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Elevated pH Conditions Associated With Microcystis spp. Blooms Decrease Viability of the Cultured Diatom Fragilaria crotonensis and Natural Diatoms in Lake Erie


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Cyanobacterial Harmful Algal Blooms (CyanoHABs) commonly increase water column pH to alkaline levels ≥9.2, and to as high as 11. This elevated pH has been suggested to confer a competitive advantage to cyanobacteria such as Microcystis aeruginosa. Yet, there is limited information regarding the restrictive effects bloom-induced pH levels may impose on this cyanobacterium’s competitors. Due to the pH-dependency of biosilicification processes, diatoms (which seasonally both precede and proceed Microcystis blooms in many fresh waters) may be unable to synthesize frustules at these pH levels. We assessed the effects of pH on the ecologically relevant diatom Fragilaria crotonensis in vitro, and on a Lake Erie diatom community in situ. In vitro assays revealed F. crotonensis monocultures exhibited lower growth rates and abundances when cultivated at a starting pH of 9.2 in comparison to pH 7.7. The suppressed growth trends in F. crotonensis were exacerbated when co-cultured with M. aeruginosa at pH conditions and cell densities that simulated a cyanobacteria bloom. Estimates demonstrated a significant decrease in silica (Si) deposition at alkaline pH in both in vitro F. crotonensis cultures and in situ Lake Erie diatom assemblages, after as little as 48 h of alkaline pH-exposure. These observations indicate elevated pH negatively affected growth rate and diatom silica deposition; in total providing a competitive disadvantage for diatoms. Our observations demonstrate pH likely plays a significant role in bloom succession, creating a potential to prolong summer Microcystis blooms and constrain diatom fall resurgence.
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fmicb-12-598736 February 19, 2021 Time: 14:22 # 1
published: 24 February 2021
doi: 10.3389/fmicb.2021.598736
Edited by:
Petra M. Visser,
University of Amsterdam, Netherlands
Reviewed by:
Jolanda Verspagen,
University of Amsterdam, Netherlands
John Beardall,
Monash University, Australia
Steven W. Wilhelm
Present address:
Lauren E. Krausfeldt,
College of Natural Sciences
and Oceanography, Nova
Southeastern University, Dania Beach,
FL, United States
Specialty section:
This article was submitted to
Aquatic Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 25 August 2020
Accepted: 20 January 2021
Published: 24 February 2021
Zepernick BN, Gann ER,
Martin RM, Pound HL, Krausfeldt LE,
Chaffin JD and Wilhelm SW (2021)
Elevated pH Conditions Associated
With Microcystis spp. Blooms
Decrease Viability of the Cultured
Diatom Fragilaria crotonensis
and Natural Diatoms in Lake Erie.
Front. Microbiol. 12:598736.
doi: 10.3389/fmicb.2021.598736
Elevated pH Conditions Associated
With Microcystis spp. Blooms
Decrease Viability of the Cultured
Diatom Fragilaria crotonensis and
Natural Diatoms in Lake Erie
Brittany N. Zepernick1, Eric R. Gann1, Robbie M. Martin1, Helena L. Pound1,
Lauren E. Krausfeldt1, Justin D. Chaffin2and Steven W. Wilhelm1*
1Department of Microbiology, The University of Tennessee, Knoxville, Knoxville, TN, United States, 2F.T. Stone Laboratory
and Ohio Sea Grant, The Ohio State University, Put-in-Bay, OH, United States
Cyanobacterial Harmful Algal Blooms (CyanoHABs) commonly increase water column
pH to alkaline levels 9.2, and to as high as 11. This elevated pH has been suggested
to confer a competitive advantage to cyanobacteria such as Microcystis aeruginosa.
