobacterium,
Trichodesmium, grown at 900ppm CO2 was ascribed to
down regulation of the CCM, thereby reducing the energy
demands on the cell (Kranz etal. 2011). Under Fe- limiting
conditions, decreasing the medium pH reduced N2 fixation
rates in Trichodesmium, with the reduced N2 fixation rates
corresponding to reduced nitrogenase efficiency at lower
pH (Kranz etal. 2011). Exposing cultures of the freshwater
diazotroph, Nostoc muscorum, to raised HCO3− concentra-
tions under diazotrophic conditions resulted in enhanced
growth, O2 and pigment production and nitrogenase activi-
ties (Bhargava etal. 2013). The brackish diazotroph, Nodu-
laria spumigena sp. KAC12, when grown at elevated CO2
of 960ppm, demonstrated increased photochemical yield
after 5days exposure (Karlberg and Wulff 2013), suggest-
ing higher potential net primary productivity rates. Nodu-
laria spumigena CCY9414, grown under elevated CO2
conditions (548ppm), exhibited increased C fixation rates
compared to control cultures, with increased carbon to nitro-
gen (POC:PON) and nitrogen to phosphate ratios recorded
(Wannicke etal. 2012). Only a slight increase was observed
in the C:N ratios in three Cyanobacterial cultures grown
at elevated CO2 (~ 900ppm) in continuous culture in bub-
ble reactors, namely Cyanothece sp. ATCC51142, Nodu-
laria spumigena IOW-2000/1 and Calothrix rhizosoleniae
sp. SC01 (Eichner etal. 2014). This study emphasised the
need to generate more data on the effects of elevated CO2
levels on Cyanobacterial BNF, and highlighted the diver-
sity in observed responses of marine Cyanobacterial species
to elevated atmospheric CO2. Wannicke etal. (2018b), in
their metadata study, found indications that ocean acidifi-
cation would benefit BNF in the future ocean. They also
drew attention to the fact that these studies were mostly con-
ducted on only two species, the filamentous Trichodesmium
and unicellular Crocosphaera. Very few studies were pub-
lished on filamentous heterocystous Nodularia, Calothrix
and Anabaena (alias: Dichlospermum) species (reviewed by
Wannicke etal. 2018b). A more recent study suggested that
growth of the diazotrophic Dolichospermum circinale might
benefit from increased CO2 levels of 1700ppm (Symes and
van Ogtrop 2019).
Studies investigating the effect of climate change on fila-
mentous diazotrophic Cyanobacteria in terrestrial habitats
are rare too. Terrestrial surfaces are often inhabited by cryp-
togrammic covers, including Cyanobacteria that contribute

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
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Page 3 of 17 33
a significant amount to global net primary productivity
(Elbert etal. 2012). Specifically, it is estimated that N2
fixation by cryptogrammic covers may account for almost
half of biological nitrogen fixation on land, ~ 49Tg per year
(Elbert etal. 2012). Biological soil crusts showed a decrease
in Cyanobacterial abundance when grown under elevated
atmospheric CO2 for 10years, suggesting a negative impact
of climate change on arid soil crusts (Steven etal. 2012).
The ability of Cyanobacterial soil crusts to increase net pri-
mary production under high CO2 (HC) exposure was shown
to be dependent on water availability (Lane etal. 2013). This
was in agreement with previous research demonstrating that
terrestrial Nostoc flagelliforme exhibited its highest relative
growth rate under conditions of high CO2 (1500ppm) in
moist conditions when compared to mats grown at 350ppm
CO2 (Gao and Yu 2000). Rodriguez-Caballero etal. (2018)
suggested that dryland soil crusts are under threat due to
anthropogenically induced climate change. Their projected
loss of 25–40% cryptogrammic coverage will result in
reduced microbial contributions to nitrogen cycling and soil
surface stabilisation. Additionally, little information exists as
to how Cyanobacterial biocrusts specifically, will respond to
increasing atmospheric CO2 levels (Reed etal. 2016).
Given the evolutionary history of Cyanobacteria already
existing under raised CO2 levels, researchers (Gehringer and
Wannicke 2014; Sandrini etal. 2016; Visser etal. 2016;
Buratti etal. 2017) have voiced concerns for increased
Cyanobacterial bloom occurrence and toxin production
under the elevated levels of CO2 proposed by current climate
change scenarios, particularly in eutrophic waters (Ma etal.
2019). Especially toxin producing Cyanobacteria capable
of fixing atmospheric N2 would offer a potential threat to
human safety, as they could thrive in otherwise nitrogen-
limited habitats (O’Neil etal. 2012). The most commonly
occurring Cyanobacterial toxins are microcystin and nodu-
larin, both strong protein phosphatase inhibitors, capable of
inducing extensive hepatocellular bleeding and collapse in
exposed individuals and animals (Gehringer 2004; Ibelings
etal. 2015; Buratti etal. 2017). Microcystin and nodula-
rin are synthesized by non-ribosomal peptide synthetases
(Dittmann et. al. 2001; Moffit and Neilan 2004) for which
the control mechanisms remain largely unknown. The levels
of toxin production within Cyanobacterial blooms is largely
determined by several abiotic factors such as light inten-
sity and quality, pH and nutrient availability (Reviewed by
Gehringer and Wannicke 2014; Visser etal. 2016; Buratti
etal. 2017). Raised temperatures and elevated CO2 levels
in the range of those proposed under climate change, are
linked to increased primary production (Paerl and Huisman
2009) and toxin production by Cyanobacteria (El-Shehawy
etal. 2012; Kleinteich etal. 2012). Increased production of
the secondary metabolite, microcystin, is linked to main-
taining the C:N balance in the cell in the non-diazotrophic
Microcystis aeruginosa (Downing etal. 2005), particularly
when N uptake exceeds the relative growth rate. Elevated
CO2 levels have the capacity to affect the community com-
position and toxicity of Microcystis blooms significantly
(Liu etal. 2016; Van De Waal etal. 2011; Sandrini etal.
2016; Buratti etal. 2017). Microcystin synthesis requires
active photosynthesis (Sevilla etal. 2012) and, like nodula-
rin synthesis, is regulated by the global N uptake regulator,
NtcA, supporting the proposed importance of the C:N bal-
ance on toxin production (Neilan etal. 2013). This agrees
with observed anthropogenically induced alterations in envi-
ronmental N:P ratios, resulting in the appearance of Cyano-
bacterial blooms (Beversdorf etal. 2013) and increased toxin
production (Horst etal. 2014). Inorganic nitrogen limita-
tion was thought to induce a shift to N2 fixing, diazotrophic
Cyanobacteria, thereby increasing organic N availability
and a subsequent increase in toxin production (Posch etal.
2012; Gehringer and Wannicke 2014). Recent investigations
of bloom dynamics in Lake Müggelsee suggest that the pre-
dominant Cyanobacterial diazotrophs, Aphanizomenon sp.
and Anabaena sp (Dichlospermum sp.), do not proportion-
ally increase in numbers relative to non-nitrogen producers
under conditions of reduced N availability (Shatwell and
Köhler 2019). The changes in Ci availability, nitrogen fixa-
tion rates and potential cyanotoxin production levels were
not reported. Transcription of the nda cluster in Nodularia
spumigena AV1 was found to be altered in response to
changes in ammonia and phosphate availability, however,
the levels of intra- and extracellular nodularin were not sig-
nificantly altered (Jonasson etal. 2008). Production of cylin-
drospermopsin and microcystin is thought to be constitutive,
with cell cyanotoxin quotas being relatively fixed (Orr etal.
2018; Pierangelini etal. 2015). Orr etal. (2018) furthermore
argued that toxicity is not affected through any stimulatory
or trigger effect on the toxin production pathway itself, but
via changes in rates of cell division and growth of different
strains with genetically different cyanotoxin cell quotas. If
the Cyanobacterial specific cyanotoxin rate matches the spe-
cific cell division rate, the overall cell cyanotoxin remains
fixed. Dense Cyanobacterial blooms require excessive CO2
to support their continued growth (Paerl and Huisman 2009)
with CO2 availability often limiting bloom growth, a restric-
tion that could be removed under increased atmospheric CO2
levels. Only aquatic Cyanobacteria carrying the high flux,
low affinity BicA HCO3− receptor were able to benefit from
elevated CO2 levels and increase their growth rates (Sandrini
etal. 2015; 2016; Visser etal. 2016).
Most studies on elevated CO2 effects reported for toxin
producing Cyanobacteria have focused on the production
of the heptapeptide toxin, microcystin, in freshwater uni-
cellular non-diazotrophic Microcystis aeruginosa species.
The microcystin content of Microcystis aeruginosa HUB
5-2-4 grown at elevated CO2 was raised, while growth rates