Yet, there is limited information regarding the restrictive effects bloom-induced pH
levels may impose on this cyanobacterium’s competitors. Due to the pH-dependency
of biosilicification processes, diatoms (which seasonally both precede and proceed
Microcystis blooms in many fresh waters) may be unable to synthesize frustules at these
pH levels. We assessed the effects of pH on the ecologically relevant diatom Fragilaria
crotonensis in vitro, and on a Lake Erie diatom community in situ.In vitro assays
revealed F. crotonensis monocultures exhibited lower growth rates and abundances
when cultivated at a starting pH of 9.2 in comparison to pH 7.7. The suppressed
growth trends in F. crotonensis were exacerbated when co-cultured with M. aeruginosa
at pH conditions and cell densities that simulated a cyanobacteria bloom. Estimates
demonstrated a significant decrease in silica (Si) deposition at alkaline pH in both in vitro
F. crotonensis cultures and in situ Lake Erie diatom assemblages, after as little as 48 h
of alkaline pH-exposure. These observations indicate elevated pH negatively affected
growth rate and diatom silica deposition; in total providing a competitive disadvantage
for diatoms. Our observations demonstrate pH likely plays a significant role in bloom
succession, creating a potential to prolong summer Microcystis blooms and constrain
diatom fall resurgence.
Keywords: microcystis blooms, CyanoHABs, lake alkalinity, biogenic silica, diatoms, Lake Erie
Toxin-producing cyanobacteria of the genus Microcystis have inundated fresh waters in recent
decades (Steffen et al., 2014). Blooms have detrimental ecological and economic effects due to the
production of secondary metabolites and the formation of extensive biomass that, upon bloom
termination, can drive anoxia (Anderson, 2009). To this end, there is a crucial need to determine
the factors responsible for the ecological success of Microcystis.
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Zepernick et al. Alkaline pH Decreases Diatom Viability
The mechanisms by which Microcystis displaces other
phytoplankton in fresh waters, including Lake Erie
(United States/Canada), Lake Okeechobee (United States)
and Lake Tai (China) remain unclear, but are likely multifaceted.
In these lakes, a seasonal pattern of phytoplankton taxa
succession has emerged. Non-toxic diatoms and other algae
dominate throughout fall, winter, and spring, only to be
displaced by Microcystis blooms mid-summer into fall (Ke
et al., 2008;Reavie et al., 2014). This successional trend has
been evidenced in Lake Erie’s paleolimnological record, which
traces the emergence of eutrophication back to the 1930’s
(Allinger and Reavie, 2013). Monitoring efforts of a 2015
Microcystis bloom in Lake Eries western basin further confirmed
this succession, demonstrating diatoms dominated the early
summer period prior to their succession by cyanobacteria in
mid-summer (Figure 1A).
Several factors contribute to the ecological success of
Microcystis. Summer dominance in Lake Eries western basin
has been attributed to nutrient loading (Michalak et al.,
2013;Paerl et al., 2016), predation (Vanderploeg et al., 2001;
Steffen et al., 2015), and temperature (Andersson et al.,
1994;Peng et al., 2018). Likewise, spring diatom decline has
been linked to silica limitation and temperature intolerance
(Twiss et al., 2012;Reavie et al., 2016). While these factors
each contribute to Microcystis growth during cyanobacterial
bloom years, non-cyanobacterial bloom years have demonstrated
that diatoms, such as the temperature tolerant Fragilaria
crotonensis, can persist and even dominate the summer water
column in Lake Erie (Hartig and Wallen, 1986;Saxton
et al., 2012;Reavie et al., 2014). Indeed, F. crotonensis
summer blooms were a frequent occurrence in the western
basin of Lake Erie throughout the 1960–1980’s during lake
remediation efforts (Hartig, 1987). Furthermore, monitoring
data from the 2015 Lake Erie Microcystis bloom indicates
dissolved silica concentrations, though lowest during the peak
diatom bloom, were non-limiting during Microcystis succession
(Supplementary Figure 1). These observations suggest there
are additional and multiple factors contributing to Microcystis
succession of spring-summer diatoms (Wilhelm et al., 2020).
Amongst these factors playing a potential role in succession
dynamics is pH. For example, during the 2015 M. aeruginosa
bloom monitoring efforts, a sharp rise in water column pH
was found to co-occur with cyanobacterial bloom formation
(Figures 1A,B). While pH can have multiple effects on
cellular physiology and biogeochemistry, in the present study,
we investigated the response of one physiological aspect of
diatoms – silicification – to the shifts in pH that occur during
Microcystis blooms.
Microcystis blooms increase water column pH above 9.2
as CO2is consumed during photosynthesis (Verspagen et al.,
2014;Bullerjahn et al., 2016;Krausfeldt et al., 2019). This
alkaline pH is considered advantageous to cyanobacteria (Wilson
et al., 2010;Shruthi and Rajashekhar, 2014), due to their
unique carbon concentrating mechanisms (CCMs) which confer
a competitive advantage during growth at low CO2/ high
pH conditions (Shapiro, 1990;Raven, 2010;Sandrini et al.,
2016). Yet freshwater and marine diatoms have been shown
to possess a competitive array of CCMs themselves which
optimize CO2and HCO3acquisition (Clement et al., 2017).
While this may allow diatoms to evade pH-induced carbon-
limitation, elevated pH has been shown to decrease carbon
uptake, growth rate and metabolic processes in various marine
diatoms (Raven, 1981;Taraldsvik and Myklestad, 2000). While
these effects of pH on diatom carbon acquisition have been
well characterized, pH-induced effects on other metabolic
processes have been widely unstudied to date, particularly in
freshwater diatoms.
One metabolic process that serves as a distinctive metric for
diatom viability is silica deposition. Diatoms possess siliceous cell
walls (i.e., frustules) which may pose a unique disadvantage in
alkaline bloom conditions. Silica deposition relies on the uptake
of dissolved silica (dSi) in the form of silicic acid (Si[OH]4) to
synthesize biogenic silica (bSi) frustules (Vrieling et al., 1999;
Otzen, 2012;Hildebrand et al., 2018). In marine and estuarine
systems, diatom viability has been strongly correlated to pH, with
studies demonstrating marine diatoms are unable to survive at
pH >8.7 due to silica solubility dynamics and the inhibition of
biosilicification (Hansen, 2002;Hervé et al., 2012). Yet, to our
knowledge, the effect of pH in freshwater diatom Si deposition
remains unassessed.
In this study, we combined lab and field-based experiments
to assess the effect of pH on diatom growth rate and
silica deposition. As part of this effort, we assessed the
effect of pH on F. crotonensis growth rate in monoculture
and ecologically relevant co-cultures with M. aeruginosa.
Effects of pH on silica deposition were assessed using
a fluorescent dye (PDMPO) which intercalates into newly
formed frustules. Laboratory and field-based results indicate
pH conditions consistent with M. aeruginosa blooms (i.e.,
pH 9.2) decreased diatom growth rate, abundance, and
silica deposition. In total the pH shift reduces diatom
viability and the ability to compete for valuable niche-space
with cyanobacteria.
Assessing Successional Trends of a 2015
Lake Erie Microcystis Bloom
A temporal dataset collected at the Ohio State University
Stone Laboratory was used to preliminarily evaluate the
dynamics of cyanobacteria, diatoms, and pH during the
summer of 2015, which was the largest M. aeruginosa bloom
observed to date (Davis et al., 2019). Water column pH
was recorded every 30 min via a Yellow Spring Instruments
6600v2 multiprobe sonde suspended at 1 m depth from
a buoy located between South Bass and Gibraltar Islands
(N 41.66, W 82.92). Water samples for phytoplankton
community composition were collected next to the buoy
several times a week. Diatom-specific and cyanobacteria-
specific chlorophyll aconcentrations were recorded via a bbe
Moldaenke FluoroProbe (Beutler et al., 2002). Total chlorophyll
aconcentrations corresponding to the sampling period have
been provided (Supplementary Figure 2), with the complete
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Zepernick et al. Alkaline pH Decreases Diatom Viability
FIGURE 1 | Environmental data corresponding to a 2015 Lake Erie M. aeruginosa bloom. (A) Relative abundance (reported as percentage of total chlorophyll a) of
diatoms (closed blue squares) and cyanobacteria (open blue circles) within the Lake Erie water column. (B) Average daily pH of the Lake Erie water column (closed
blue triangles).
details of this dataset found in the original publication
(Chaffin et al., 2018).