N.Wannicke et al.
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33 Page 4 of 17
were kept constant (Van de Waal etal. 2009). Sandrini etal.
(2015) reported that the shift of Microcystis aeruginosa PCC
7806 from 200ppm pCO2 to 1450ppm pCO2 in a continu-
ous culture, resulted in a ∼2.7-fold increase of Cyanobacte-
rial biomass and ∼2.5-fold elevation in microcystin per cell.
Moreover, at high pCO2, gene expression of the high flux,
low affinity BicA HCO3− receptor was down-regulated and
cells shifted to CO2 and low-affinity, high flux receptors for
bicarbonate uptake. Interestingly, the expression of the mcy
genes involved in microcystin synthesis remained constant,
suggesting additional regulatory steps are involved in toxin
synthesis under elevated CO2 conditions. Studies investi-
gating the competition of microcystin and non-microcystin
producing strains of Microcystis at low and elevated pCO2
levels found that non-toxic strains outcompete toxic strains
under conditions of low light and high CO2 availability (Van
De Waal etal. 2011; Yu etal. 2015). On the other hand,
toxin-producing strains display a better fitness under growth-
limiting conditions suggesting that the benefit of producing
the toxin outweighs its costs under unfavourable conditions
(Briand etal. 2008; Van De Waal etal. 2011).
To our knowledge, there is no peer- reviewed publication
concerning the effect of elevated CO2 on nodularin produc-
tion in Cyanobacteria. This study is directed at studying
diazotrophic Cyanobacterial species from both terrestrial
and aquatic environments to investigate the effect of elevated
CO2 levels on net photosynthesis, toxin production, growth
and N2 fixation rates in a multiple matrix approach. To do
so, seven different species were chosen of which three are
able to produce the toxin nodularin and four are non-nod-
ularin producers. Finally, we analysed the data set gained
in this study by applying weighted mean effect sizes to test
the hypothesis that Cyanobacteria react differently towards
elevated CO2 depending on the whether they produce nodu-
larin or not.
Materials andmethods
Culture conditions andexperimental design
The experimental design contained a multiple matrix
approach with different factorial designs for the Cyanobac-
teria tested, using a combination of different atmospheric
CO2 treatments of high CO2 (HC), low CO2 (LC), culture
medium containing nitrogen in the form of NaNO3 or not
(N+ and N−) and the ability to produce the hepatotoxin,
nodularin (+ and −) (Fig.1a).
In total, six species of heterocystous filamentous Cyano-
bacteria belonging to two families, Nostocaceae and Apha-
nizomninaceae, within the order Nostocales, were selected
for investigation. Four representative species of the genus
Nostoc were analysed, with two being nodularin producers,
ie Nostoc punctiforme sp. 73.1 and Nostoc muscorum sp.
65.1 and two non-nodularin producing species: Nostoc punc-
tiforme sp. 40.5 and Nostoc entophytum sp. C1.8 (Gehringer
etal. 2010, 2012). Two representative species of the genus
Nodularia, were investigated, with one nodularin producing
species, Nodularia spumigena CCY9414 (Voss etal. 2013)
and its related non-toxic mutant strain Nodularia spumigena
NSBL06 [analogous to N. spumigena NSBL05 (Bolch etal.
1999; Moffit etal. 2001)] and the non-toxic benthic spe-
cies of Nodularia harveyana SAG 44.85 (Lyra etal. 2005;
Řeháková etal. 2014). The combination of the three variable
factors, atmospheric CO2 levels, nitrogen content and toxin
production, generated four different factorial designs for the
species tested (Fig.1b).
The terrestrially isolated Nostoc species were maintained
since their isolation on the nitrogen free medium, BG110,
medium with ferric ammonium citrate replaced with ferric
citrate (Gehringer etal. 2010), thereby ensuring their ability
to fix nitrogen was not lost. Three months prior to this study,
the Nostoc species were also subcultured into BG11 medium
containing NaNO3 (17.6mM). The aquatic diazotrophic spe-
cies Nodularia spumigena CCY9414 (Culture Collection
Yerseke), Nodularia spumigena NSBL 06 (kindly provided
by Hanna Mazur-Marzec, University of Gdansk) and the
benthic Nodularia harveyana SAG 44.85 (Culture Collec-
tion of Algae, SAG, Georg August University, Göttingen)
were cultivated in nitrogen free brackish sea water medium
(F/2) with a salinity of 10 containing vitamins (UTEX, Aus-
tin). The benthic species, Nodularia harveyana SAG 44.85,
required the addition of 5ml l−1 of soil extract.
Fifty ml of stationary phase cultures were inoculated into
150ml of the appropriate media in a Fernbach flask (Duran,
d = 45mm) for maximal volume to surface area ratio, and
placed at the control or experimental conditions for 14days
to allow them to adjust to their new conditions (Eichner etal.
2014). The inoculum cultures were then diluted 1:1 with
fresh medium and divided into two ventilated T175 polysty-
rene cell culture flasks (Greiner). In this manner three toxin
producing species (n = 3), namely Nodularia spumigena
CCY9414, Nostoc punctiforme sp. 73.1 and Nostoc mus-
corum sp. 65.1 and four control, non-toxin producing spe-
cies (n = 4), namely Nodularia spumigena NSBL06, Nodu-
laria harveyana SAG 44.85, Nostoc punctiforme sp. 40.5
and Nostoc entophytum sp. C1.8, were studied (Fig.1). The
flasks were laid flat to minimise shading effects, resulting
in a culture depth of 1cm that maximised gas exchange at
the culture surface (Herrmann and Gehringer 2019). Experi-
mental cultures were exposed to elevated CO2 of 2000ppm
(defined here as “High CO2”—HC), 10:14h light:dark cycle,
22°C, 60% humidity and 130µmol photons m−2 s−1 (Plant
growth chamber E-22L, Percival, USA). Control cultures
were exposed to CO2 at present day level, ~ 440ppm in Kai-
serslautern, Germany (defined here as “Low CO2”—LC),

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
1 3
Page 5 of 17 33
with the same culture conditions as above. Different param-
eters were sampled on days 7- and 14-post inoculation. The
numbers of replicates for each sampling day and total num-
bers are provided in Table1. In our probe study, a trade
off was made between using a variety of species (toxic and
non-toxic) from aquatic and terrestrial origin versus increas-
ing the number of replicated incubation bottles per sampling
time point. We chose to use a repeated measure approach for
most of the parameters with two replicate incubation bottles
and two sampling time points. In the case of N2 fixation, we
ended up with technical replicated sampling from each bot-
tle at one sampling time point, applying pseudo-replication
in this case.
Determination of growth curves based on optical density
were set up separately from the experimental bottles after
the adjustment phase with a start inoculum of OD650 of ~ 0.1
and 200µl pipetted into 6 wells of a 96 well microtiter plate
for each Cyanobacterial culture. Individual Cyanobacteria
were grown in their appropriate medium, both diazotrophi-
cally and in nitrogen replete medium, at LC and HC. Cul-
tures were resuspended by pipetting and shaking just before
the OD650 was read in a Multiscan Microtitre plate reader
(Thermo Scientific) over 14days post inoculation at exper-
imental conditions identical to the cell culture flasks. To
ensure growth rates based on optical density measurements
in the 96 well microtiter plate were not biased compared to
Fig. 1 Species characteristics
and three factorial design cho-
sen for the seven Nostocaceae
species tested (a). Factors
include the ability to produce
nodularin (Factor A); the CO2
treatment (Factor B) with
present day levels (440ppm—
“Low CO2” LC) and elevated
CO2 (2000ppm- “High
CO2”-HC), and nitrogen avail-
ability (Factor C) with cultures
grown either diazotrophically
or in N-replete medium (N + an
N−). The different factorial
designs in a) result from the
combination of factors A–C for
the different species illustrated
in (b)

N.Wannicke et al.
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33 Page 6 of 17
the actual experimental set-up in culture flasks, we reputed
growth rates determination in culture flasks for 14days of
incubation. Growth curves were therefore set up in culture
flasks, in triplicate for terrestrial Nostoc species Nostoc
punctiforme sp. 73.1, Nostoc muscorum sp. 65.1, Nostoc
punctiforme sp. 40.5 and Nostoc entophytum sp. C1.8 in
freshwater growth medium under both diazotrophic (N−)
and non—diazotrophic (N+) conditions at LC and HC
atmospheric conditions. Similarly, growth curves were
established in triplicate for Nodularia spumigena CCY9414
and Nodularia spumigena NSBL06 in brackish sea water
growth medium, in both N-replete and N-free medium. T75
ventilated suspension culture flasks (Sarstedt, Germany)
containing 75ml of the appropriate medium, were inoc-
ulated with stationary phase cultures from the respective
atmospheres to give a starting Chl a content of 0.1µg ml−1.
Table 1 Number of replicate
incubation bottles and sampling
per day for the seven species
tested for the LC (440ppm) and
HC (2000ppm) treatment. 2 + 2
indicates technical replicates
from 2 incubation bottles at one
time point
Investigated
Parameter
Treatment Sampling day No. of replicates
per sampling
day
Total no. of
replicates
714714
CO2uptakeLC 22 4
HC  22 4
Dark
respiration
LC  22 4
HC  22 4
Chlorophyll aLC 22 4
HC  22 4
NodularinLC
33
HC 
33
PON/POCLC2+
24
HC 2+
24
N2fixation LC Nostoc sp.* 
22
Nodularia sp. 2+
24
HC Nostoc sp.* 
22
Nodularia sp. 2+
24
Incubation and sampling in 96 well-microtiter plates
Growth
based on
OD650**
LC
Read on days 3 –7 and10, 11, 12 and 14, 6
replicates each
HC
Incubation and sampling in cell culture flasks
Growth
based Chl a.
LC
Sampling at day 3, 5, 7, 10, 12, 14
3 replicates eachHC
* Determined only for Nostoc punctiforme sp. 73.1 (−N)
**Readings were repeated on days listed on cultures resuspended in 96 well- microtiter plates every over
a period of 14days. Calculation was done for the period of day 3–14 (10 absorbance readings)