Effect of pH on Growth in F. crotonensis
and M. aeruginosa Monocultures
To assess the effects of this environmentally observed pH
on diatom and cyanobacteria growth, in vitro monoalgal
experiments were performed using 2 model taxa. F. crotonensis
SAG 28.96 (acquired from the Culture Collection of Algae at
the University of Göttingen, Germany) and M. aeruginosa NIES
843 (acquired from the National Institute for Environmental
Studies, Japan) were maintained in batch cultures using CT
medium (Wilhelm, 2017) at respective optimal pH levels of
pH 7.7 (Guillard and Lorenzen, 1972;Hervé et al., 2012)
and 8.2 (Watanabe et al., 2000;Krausfeldt et al., 2019). To
initiate in vitro monoculture experiments, F. crotonensis and
M. aeruginosa cultures were filter-concentrated, respectively,
using a 2.0-µm and 1.0-µm nominal pore-size 47-mm diameter
polycarbonate filter and inoculated into sterile 250 mL filter-
vented, baffled polycarbonate flasks (Corning) at a starting
concentration of 700 cells/mL. Monocultures were maintained
in 125 mL CT medium containing a non-limiting concentration
of silicic acid (176 µM Na2SiO39H2O) (Hervé et al.,
2012) and adjusted to an initial pH of 7.7 (optimal pH
for diatom growth) or 9.2 (pH observed during Microcystis
blooms). pH conditions in the lab study were maintained
by adding TAPS buffer as described previously (Zepernick
et al., 2020). Cultures were monitored for 30 days at 26C,
with orbital shaking at 70 rpm, and a light intensity of
approximately 55–60 µmol photons m2s1on a 12:12 light:
dark photoperiod cycle.
Abundances were measured every 2-d via flow cytometry
(BD FACSCalibur). Populations of each species were gated and
counted based on forward scatter (FSC), a proxy for size,
and chlorophyll afluorescence (FL3) using FlowJoTM software
(Becton, Dickinson and Company). Due to the filamentous
nature of F. crotonensis, direct estimates of individual cell
abundance are challenging (Bramburger et al., 2017). In
this study, F. crotonensis abundances are estimated based
on filaments/mL, which form a tight cluster (Supplementary
Figure 3). Exponential growth rates (µ)were calculated as the
slope of log-scaled data, and were reported in filaments/mL for
F. crotonensis, and cells/mL for M. aeruginosa. Specifically, log-
scaled growth data for each replicate were fitted with a linear
regression to select time points to be used for µcalculations.
Time points demonstrating the logarithmic growth phase with
a linear regression R2value of 0.95 (i.e., the most linear data
points) were subsequently used to calculate average growth rate).
Culture pH was checked every 10-d using a sterilized pH probe
(Mettler Toledo Seven CompactTM pH/Ion meter S220 fitted with
a Mettler InLab Expert Pro-ISM electrode with a temperature
range and correction of up to 100C). Growth experiments
were performed in biological triplicate. We note all results will
be referred to in this study based on the initial pH condition
of the treatment.
Effect of pH on Growth in F. crotonensis
and M. aeruginosa Co-cultures
To evaluate the effects of ecologically relevant pH conditions
on diatom growth, in vitro co-culture assays were performed.
Concurrent with the monoculture assays, co-cultures of
F. crotonensis SAG 28.96 and M. aeruginosa NIES 843
were inoculated. To initiate in vitro co-culture experiments,
M. aeruginosa and F. crotonensis batch cultures were filter-
concentrated and inoculated into the same experimental
media and initial pH levels as previously described. Taxa were
inoculated at 3 ratios (reported as F. crotonensis:M. aeruginosa)
based on the succession patterns observed (Figure 1): 10:1
ratio simulating a spring diatom bloom, 1:1 ratio simulating
the onset of the summer M. aeruginosa bloom, and a 1:10 ratio
simulating the peak M. aeruginosa bloom. All co-cultures were
inoculated at net starting concentrations of 7,000 cells/mL.
Hereafter, co-culture treatments will be referred to by the
F. crotonensis:M. aeruginosa ratio. Co-cultures were subjected
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Zepernick et al. Alkaline pH Decreases Diatom Viability
to the same incubation conditions and procedures as described
above. All experiments were performed in biological triplicate.
Effect of pH on in vitro F. crotonensis
Silica Deposition
To determine the effect of pH on silica deposition in vitro,
batch cultures of F. crotonensis SAG 28.96 were inoculated
with the fluorescent dye PDMPO [2-(4-pyridyl)-5-((4-(2-
(Lysosensor DND 160 Yellow/Blue; Invitrogen, Carlsbad, CA,
United States). Diatom cultures were acclimated to pH conditions
of 7.7 and 9.2 for a 6-d period (i.e., approximately 2 doubling
times). Acclimated cultures were filter-concentrated using a
2.0-µm nominal pore-size 47-mm diameter polycarbonate
filter and inoculated in acid-clean, sterilized 50 mL glass
culture tubes containing 25 mL of CT medium with 176 µ
M Na2SiO39H2O. Tubes were inoculated at an initial
concentration of 1500 filaments/mL. PDMPO was added at a
final concentration of 0.125 µM (Leblanc and Hutchins, 2005).
Cultures were incubated at 26C and approximately 55–60 µmol
photons m2s1on a 12:12 light: dark photoperiod cycle
for 48 h. Abundances were determined via flow cytometry as
described above.
Si deposition was assessed using microscopic and fluorometric
approaches that detect freshly incorporated PDMPO. Bulk Si
deposition into individual cells was assessed via epifluorescence
microscopy. After 48 h, 2 mL of each culture was filtered onto
0.2-µm nominal pore-size 25 mm diameter black polycarbonate
filters (Millipore), mounted onto glass slides, treated with anti-
fade (Suttle and Fuhrman, 2010), and a coverslip applied
prior to storage (80C). F. crotonensis killed controls (0.5%
glutaraldehyde-fixed) were performed according to previous
studies (Saxton et al., 2012) to assess abiotic incorporation.
Slides were viewed on a Leica DM5500 (Wetzlar, Germany)
epifluorescence microscope equipped with a Hamamatsu ORCA-
ER camera (Sewickley PA) according to previous methods
(Saxton et al., 2012). A “Texas red” filter cube set (λex = 520–
600 nm; λem = 570–720 nm) was used to view chlorophyll
aautofluorescence, and a “DAPI filter” cube set (λex = 340–
380 nm; λem >425 nm) to view PDMPO fluorescence,
indicative of Si deposition during the experimental period.
Quantitative assessment of epifluorescence microscopy data
was achieved by randomized scoring of 100 F. crotonensis
filaments per pH treatment: analyses included chlorophyll a
fluorescing cells per filament, PDMPO fluorescing cells per
filament, and the proportion of filaments demonstrating one
PDMPO fluorescing cells. Total Si deposition was quantitatively
measured fluorometrically after HCl-Milli Q lysis to remove
unincorporated PDMPO from the silica deposition vesicle (SDV),
followed by frustule digestion with hot-NaOH (Saxton et al.,
2012;Zepernick et al., 2019). After 48 h of growth, 20 mL of
culture was collected on a 0.2-µm nominal pore-size 47-mm
diameter polycarbonate filter and subjected to HCl-Milli Q lysis.