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
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Page 7 of 17 33
Two ml samples were collected from agitated cultures on
day 3, 5, 7, 10, 12 and 14 for Chl. a determinations (below).
Two ml of culture material was harvested for nodularin anal-
ysis on day 14 (below) and a 20ml volume was centrifuged,
drained and dried in a 60°C oven to obtain the biomass per
volume.
Carbonate chemistry
The pH was determined on day 14 from sample filtrates
using an electrode (Radiometer analytical PHM210, France)
calibrated with a three-point calibration using NBS (National
Bureau of Standards) buffers giving values of pH relative to
the NBS scale. Total alkalinity (AT) was determined using
the colorimetric SOMMA system according to Johnson etal.
(1993). The system was calibrated with carbon reference
material provided by A. Dickson (University of California,
San Diego) and yielded a precision of about ± 2μmol kg−1.
Total carbon (CT) and pCO2 in the growth media were calcu-
lated using CO2SYS (Lewis etal. 1998). Media control car-
bonate chemistry was similarly assessed with experimentally
obtained values for pH and total alkalinity (TA) determined
by manual titration (Dickson etal. 2007) and calculated
using the Seacarb package in RStudio version 1.0.153, as
input data (Hermann and Gehringer 2019).
Net CO2 uptake/net photosynthesis (NP)
Culture material was removed by pipetting under sterile con-
ditions in a clean bench in LC and HC conditions on days 7
and 14 representing mid to late exponential growth phases.
Harvested Cyanobacteria were filtered onto a 3µM SSWP
(Millipore) glass fibre filter (Ritchie 2008), placed onto an
appropriate, moist agar plate and incubated under experi-
mental conditions until CO2 uptake determinations (between
1 and 4h after sampling) by means of CO2 gas exchange
measurements (GFS 3000, WALZ, Effeltrich, Germany).
Bacterial covered filters of LC acclimated cultures were
placed in the sample cuvette and CO2 uptake determined at
80% humidity at 440ppm (Herrmann and Gehringer, 2019),
while for HC acclimated cultures measurements was done at
1500ppm CO2 (readings at 2000ppm were too unstable at
80% humidity in the sample cuvette of the GFS 3000). The
respiration rate was determined for each filter after 5min
dark incubation at the start and end of the measuring period
to ensure the cultures were not stressed. CO2 assimilation
rates were determined at 500µmol photons m−2 s−1 (approx-
imate light saturation point for all Cyanobacteria used in this
study, determined from light curves), and expressed per µg
chlorophyll a. Net photosynthesis (NP) rates, representing
the total assimilation of CO2 minus the CO2 released during
respiration, were calculated.
Chlorophyll adetermination
Chlorophyll a was extracted from the bacterial filter discs
used for the gas exchange experiments above. After CO2
uptake measurements, each filter was placed in a 2ml cen-
trifuge tube containing 100mg of 0.1mm zirconia silica
beads (BioSpec) to which 1.5ml 90% HPLC grade methanol
was added (Meeks and Castenholz 1971). The samples were
bead-beated (Retch, Germany) for 1min at 30 beats per min
and incubated at 4°C in the dark overnight. Samples were
subsequently centrifuged at 10 000 rcf for 5min at 20°C and
the OD665 was determined (Lambda 35 UV/VIS spectrom-
eter, Perkin-Elmer). Chlorophyll a content was calculated
using the equation: Chl a µg ml−1 = OD665 × 12.7 (Meek s
and Castenholz 1971). Cell pellets obtained from centrifug-
ing two ml of culture were extracted in the same manner to
generate the Chl a based growth rates.
Nodularin analysis
Samples for toxin determinations were obtained on day 14
of the growth curves for nodularin producing Cyanobacterial
cultures. A 2ml volume of culture material was centrifuged
and the cell pellet drained. One hundred mg of 0.1mm zir-
conia silica beads (BioSpec) were added to the pellet with
1.5ml 70% HPLC grade methanol (Gehringer etal., 2012).
The samples were lysed by bead beating as above and incu-
bated at room temperature in the dark overnight. The fol-
lowing morning the samples were vortexed, the lysed cell
material removed by centrifugation as above, and the super-
natant fluid used in a competitive ELISA assay (Abraxis
#522015, Eurofins, Luxembourg) following the manufac-
turer’s instructions. The amount of toxin extracted for each
nodularin producing Cyanobacterium under diazotrophic
and non-diazotrophic conditions was calculated from the
standard curve (R2 = 0.9937) and expressed as total soluble
cellular nodularin content per dry biomass [ng nodularin.
µg dry weight−1].
Particulate organic matter andN2 xation
The PON and POC content were measured for Nodularia
cultures and Nostoc punctiforme sp. 73.1 grown in N-free
medium on day 14. Due to budget constraints, the remaining
Nostoc cultures were not studied. Filters containing culture
samples were trimmed, sectioned, then loaded into tin cap-
sules and palletised for isotopic analysis. Measurement was
done by means of flash combustion in a Carlo Erba EA 1108
at 1020°C in a Thermo Finnigan Delta S mass-spectrometer.
Calibration material for N and C analysis was acetanilide
(Merck). N2 fixation activity was determined by incubating
cultures in two replicates per treatment with bubble addition
of 15N–N2 enriched gas (99% 15N2) for 24h, guaranteeing

N.Wannicke et al.
1 3
33 Page 8 of 17
sufficient dissolution of the 15N gas in the incubation bot-
tle (Wannicke etal. 2018a). Tracer incubations were termi-
nated by gentle vacuum filtration (< 200mbar) of the culture
material through pre-combusted GF/F filters (Whatman) that
were then dried at 60°C, analysed and the N2 fixation rates
calculated (Montoya etal. 1996). Two technical replicates
were conducted per bottle.
Statistical analysis
Statistical analyses were done either by using the Student´s
t test or Mann–Whitney Rank Sum Test comparing the
mean effect sizes and mean values or by using one-way and
repeated measures ANOVA to determine the CO2 treat-
ment effect. It has to be noted that in the case of Nostoc
punctiforme sp. 73.1, only two samples for N2 fixation were
successfully measured. Two replicates were lost due to an
autosampler error during processing. No statistical analysis
comparing the treatment groups was applied in this case.
Prior to statistical analysis, data were tested for normality
and homogeneity of variances using Wilk-Shapiro and Lev-
ene’s tests. All analyses were performed using the software
SigmaPlot 13 (Systat Software Inc., San Jose, CA, USA).
Response ratios andweighted mean effect sizes
To investigate a possible modulating effect of toxin produc-
tion in response to elevated CO2 and of elevated CO2 on
volume and dry weight specific nodularin production, we
determined the response ratio, i.e.
lnRR
=
(
xT
xC
)
for net pho-
tosynthesis, dark respiration, growth and N2 fixation in
selected Cyanobacteria. Here lnRR is the natural-log pro-
portional change in the means (
x
) of the CO2 treatment (T,
i.e. HC) and control group (C, i.e. LC). Negative values of
lnRR denote lower rates/ growth at elevated CO2 compared
to control, and vice versa.
To examine the modulating effect of nodularin production
over all species tested, pooled lnRR values were combined
to give a mean effect size (i.e. Cohen’s d). A weight was
assigned to each lnRR obtained from individual species
which was inversely proportional to its sampling variance
(DerSimonian and Laird 1986) as represented by the follow-
ing equation:
d
=
xT−xC
SampleSDpooled
. Sub-group calculations were
done for the groups “nodularin producer” and “non-nodula-
rin producer” (see Fig.1 for the toxin status of each Cyano-
bacterium investigated) and for the nodularin production per
volume and per dry weight. To calculate the weighted mean
effect sizes, their significance and 95% confidence intervals,
a random effect model was applied (DL = DerSimo-
nian–Laird estimator) using Meta-Essential (Suurmond etal.
2017a, b).
Results
Carbonate chemistry
Carbonate chemistry of the media confirmed that experi-
mental application of a continuous atmospheric gas supply
ensured enrichment with CO2 in cultures grown at elevated
CO2 levels. The pCO2 in the growth media of cultures
incubated at HC in the Nostoc cultures was determined
to be 1987 ± 42 µatm for N-free media, while the pCO2 in
the LC treatment was 293 ± 38 µatm. Nodularia cultures
displayed a mean pCO2 of 1701 ± 83 µatm in N-replete
brackish seawater medium (Suppl. Table1). Control cul-
tures at LC conditions revealed significantly reduced pCO2
availability at 232 ± 26 µatm. Control medium at HC was
2728 ± 323µatm and 2203 ± 405 µatm for fresh and brack-
ish N-free media respectively. Also, carbonate chemistry
determined in the experimental bottles showed signifi-
cant differences between LC and HC treatments (Suppl.
Table1). The N-free media TA values determined agreed
with previously published data (Wannicke etal. 2012)
in low nutrient media. The TA values for experimental
cultures grown at HC in media containing N were also
exceedingly high, suggesting interference of biologically
synthesised compounds interfering with the TA assess-
ment. These values were therefore not reported.
Eect ofelevated CO2 onCyanobacterial growth
The growth curves for each species grown under LC and
HC conditions highlight the different responses between
Cyanobacterial cultures (Suppl. Fig.1, 2, 3) to atmos-
pheric CO2 and / or nitrogen availability. The data for
Nostoc species 65.1, 40.5 and C1.8, under N limitation,
are not presented as the NP readings fell below the level
of detection in the gas exchange measurements.
Nostoc punctiforme sp. 73.1 exhibited higher growth
rates at HC conditions than at LC, with higher growth rates
observed under N replete conditions. Nostoc punctiforme
sp. 40.5 did not show a significant response towards HC.
On the other hand, both Nostoc muscorum sp. 65.1 and
Nostoc entophytum sp. C1.8 displayed lower growth rates
at HC (Suppl. Table2).
Nodularia harveyana SAG 44.85 and Nodularia spu-
migena CCY9414 showed increased growth at HC com-
pared to LC grown cultures for the time interval 0–14days
(Suppl. Table3), while Nodularia spumigena NSBL206
displayed lower growth rates at HC.
Growth rates determined in 96-well microtitre plates
and culture flasks are mostly in agreement (Suppl. Fig.4).
In Nostoc species, growth rates calculated from optical

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
1 3
Page 9 of 17 33
density in the 96-well microtiter plate were mostly lower
than those derived from tracking Chl a content in the cul-
ture flasks, with the largest deviation of ~ 35% in Nostoc
entophytum sp. C1.8 and Nostoc punctiforme sp. 73.1
grown at N-replete conditions, at LC and HC respectively.
In Nodularia species, the largest deviations in recorded
growth rates were 28% for N. spumigena NSBL206 and
17% for N. spumigena CCY9414, grown diazotrophically,
at LC and HC respectively, with higher growth rates deter-
mined when optical density was used to track growth.
The endpoint biomass dry weight (µg/ml) indicates a
general trend of increasing biomass under HC conditions
compared to LC growth controls (Supp. Table1). Nostoc
species increased their biomass significantly under HC
conditions under both diazotophic (170.7 ± 52.2µg ml−1)
and N-replete (288.0 ± 94.6 µg ml−1) conditions, while
Nodularia species. showed a non-significant trend
towards elevated biomass under HC conditions under
both diazotophic (534.0 ± 343.0µg ml−1) and N-replete
(622.0 ± 333.7µg ml−1) conditions.
Eect ofelevated CO2 onphotosynthesis
A statistically significant increase in net photosynthesis
was observed in all species tested at HC compared to the
LC control cultures (Fig.2), except for Nodularia har-
veyana SAG 44.85 which displayed a significant reduction
(p ≤ 0.005) in photosynthesis at HC. This effect was inde-
pendent of inorganic nitrogen in the growth media being
present or absent in Nostoc punctiforme sp. 73.1. Overall,
photosynthesis rates pooled for all Nostoc species over all
treatments was significantly higher than pooled for Nodu-
laria species (F = 36.3, n = 92/22, p ≤ 0.001). There was
a trend towards elevated dark respiration at HC in Nostoc
punctiforme sp. 73.1 and Nodularia harveyana SAG 44.85
grown diazotrophically (Suppl. Fig.5). For Nodularia spu-
migena NSBL206 this trend was statistically significant
(Suppl. Fig.5).
(a)
Net photosynthesis
[µmol C (µg chl a -1 s-1)]
0.0
0.5
1.0
1.5
2.0
Net photosynthesis
Nodularia harveyana 44.85_N-
[µmol C (µg chl a -1 s-1)]
0.00
0.01
0.02
0.03
0.04
0.05
LC
HC
**
**
***
*
*
*
(b)
Nostoc punctiforme 73.1_N-
Nostoc punctiforme 73.1_N+
Nostoc punctiforme 40.5_N+
Nostoc muscorum 65.1_N+
Nostoc entophytum C1.8_N+
Nodularia spumigena CCY9414_
N-
Nodularia spumigena NSBL206_N
-
Nodularia harveyana 44.85_N-
Response ratio (lnRR) of
net photosynthesis
-1
0
1
2
3
4
**
Fig. 2 a Net photosynthesis (NP) in control cultures (LC-440ppm)
and cultures grown at elevated CO2 (HC-2000ppm). Bars represent
mean and standard deviation of four measurements (day 7 + day 14).
Second y-axis (RHS) refers to Nodularia harveyana SAG 44.85. Sig-
nificant differences between measurements are indicated by *p ≤ 0.05,
** p ≤ 0.005, ***p ≤ 0.001 according to repeated -measure ANOVA
and b Response ratio of net photosynthesis was calculated by deter-
mining the natural logarithm of the ratio of the NP rate of HC cul-
tures by those of cultures grown at LC (lnRR). Data is presented as
means and 95% confidence interval (CI). The horizontal grey line
indicates lack of response to the CO2 treatment (i.e. RR = 0). If the CI
crossed the 0 response line, the effect of elevated CO2 is considered
as non-significant. Mean and CI > 0 indicate a stimulation by elevated
CO2. Mean and CI < 0 indicate a negative effect of elevated CO2

N.Wannicke et al.
1 3
33 Page 10 of 17
Eect ofelevated atmospheric CO2 onnodularin
production perculture volume
Comparing dry weight volume specific biomass nodularin
content revealed a statistically significant impact of HC
growth conditions in two of the three toxin producing spe-
cies, Nostoc punctiforme sp. 73.1 and Nodularia spumigena
CCY9414. A significant decrease in nodularin content per
dry weight was observed at elevated CO2 in Nostoc punc-
tiforme sp. 73.1 when grown diazotrophically, while there
was no significant impact in nitrogen containing growth
media (Fig.3a). No significant impact of HC on nodularin
content was detectable in Nostoc muscorum sp. 65.1, regard-
less of N availability (Fig.3b). A significant decrease in dry
weight specific nodularin content was apparent for Nodu-
laria spumigena CCY9414 when grown under diazotrophic
or N-replete conditions (Fig.3c) at HC.
The effect of elevated CO2 on nodularin production was
strongly influenced by the normalisation of actual toxin
concentrations. When comparing volume specific nodularin
concentration (ng nodularin per culture volume analysed) a
strong increase in nodularin production was observed at ele-
vated CO2 for Nostoc punctiforme sp. 73.1 under N-replete
conditions (N+) when compared to diazotrophically grown
Nostoc punctiforme sp. 73.1, while there was only a slight
increase for Nodularia spumigena CCY9414 (Suppl. Fig.6)
cultured diazotrophically. Dry weight specific nodularin
content was decreased in diazotrophically grown Nostoc
punctiforme sp. 73.1 in contrast to the decrease observed in
nodularin levels of Nodularia spumigena CCY9414 grown
under both nutrient conditions (Fig.3).
Eect onN2 xation
N2 fixation rates in both Nodularia species were signifi-
cantly elevated by a factor of 6 for biomass specific rates
(t = 3.85, p = 0.001, n = 24 for Nodularia/n = 4 for Nostoc)
and 17 for volume specific rates (t = 2.83, p = 0.008, n = 24
for Nodularia/n = 4 for Nostoc, Fig.4) when compared to
rates determined for Nostoc punctiforme sp. 73.1. These
rates should be interpreted carefully, especially for Nostoc
punctiforme sp. 73.1, due to the low number of replicates
(n = 2). Moreover, volume specific N2 fixation rates were
significantly higher for Nodularia spumigena CCY9414 and
Nodularia harveyana SAG 44.85 under HC growth condi-
tions when compared to the LC cultures (F = 34.9, p = 0.01,
n = 4 and F = 11.7, p = 0.04, n = 4), respectively (Fig. 4a).
There was no trend in N2 fixation in Nodularia spumigena
NSBL206.
N2 fixation rates normalized to particulate organic nitro-
gen were significantly decreased in the two non-toxin pro-
ducing Nodularia species, Nodularia harveyana SAG 44.85
and Nodularia spumigena NSBL206 at elevated pCO2 (HC)
(Fig.4a). This may reflect the significant increase (p ≤ 0.05)
in PON levels recorded for all three Nodularia cultures under
HC conditions, which were elevated by a factor of 3–6.5 under
HC conditions (Suppl. Fig.7a). The BNF rates determined for
the Nostoc punctiforme sp. 73.1 (n = 2) showed no definitive
trend at HC growth conditions as seen for Nodularia spumi-
gena CCY9414 and Nodularia harveyana SAG 44.85, at HC.
Modulation ofCO2 response innodularin
andnon‑nodularin producer
Weighted mean effect sizes for the subgroups, nodularin
producer and non-nodularin producer, were positive for NP
Nodularin per biomass [ng x µg dry weight -1]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
LC_N+
H
C_N+
LC N-
HCN-
0.0
0.2
0.4
0.6
0.8
1.0
1.2
(a)
Nostoc sp. 73.1
(b) Nostoc sp. 65.1
(c) Nodularia spum igena CCY9414
n.d.
a
a
b
c
b
a
b
a
aa
a
Fig. 3 Total soluble cellular nodularin content per µg dry weight for
control cultures (LC-440ppm) and cultures grown at elevated CO2
(HC-2000ppm). Values represent mean and standard deviation deter-
mined after 14days of incubation (n = 3). Significant differences are
indicated by different letters to the level of p ≤ 0.05 according to one-
way ANOVA

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
1 3
Page 11 of 17 33
indicating a positive effect of the high CO2 treatment on
net photosynthesis. Nodularin producing Cyanobacteria dis-
played a significantly higher positive response towards HC
compared to non- nodularin producers (p ≤ 0.006, Student’s
t test, Fig.5). Weighted mean effect sizes of dark respiration
differed in the two subgroups. Nodularin producers showed
no significant effect of HC for dark respiration (overlap
of confidence interval with 0-response line, Fig.5), while
non-nodularin producer displayed a positive mean effect
size. Weighted mean effect sizes of growth rates showed a
significant difference in-between the two subgroups. Mean
effect size of non-nodularin producing Cyanobacteria did
not deviate from the 0-response line indicating no effect of
CO2 treatment on growth, while the nodularin producers
displayed a significant positive response to HC. Contrast-
ing patterns were visible for N2 fixation, depending on the
mode of normalisation of rates, namely volume or PON.
Weighted mean effect sizes of biomass specific N2 fixation
showed opposing directions and a significant difference in
the subgroups (p ≤ 0.001, Student’s t test) with a significant
decrease in N2 fixation at HC in the non-nodularin producer
and a no effect on nodularin producer (overlap of confidence
interval with 0- response line, Fig.5). Mean effect sizes of
volume specific N2 fixation rates showed an opposing trend.
Non-nodularin producers displayed significantly elevated
BNF rates at HC, while nodularin producers displayed a
non-significant increase at HC.
Discussion
The recent report by the Intergovernmental Panel on Cli-
mate Change indicates that CO2 emission rates are not
being reduced as rapidly as desired, suggesting levels of
CO2 which will most likely exceed 1000ppm by the year
2100 for the worst- case-scenario (RCP8.5) (IPCC 2019).
Nostoc punctiform e 73.1_N-
LC HC
0.00
0.03
0.06
0.09
0.12
0.15
0.18
NodulariaspumigenaCCY9414_N-
NodulariaharveyanaSAG44.85_N
-
NodulariaspumigenaNSBL206_N-
N2 fixation [nmol N (µmol PON-1 h-1)]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
LC
HC
(d)
(c)
*
*
0
5
10
15
20
25
N2 fixation [nmol N (L-1 h-1)]
0
200
400
600
800
*
(b)
(a)
*
Fig. 4 Volume specific and biomass specific N2 fixation rates deter-
mined for Nodularia sp. (a, c) and Nostoc punctiforme sp. 73.1 (b,
d) grown at LC (440ppm) and HC (2000ppm). Bars in a and c rep-
resent mean and standard deviation of four replicates, bars in b and
d represent single measurements. Significant differences between
treatments are indicated by *p ≤ 0.05 according to repeated -measure
AN OVA

N.Wannicke et al.
1 3
33 Page 12 of 17
Recent reviews have summarised the literature regarding the
response of marine and freshwater bloom forming Cyano-
bacteria to elevated CO2 levels (Huisman etal. 2018; Visser
etal. 2016; Wannicke etal. 2018b), suggesting an increase
in bloom formation, possibly favouring diazotrophs, as
they would be less susceptible to N-limitation (Gehringer
and Wannicke 2014). While numerous studies have been
conducted on toxin and non-toxin producing Microcys-
tis aeruginosa strains under elevated CO2 conditions, lit-
tle data has been accumulated as to the effects on diazo-
trophic Cyanobacteria, especially terrestrial Nostoc species
(Gehringer and Wannicke 2014). In this study, the effects
of elevated atmospheric CO2 on various growth parameters
of six species of diazotrophic, heterocystous Cyanobacteria
of two families of the order Nostocales were assessed. The
weighted mean effect sizes (Cohens’s d) of three nodularin
producing Cyanobacteria were compared to four non-nodu-
larin synthesising Cyanobacteria with respect to NP, growth
and BNF rates (Fig.5). Nodularin producers tended to have
higher NP rates under HC conditions than non-toxin pro-
ducers, accompanied by increased growth rates. Non-toxic
Cyanobacteria in contrast showed increased respiration at
HC conditions, suggesting increased respiratory or oxida-
tive stress. This effect was largely driven by the negative
response of Nostoc punctiforme sp. 73.1. Due to the lack of
individual species replication, these investigations need to be
repeated to make statistically robust conclusions. The BNF
rates of both nodularin producers and non -toxic species
were unaffected by HC growth conditions when normalised
against PON given that the 95% confidence interval crosses
the zero line in the Cohens’s d plot (Fig.5), while toxin
producers had slightly lower rates. This trend appeared
inversed if BNF were normalised against culture volume,
where non-nodularin producers were negatively affected
by HC, while nodularin producers showed no effect. These
trends suggested that nodularin producing diazotrophs did
indeed respond differently to changing elevated CO2 levels
compared to non-nodularin producers. We then proceeded
to investigate these results in more detail at the individual
genus, and species level.
Response ofelevated CO2 onNP andgrowth
All aquatic and terrestrial diazotrophic Cyanobacteria inves-
tigated in this study fixed atmospheric CO2 in the range of
0.1–1.5µmol C. ng Chl a−1 s−1, whether under N-replete or
diazotrophic conditions. Additionally, our study has con-
firmed that most Nostoc species and Nodularia spumigena,
grown at 2000ppm CO2, have the capacity for significantly
higher NP at HC conditions (Fig.2), indicating that they are
not functioning at saturation under current atmospheric lev-
els of CO2. The benthic species, N. harveyana SAG 44.85,
however showed a significant reduction in NP with HC. A
similarity search conducted using BLASTn (Altschul etal.
1990) on the genome of Nodularia spumigena CCY9414,
found that this strain carries a gene (NSP_RS09630) with
75% identitity to the BicA gene of Microcystis aerugi-
nosa PCC7806, thereby suggesting that it can benefit from
increased HCO3− in the media and thus, increase its NP
rates accordingly (Visser etal. 2016). Gas exchange meas-
urements were used to assess NP (Herrmann and Gehringer
2019) as most Cyanobacteria are known to encode the CO2
converting enzymes, NDH-I4 and NDH-I3 (Visser etal.
2016), necessary for the direct, non-energy demanding con-
version of CO2 to bicarbonate for transport to the carboxy-
some. As non-sequenced environmental isolates were used,
there was no information regarding the status of bicarbonate
transporters of all the Cyanobacteria under investigation.
Alkalisation of the culture medium of Nostoc species
grown under N-replete conditions at both HC and LC cul-
ture conditions was recorded, whereas alkalisation was only
seen for Nodularia grown under N-replete conditions at LC
conditions (Supp. Table1). The changes in pH do not follow
the observed changes in biomass, suggesting species specific
responses to changes in pH and dissolved inorganic carbon
availability.
In our study, a CO2 enriched atmosphere led to an
increase in inorganic carbon in the control media flasks,
while alkalinity was kept constant and pH decreased.
Often discussed is the effect of reduced pH by adding acid
to keep inorganic carbon stable while total alkalinity and
pH decrease. For example, a study by Berge etal. (2010)
NP
Dark resp.
N2fix.pervol
N
2fix.perPON
µ
Mean effect size-
Cohen´s d
-1
0
1
2
3
Nodularin producer
Non -nodularin producer
Fig. 5 Weighted mean effect sizes (Cohen’s d) of net photosynthe-
sis (NP), respiration, growth and N2 fixation for the two subgroups
“Nodularin producer” (black circles, for details on Cyanobacteria
see Fig.1a) and “Non-nodularin producer” (white circles, for details
on Cyanobacteria see Fig.1a). Data is presented as means and 95%
confidence interval (CI). The horizontal grey line indicates lack of
response to the CO2 treatment (i.e. mean effect size = 0). If the CI
crossed the 0 response line, the effect of elevated CO2 is considered
as non-significant. Mean and CI > 0 indicate a stimulation by elevated
CO2. Mean and CI < 0 indicate a negative effect of elevated CO2

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
1 3
Page 13 of 17 33
showed that phytoplankton of the genera dinoflagellates,
cryptophytes, diatom and prymnesiophyte were resistant
in terms reduced pH and did not increase or decrease their
growth rates according to ecological relevant ranges of pH
from 7.0 to 9.0. More recently, the response of Raphidiop-
sis raciborskii to changes in pH and inorganic carbon in
water was assessed (Vilar and Molica 2020). The growth of
the Cyanobacterium, R. raciborskii was increased with the
addition of sodium carbonate and air bubbling, however,
saxitoxin production was reduced. Additionally, the authors
observed that pH changes were related to significant changes
in cellular saxitoxin levels. In general, the potential effect of
pH changes on neither the growth, nor nodularin production,
of the Nostocales strains investigated in our study, have been
characterised and published in the past.
Especially the Baltic Sea is depleted with CO2 during
summer time due to the high draw down of dense phyto-
plankton blooms with high photosynthetic activity (e.g.
Huisman etal. 2018), as well as poor diffusion of CO2 in
water and the slow equilibrium between CO2 and HCO3 (e.g.
Ibelings and Maberly 1998). Photosynthesis in biocrusts
is also often limited by CO2 availability, especially when
flooded or desiccated (Tuba etal. 1998; Jauhiainen and Sil-
vola 1999; Lange 2002; Botting and Fredeen 2006; Toet
etal. 2006).
The reduction in NP seen for the benthic Nodularia har-
veyana SAG 44.85, suggests that it may tightly regulate
its Ci uptake mechanisms, as elevated CO2 levels of up to
3000µatm can occur in benthic layers due to high organic
matter decomposition and remineralisation (Haynert etal.
2012). The capacity to tightly regulate C assimilation is an
important prerequisite for reducing respiratory stress in this
environment. The increase in dark respiration in N. harvey-
ana SAG44.85 suggests it may indeed be under respiratory
stress at HC conditions.
Nodularin production underelevated atmospheric
CO2 exposure
Intracellular nodularin content showed significant variation
dependent on the Cyanobacterium and/ or atmospheric CO2
content (Fig.3). Nostoc sp. 65.1 appears to constitutively
produce nodularin at low levels, independent of medium N
content and atmospheric CO2 levels. This Nostoc species
also exhibited the lowest growth rate overall (Supplementary
Table2), which prevented the generation of sufficient bio-
logical biomass under diazotrophic growth conditions for NP
and BNF determinations. The nodularin content of Nostoc
sp. 73.1 was significantly raised under diazotrophic growth
conditions at both atmospheres. NP rates (Fig.2) and growth
rates (Supplementary Table2) of Nostoc sp. 73.1 exceeded
those of Nostoc sp. 65.1 under N-replete conditions, factors
that may contribute to its higher levels of intracellular toxin
production under diazotrophic conditions.
Nodularia spumigena CCY 9414 exhibited a very dif-
ferent nodularin synthesis profile, with cultures grown at
LC conditions containing significantly more nodularin per
dry weight than the cultures in HC conditions, irrespective
of medium N content (Fig.3). The high NP rates observed
under HC conditions (Fig.2), combined with the increased
growth rates at HC under diazotrophic conditions sug-
gest that the cells were N depleted and thus not expending
resources to produce nodularin. However, the particulate
organic nitrogen levels of the HC grown cultures (Supple-
mentary Fig.7) suggest that the cells were not N-depleted
and that some other regulatory mechanism was suppressing
nodularin synthesis, compared to the LC cultures.
Biological nitrogen fixation
All Nodularia investigated exhibited significant increases
in their culture volume PON content under HC culture con-
ditions (Supplementary Fig.7). However, increased BNF
rates per volume were only significantly raised (p ≤ 0.05)
for Nodularia spumigena CCY 9414 and Nodularia har-
veyana SAG 44.85 under HC, while those for N. spumi-
gena NSBL06 remained unaffected. If the BNF rates are
expressed per PON content, the LC exposed cultures of Nod-
ularia harveyana SAG 44.85 and N. spumigena NSBL06
are significantly higher (p ≤ 0.05) than HC grown cultures
(Fig.4), suggesting inhibition of BNF under HC conditions.
A recent study (Boatman etal. 2019) found that dark res-
piration rates were up to 5 times higher in Trichodesmium
erythraeum IMS101cultures exposed to elevated levels of
CO2 (720μmol mol–1). While increases in dark respiration
were observed for the non-toxin producing Nodularia spu-
migena NSBL06 and Nodularia harveyana SAG 44.85 (Sup-
plementary Fig.5), no increase in BNF rates per PON were
recorded (Fig.4).
Nodularia spumigena CCY9414 and Nodularia har-
veyana SAG 44.85 exhibited significantly raised volume
specific N2 fixation rates under HC culture conditions, as
observed for N. spumigena CCY9414 at 548ppm CO2
by Wannicke etal. (2012). Czerny etal. (2009) reported
negative effects of HC on cell specific N2 fixation rates
for Nodularia spumigena IOW-2000/1 at 16°C, whereas
Eichner etal. (2014) found no significant changes in the
same nodularin producing species exposed to elevated
CO2 through continual bubbling of the cultures. Whether
these inconsistencies reflect differences in species selec-
tion, culture conditions or method of monitoring of N2
fixation rates can only be determined with repeat experi-
ments under the identical conditions, preferably related to
the ecological environment being investigated. A review of
published data of N2 fixation rates in relation to CO2 gave

N.Wannicke et al.
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33 Page 14 of 17
evidence for a global positive but non- significant mean
effect size for heterocystous species from marine, brackish,
and limnic environments (Wannicke etal. 2018b).
When combined with the C assimilation data previously
presented, we propose that both the Nodularia spumigena
strains are capable of immediately responding to elevated
CO2 levels and rapidly increasing their NP rates (Fig.2).
Additionally, they are capable of increasing their biomass
with respect to particulate to PON (Supplementary Fig.7)
in the system under HC conditions, thereby making a sig-
nificant contribution to the primary productivity in the sys-
tem. Nodularia harveyana SAG 44.85 appears to be able
to regulate NP under HC conditions (Fig.2), a trait essen-
tial for survival in the organic rich benthic zone (Haynert
etal. 2012), but still exhibits a significant contribution
to the PON (Supplementary Fig.7) at the elevated CO2
conditions investigated in this study.
Significantly, the average N2 fixation rate of the Nostoc
punctiforme sp. 73.1 at both LC and HC was 6–17 times
lower than that recorded for the Nodularia species inves-
tigated, although the low number of repetitions prevents a
generalisation of the observed trend (Fig.4).
This study supports the observation of phenotypic
plasticity of carbon fixation rates observed for aquatic
freshwater Microcystis cultures grown under elevated
CO2 conditions of 1000ppm (Ji etal. 2020). While the
Nostoc species responded to HC with increased NP rates
(Fig.2), Nostoc punctiforme sp. 73.1 most likely did not
invest in the highly energy demanding process of N2
fixation (Fig.4) under N limitation as observed for the
aquatic Nodularia species studied. To speculate on a gen-
eral pattern, however, N2 fixation measurements have to be
repeated for all Nostoc species. The PON results (Supple-
mentary Fig.7) also suggest that Nostoc sp. sp. 73.1 would
not increase its contribution to N availability in its direct
vicinity, thereby possibly offering an explanation as to the
overall reduction in Cyanobacterial biomass observed in
dryland soilcrusts exposed to HC of 550ppm for 10years
(Steven etal. 2012). This negative effect of exposure to HC
highlights the complexity of dryland biocrust systems and
their response to climate change (Reed etal. 2016) may
have been the result of reduced BNF and supply of PON
to the system. In contrast, an increase in N2 fixation was
observed in earlier studies in cultures of the Nostoc punc-
tiforme CPCC41 when grown at elevated CO2 levels of
940ppm, about half of the CO2 used in this investigation
(Lindo etal. 2017). Further examination of the survival
strategies of these important terrestrial primary producers
will offer greater insights into the nutrient partitioning and
growth strategies, especially under elevated atmospheric
CO2 levels. Additionally, further research into the phe-
notypic plasticity of carbon fixation within the complex
filamentous diazotrophs studied here is crucial to under-
stand the effects of climate change on Cyanobacterial pri-
mary productivity under future climate change scenarios.
Conclusion
Our study demonstrates species and strain specific vari-
ations to elevated atmospheric CO2 levels. Interestingly,
our data suggests that nodularin producers have, on aver-
age, higher NP rates than non-nodularin producers under
HC conditions, with lower respiration rates. HC growth
conditions induce increases in BNF rates and PON levels
per volume of cultures of Nodularia spumigena CCY9414
and N. harveyana SAG 44.85 species, while Nostoc BNF
rates are seemingly unaffected. Unexpectedly, the com-
bined BNF of all Nostoc sp. sp. 73.1 determined for LC
and HC are significantly lower than those for all Nodularia
species tested.
A correlation was observed between HC growth condi-
tions and a decrease in nodularin production under diazo-
trophic conditions for Nodularia CCY9414 and Nostoc sp.
sp. 73.1 (Fig.3), with Nostoc sp. 73.1 showing increased
nodularin content under diazotrophic conditions and Nodu-
laria spumigena CCY9414 under LC conditions. Future
studies using similar toxin and non-toxin producing Cyano-
bacteria for which genomic sequence data exists, need to
be undertaken under identical conditions to further eluci-
date the effects of elevated CO2 on Cyanobacterial cellu-
lar metabolism, and the role of secondary metabolites, like
nodularin, in mediating the cellular responses to future cli-
mate change conditions. This study would suggest that toxin-
producing diazotrophs may be less advantaged under cur-
rent climate change predictions in diazotrophic conditions,
due to impaired N2 fixation under elevated CO2 conditions,
when compared with similar non-toxin producing species of
Cyanobacteria. On the other hand, a higher positive response
in NP may outbalance this effect at elevated CO2.
Supplementary Information The online version contains supplemen-
tary material available at https ://doi.org/10.1007/s0002 7-021-00788 -6.
Acknowledgements N.W. thankfully acknowledges the financial sup-
port by the Project BIOACID of the German Federal Ministry of Edu-
cation and Research [BMBF, FKZ 03F0728F]. M. G. funded by the
German Research Foundation [DFG GE 2558/3-1 under the SPP1833].
We wish to convey our gratitude to B. Büdel, C. Colesie and E. Neu-
haus (TU Kaiserslautern, Germany) for providing experimental facili-
ties and expertise, and to Iris Liskow (The Leibniz Institute for Baltic
Sea Research, Germany) for determination of stable isotopes and POM
concentrations.
Funding Open Access funding enabled and organized by Projekt
DEAL.

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
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Page 15 of 17 33
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Conflict of interest The authors have no conflicts of interest to declare.
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References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic
local alignment search tool. J Mol Biol 215:403–410
Berge T, Daugbjerg N, Andersen BB, Hansen PJ (2010) Effect of
lowered pH on marine phytoplankton growth rates. Mar Ecol
Prog Ser 416:79–91
Beversdorf LJ, Miller TR, McMahon KD (2013) The role of nitro-
gen fixation in Cyanobacterial bloom toxicity in a temperate,
Eutrophic Lake. PLoS ONE 8:e56103
Bhargava S, Chouhan S, Kaithwas V, Maithil R (2013) Carbon diox-
ide regulation of autotrophy and diazotrophy in the nitrogen-
fixing Cyanobacterium Nostoc muscorum. Ecotox Environ Safe
98:345–351
Boatman TG, Davey PA, Lawson T, Geider RJ (2019) CO2 modu-
lation of the rates of photosynthesis and light-dependent O2
consumption in Trichodesmium. J Exp Bot 70:589–597
Bolch CJS, Orr PT, Jones GJ, Blackburn SI (1999) Genetic, morpho-
logical, and toxicological variation among globally distributed
strains of Nodularia Cyanobacteria. J Phycol 35:339–355
Botting RS, Fredeen AL (2006) Net ecosystem CO2 exchange for
moss and lichen dominated forest floors of old-growth sub-boreal
spruce forests in central British Columbia, Canada. Forest Ecol
Manage 235(1–3):240–251
Briand E, Yepremian C, Humbert JF, Quiblier C (2008) Competition
between microcystin-and non-microcystin-producing Plankto-
thrix agardhii (Cyanobacteria) strains under different environ-
mental conditions. Environ Microbiol 10:3337–3348
Buratti FM, Manganelli M, Vichi S, Stefanelli M, Scardala S, Testai
E, Funari E (2017) Cyanotoxins: producing organisms, occur-
rence, toxicity, mechanism of action and human health toxico-
logical risk evaluation. Arch Toxicol 91:1049
Burnap RL, Hagemann M, Kaplan A (2015) Regulation of CO2 con-
centrating mechanism in Cyanobacteria. Life 5(1):348–371
Czerny J, Barcelos e Ramos J, Riebesell U (2009) Influence of ele-
vated CO2 concentrations on cell division and nitrogen fixation
rates in the bloom-forming Cyanobacterium Nodularia spumi-
gena. Biogeosciences (BG) 6:1865–1875
DerSimonian R, Laird N (1986) Meta-analysis in clinical trials. Con-
trol Clin Trials 7:177–188
Dickson AG, Sabine CL, Christian JR (2007) Guide to best practices
for ocean CO2 measurements. North Pacific Marine Science
Organization (PICES Special Publication, 3), Sidney, p 176
Dittmann E, Erhard M, Kaebernick M, Scheler C, Neilan BA,
von Döhren H, Börner T (2001) Altered expression of two
light-dependent genes in a microcystin-lacking mutant of Micro-
cystis aeruginosa PCC 7806. Microbiol 147(11):3113–3119
Downing TG, Sember CS, Gehringer MM, Leukes W (2005) Medium
N:P ratios and specific growth rate comodulate microcystin and
protein content in Microcystis aeruginosa PCC7806 and M. aer-
uginosa UV027. Microbial Ecol 49:468–473
Eichner M, Rost B, Kranz SA (2014) Diversity of ocean acidification
effects on marine N2 fixers. J Exp Mar Biol Ecol 457:199–207
Elbert W, Weber B, Burrows S, Steinkamp J, Büdel B, Andreae
MO, Pöschl U (2012) Contribution of cryptogamic covers to
the global cycles of carbon and nitrogen. Nat Geosci 5:459–462
El-Shehawy R, Gorokhova E, del Fernandez-Pinas F, Campo FF
(2012) Global warming and hepatotoxin production by Cyano-
bacteria: what can we learn from experiments? Water Res
46:1420–1429
Gao K, Yu A (2000) Influence of CO2, light and watering on growth
of Nostoc flagelliforme mats. J Appl Phycol 12:185
Gehringer MM (2004) Microcystin-LR and okadaic acid-induced
cellular effects: a dualistic response. FEBS Lett 557:1–8
Gehringer MM, Wannicke N (2014) Climate change and regulation
of hepatotoxin production in Cyanobacteria. Fems Microbiol
Ecol 88:1–25
Gehringer MM, Pengelly JJL, Cuddy WS, Fieker C, Forster PI, Nei-
lan BA (2010) Host selection of symbiotic Cyanobacteria in
31 species of the Australian cycad genus: Macrozamia (Zami-
aceae). Mol Plant Microbe Interact 23:811–812
Gehringer MM, Adler L, Roberts AA, Moffitt MC, Mihali TK, Mills
TJ, Fieker C, Neilan BA (2012) Nodularin, a Cyanobacterial
toxin, is synthesized in planta by symbiotic Nostoc sp. The
ISME J 6:1834–1847
Haynert K, Schönfeld J, Polovodova-Asteman I, Thomsen J (2012)
The benthic foraminiferal community in a naturally CO2-rich
coastal habitat in the southwestern Baltic Sea. Biogeosciences
(BG) 9:4421–4440
Herrmann AJ, Gehringer MM (2019) An investigation into the
effects of increasing salinity on photosynthesis in freshwater
unicellular Cyanobacteria during the late Archaean. Geobiol-
ogy 17:343–359
Ho JC, Michalak AM, Pahlevan N (2019) Widespread global increase
in intense lake phytoplankton blooms since the 1980s. Nature
574:667–670
Horst GP, Sarnelle O, White JD, Hamilton SK, Kaul RB, Bressie JD
(2014) Nitrogen availability increases the toxin quota of a harmful
Cyanobacterium, Microcystis aeruginosa. Water Res 54:188–198
Huisman J, Codd GA, Paerl HW, Ibelings BW, Verspagen JMH,
Visser PM (2018) Cyanobacterial blooms. Nat Rev Microbiol
16:471–483
Ibelings BW, Maberly SC (1998) Photoinhibition and the availability
of inorganic carbon restrict photosynthesis by surface blooms of
Cyanobacteria. Limnol Oceanogr 43(3):408–419
Ibelings BW, Backer LC, Kardinaal WEA, Chorus I (2015) Current
approaches to cyanotoxin risk assessment and risk management
around the globe. Harmful Algae 49:63–74
IPCC. (2019) Intergovernmental panel on climate change. Special
report on global warming of 1.5°C (SR15)
Jauhiainen J, Silvola J (1999) Photosynthesis of Sphagnum fuscum at
long-term raised CO2 concentrations. In: Annales Botanici Fennici
(pp. 11–19). Finnish Zoological and Botanical Publishing Board
Ji X, Verspagen JM, Van de Waal DB, Rost B, Huisman J (2020) Phe-
notypic plasticity of carbon fixation stimulates Cyanobacterial
blooms at elevated CO2. Sci Adv 6(8):eaax2926
Johnson KM, Wills KD, Butler DB, Johnson WK, Wong CS (1993)
Coulometric total carbon dioxide analysis for marine studies:
maximizing the performance of an automated gas extraction sys-
tem and coulometric detector. Mar Chem 44:167–187

N.Wannicke et al.
1 3
33 Page 16 of 17
Jonasson S, Vintila S, Sivonen K, El-Shehawy R (2008) Expression
of the nodularin synthetase genes in the Baltic Sea bloom-former
Cyanobacterium Nodularia spumigena strain AV1. FEMS Micro-
biol Ecol 65:31–39
Karlberg M, Wulff A (2013) Impact of temperature and species interac-
tion on filamentous Cyanobacteria may be more important than
salinity and increased pCO2 levels. Mar Biol 160:2063–2072
Kleinteich J, Wood SA, Küpper FC, Camacho A, Quesada A, Fric-
key T, Dietrich DR (2012) Temperature-related changes in polar
Cyanobacterial mat diversity and toxin production. Nat Clim
Change 2:356–360
Kranz SA, Eichner M, Rost B (2011) Interactions between CCM and
N2 fixation in Trichodesmium. Photosynth Res 109:73–84
Lange OL (2002) Photosynthetic productivity of the epilithic lichen
Lecanora muralis: long-term field monitoring of CO2 exchange
and its physiological interpretation. I. Dependence of photosyn-
thesis on water content, light, temperature, and CO2 concentration
from laboratory measurements. Flora-Morphol Distrib Funct Ecol
Plants 197(4):233–249
Lane RW, Menon M, McQuaid JB, Adams DG, Thomas AD, Hoon
SR, Dougill AJ (2013) Laboratory analysis of the effects of
elevated atmospheric carbon dioxide on respiration in biological
soil crusts. J Arid Environ 98:52–59
Lewis E, Wallace D, Allison LJ (1998) Program developed for CO2
system calculations. Environmental Sciences Division Publica-
tion No. 4735; Carbon Dioxide Information Analysis Center,
Oak Ridge National Laboratory, Oak Ridge, TN, USA
Lindo Z, Griffith DA (2017) Elevated atmospheric CO2 and warm-
ing stimulates growth and nitrogen fixation in a common forest
floor Cyanobacterium under axenic conditions. Forests 8(3):73
Lines T, Beardall J (2018) Carbon acquisition characteristics of six
microalgal species isolated from a subtropical reservoir: poten-
tial implications for species succession. J Phycol 54:599–607
Liu J, Van Oosterhout E, Faassen EJ, Lurling M, Helmsing NRV,
de Waal DB (2016) Elevated pCO2 causes a shift towards more
toxic microcystin variants in nitrogen-limited Microcystis aer-
uginosa. Fems Microbiol Ecol 92:fiv159
Lyons TW, Reinhard CT, Planavsky NJ (2014) The rise of oxygen
in Earth’s early ocean and atmosphere. Nature 506:307–315
Lyra C, Laamanen M, Lehtimäki JM, Surakka A, Sivonen K (2005)
Benthic Cyanobacteria of the genus Nodularia are non-toxic,
without gas vacuoles, able to glide and genetically more
diverse than planktonic Nodularia. Int J Syst Evol Microbiol
55:555–568
Ma J, Wang P, Wang X, Xu Y, Paerl HW (2019) Cyanobacteria in
eutrophic waters benefit from rising atmospheric CO2 concen-
trations. Sci Total Environ 691:1144–1154
Meeks JC, Castenholz RW (1971) Growth and photosynthesis in
an extreme thermophile, Synechococcus lividus (Cyanophyta).
Arch Mikrobiol 78:25–41
Moffit CM, Neilan BA (2004) Characterization of the nodularin
synthetase gene cluster and proposed theory of the evolu-
tion of Cyanobacterial hepatotoxins. Appl Environ Microb
70:6353–6362
Moffitt MC, Blackburn SI, Neilan BA (2001) rRNA sequences reflect
the ecophysiology and define the toxic Cyanobacteria of the genus
Nodularia. Int J Syst Evol Microbiol 51:505–512
Montoya JP, Voss M, Kahler P, Capone DG (1996) A simple, high-
precision, high-sensitivity tracer assay for N2 fixation. Appl Envi-
ron Microb 62:986–993
Neilan BA, Pearson LA, Muenchhoff J, Moffitt MC, Dittmann E (2013)
Environmental conditions that influence toxin biosynthesis in
Cyanobacteria. Environ Microbiol 15:1239–1253
O’Neil JM, Davis TW, Burford MA, Gobler CJ (2012) The rise of
harmful Cyanobacteria blooms: the potential roles of eutrophica-
tion and climate change. Harmful Algae 14:313–334
Orr PT, Willis A, Burford MA (2018) Application of first order
rate kinetics to explain changes in bloom toxicity—the impor-
tance of understanding cell toxin quotas. J Oceanol Limnol
36(4):1063–1074
Paerl HW, Huisman J (2009) Climate change: a catalyst for global
expansion of harmful Cyanobacterial blooms. Env Microbiol Rep
1:27–37
Pierangelini M, Sinha R, Willis A, Burford MA, Orr PT, Beardall J,
Neilan BA (2015) Constitutive cylindrospermopsin pool size in
Cylindrospermopsis raciborskii under different light and CO2 par-
tial pressure conditions. Appl Environ Microbiol 81(9):3069–3076
Posch T, Köster O, Salcher MM, Pernthaler J (2012) Harmful filamen-
tous Cyanobacteria favoured by reduced water turnover with lake
warming. Nat Clim Chang 2:809–813
Price GD (2011) Inorganic carbon transporters of the Cyanobacterial
CO2 concentrating mechanism. Photosynth Res 109:47–57
Raven JA, Johnston AM (1991) Mechanisms of inorganic-carbon
acquisition in marine phytoplankton and their implications for
the use of other resources. Limnol Oceanogr 36(8):1701–1714
Raven JA, Beardall J, Sánchez-Baracaldo P (2017) The possible evo-
lution, and future, of CO2-concentrating mechanisms. J Exp Bot
68:3701–3716
Raven JA, Gobler CJ, Hansen PJ (2020) Dynamic CO2 and pH levels
in coastal, estuarine and inland waters: theoretical and observed
effects on harmful algal blooms. Harmful Algae 91:101594
Reed SC, Maestre FT, Ochoa-Hueso R, Kuske CR, Darrouzet-Nardi A,
Oliver M, Darby B, Sancho LG, Sinsabaugh RL, Belnap J (2016)
Biocrusts in the context of global change. In: Weber B, Büdel B,
Belnap J (eds) Biological soil crusts: an organizing principle in
drylands. Springer International Publishing, Cham, pp 451–476
Řeháková K, Mareš J, Lukešová A, Zapomělová E, Bernardová K,
Hrouzek P (2014) Nodularia (Cyanobacteria, Nostocaceae): a
phylogenetically uniform genus with variable phenotypes. Phy-
totaxa 172:235–246
Ritchie RJ (2008) Fitting light saturation curves measured using modu-
lated fluorometry. Photosynth Res 96:201–215
Rodriguez-Caballero E, Belnap J, Büdel B, Crutzen PJ, Andreae
MO, Pöschl U, Weber B (2018) Dryland photoautotrophic soil
surface communities endangered by global change. Nat Geosci
11:185–189
Rost B, Riebesell U, Burkhardt S, Sültemeyer D (2003) Carbon acquisi-
tion of bloom-forming marine phytoplankton. Limnol Oceanogr
48(1):55–67
Sabine CL, Feely RA, Gruber N, Key RM, Lee K, Bullister JL
etal (2004) The oceanic sink for anthropogenic CO2. Science
305:367–371
Sandrini G, Matthijs HC, Verspagen JM, Muyzer G, Huisman J (2014)
Genetic diversity of inorganic carbon uptake systems causes vari-
ation in CO2 response of the Cyanobacterium Microcystis. The
ISME J 8:589–600
Sandrini G, Jakupovic D, Matthijs HCP, Huisman J (2015) Strains
of the harmful Cyanobacterium Microcystis aeruginosa differ in
gene expression and activity of inorganic carbon uptake systems
at elevated CO2 levels. Appl Environ Microb 81:7730–7739
Sandrini G, Ji X, Verspagen JMH, Tann RP, Slot PC, Luimstra VM,
Schuurmans JM, Matthijs HCP, Huisman J (2016) Rapid adapta-
tion of harmful Cyanobacteria to rising CO2. P Natl Acad Sci
USA 113:9315–9320
Sevilla E, Martin-Luna B, Bes MT, Fillat MF, Peleato ML (2012) An
active photosynthetic electron transfer chain required for mcyD
transcription and microcystin synthesis in Microcystis aeruginosa
PCC7806. Ecotoxicology 21:811–819
Shatwell T, Köhler J (2019) Decreased nitrogen loading controls sum-
mer cyanobacterial blooms without promoting nitrogen‐fixing
taxa: Long‐term response of a shallow lake. Limnol Oceanogr
64(S1):S166–S178

Atmospheric CO2 availability induces varying responses innet photosynthesis, toxin…
1 3
Page 17 of 17 33
Shi D, Kranz SA, Kim JM, Morel FMM (2012) Ocean acidification
slows nitrogen fixation and growth in the dominant diazotroph
Trichodesmium under low-iron conditions. P Natl Acad Sci USA
109:E3094–E3100
Steven B, Gallegos-Graves L, Yeager CM, Belnap J, Evans RD, Kuske
CR (2012) Dryland biological soil crust Cyanobacteria show
unexpected decreases in abundance under long-term elevated
CO2. Environ Microbiol 14:3247–3258
Suurmond R, van Rhee H, Hak T (2017a) Introduction, comparison
and validation of meta-essentials: a free and simple tool for meta-
analysis. Res Synth Methods 8:537–553
Suurmond R, van Rhee H, Hak T (2017b) Introduction, comparison,
and validation of meta-essentials: a free and simple tool for meta-
analysis. Res Synth Methods 8(4):537–553 (Accessed on 01 May
19)
Symes E, van Ogtrop FF (2019) The effect of pre-industrial and pre-
dicted atmospheric CO2 concentrations on the development of
diazotrophic and non-diazotrophic Cyanobacterium: Dolichos-
permum circinale and Microcystis aeruginosa. Harmful Algae
88:101536
Tuba Z, Protor CF, Csintalan Z (1998) Ecophysiological responses
of homoiochlorophyllous and poikilochlorophyllous desiccation
tolerant plants: a comparison and an ecological perspective. Plant
Growth Regul 24(3):211–217
Toet S, Cornelissen JH, Aerts R, van Logtestijn RS, de Beus M, Sto-
evelaar R (2006) Moss responses to elevated CO2 and variation in
hydrology in a temperate lowland peatland. In: Plants and climate
change. Springer, Dordrecht, pp 27–42
Van de Waal DB, Verspagen JM, Lürling M, Van Donk E, Visser
PM, Huisman J (2009) The ecological stoichiometry of toxins
produced by harmful Cyanobacteria: an experimental test of the
carbon-nutrient balance hypothesis. Ecol Lett 12:1326–1335
Van de Waal DB, Brandenburg KM, Keuskamp J, Trimborn S, Rokitta
S, Kranz SA, Rost B (2019) Highest plasticity of carbon-con-
centrating mechanisms in earliest evolved phytoplankton. Limnol
Oceanogr Lett 4(2):37–43
Van De Waal DB, Verspagen JMH, Finke JF etal (2011) Reversal in
competitive dominance of a toxic versus non-toxic Cyanobacte-
rium in response to rising CO2. ISME J 5:1438–1450
Vilar MCP, Molica RJR (2020) Changes in pH and dissolved inorganic
carbon in water affect the growth, saxitoxins production and tox-
icity of the Cyanobacterium Raphidiopsis raciborskii ITEP-A1.
Harmful Algae 97:101870
Visser PM, Verspagen JMH, Sandrini G, Stal LJ, Matthijs HCP, Davis
TW, Paerl HW, Huisman J (2016) How rising CO2 and global
warming may stimulate harmful Cyanobacterial blooms. Harmful
Algae 54:145–159
Voss B, Bolhuis H, Fewer DP etal (2013) Insights into the physiology
and ecology of the brackish-water-adapted Cyanobacterium Nod-
ularia spumigena CCY9414 based on a genome-transcriptome
analysis. PLoS ONE 8:e60224
Wannicke N, Endres S, Engel A, Grossart HP, Nausch M, Unger J,
Voss M (2012) Response of Nodularia spumigena to pCO2—
part 1: growth, production and nitrogen cycling. Biogeosciences
9:2973–2988
Wannicke N, Frey C, Law CS, Voss M (2018b) (2018b) The response
of the marine nitrogen cycle to ocean acidification. Glob Change
Biol 24:5031–5043
Wannicke N, Benavides M, Dalsgaard T, Dippner JW, Montoya JP,
Voss M (2018a) New perspectives on nitrogen fixation measure-
ments using 15N2 gas. Front Mar Sci 5:120
Yu L, Kong F, Shi X, Yang Z, Zhang M, Yu Y (2015) Effects of
elevated CO2 on dynamics of microcystin-producing and non-
microcystin-producing strains during Microcystis blooms. Int J
Environ Sci 27:251–258
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