Filters were flash frozen and stored at 80C until hot NaOH
digestion. After frustule digestion, samples were cooled in an
ice bath and neutralized using 1M HCl. PDMPO fluorescence
was quantitatively determined using a Turner Designs TD-
700 fluorometer fitted with a specialized filter set (λex = 360–
380 nm: λem = 522–542 nm, Andover Corporation, Salem, NH,
United States). A PDMPO standard curve was generated using
PDMPO and NaOH-HCl matrix (Supplementary Figure 4), with
the PDMPO concentration converted to Si using a conversion
factor of 3230:1 for Si:PDMPO (mol:mol) (Saxton et al.,
2012). Total silica deposited into frustules after 48 h (µmol)
was normalized to final abundance (filaments/mL). PDMPO
experiments were performed with five biological replicates.
Effect of pH on in situ Lake Erie Diatom
Community Silica Deposition
To evaluate the effects of pH on silica deposition in natural
populations, we queried diatom-enriched communities from
Lake Erie with PDMPO under varying pH conditions. Samples
were collected in late July of 2019 from the western basin of Lake
Erie near the Ohio State University Stone Laboratory on South
Bass Island (N 41.69; W 82.79). Water column physiochemistry
(temperature = 25.3C; dissolved oxygen = 7.60 mg/L; pH = 8.63;
turbidity = 1.46 NTU; chlorophyll a= 0.13 RFU) was recorded
prior to sampling using an EXO multiparameter sonde (YSI
xylem). Experiments were initiated by enriching for diatoms
using a 64-µm mesh phytoplankton net, which was lowered
to a depth of 7 m. Equal volumes of concentrated seston
were diluted with lake water and inoculated into acid washed,
rinsed 500 mL polycarbonate bottles. Lake water was buffered
using TRIS (4.13 mM final concentration) in accordance with
the protocol for freshwater C medium (Watanabe et al., 2000).
The experiment consisted of three pH treatments: 7.7, 9.2, and
an in situ pH control for the sample collection site (pH 8.6).
To achieve these pH conditions, samples were incrementally
titrated using 1 M HCl or NaOH. PDMPO dye was added
at a final concentration of 0.125 µM (Leblanc and Hutchins,
2005), and bottles were placed into an in situ mesh incubation
chamber for 48 h.
Sample pH and chlorophyll aconcentration were determined
at the initiation (T0) and termination (Tf; 48 h) of the incubation.
pH was assessed via immediate readings of 15 mL subsamples
using a pH probe (Mettler Toledo Seven CompactTM pH/Ion
meter S220, fitted with a Mettler InLab Expert Pro-ISM electrode
with a temperature range of up to 100C). Chlorophyll a
concentration was determined from filtration of 100 mL onto
0.2-µm nominal pore-size 47-mm diameter polycarbonate filters.
Samples were extracted in 90% acetone for 24 h at 4C
and assessed on a Turner Designs 10-AU Field Fluorometer
(Welschmeyer, 1994). To measure silica deposition, samples were
collected by filtering 100 mL of sample onto 0.2-µm nominal
pore-size 47-mm diameter polycarbonate filters, followed by the
HCl-Milli Q lysis method as described above. Samples were
flash frozen in liquid N2and stored at 80C until further
processing. Quantitative Si deposition analyses were performed
using hot-NaOH frustule digestion, fluorometry, and subsequent
calculations using a fresh standard curve (Supplementary
Figure 5;Zepernick et al., 2019). Total silica deposited per bottle
after 48 h (µmol) was normalized to chlorophyll aconcentration
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Zepernick et al. Alkaline pH Decreases Diatom Viability
(µg/L) (Saxton et al., 2012). Field experiments were performed
with four biological replicates.
Statistical Analyses
Statistical comparisons were made using unpaired two-tailed
t-tests, ordinary one-way ANOVAs, or ordinary two-way
ANOVAs, depending on experimental design. One-way and
two-way ANOVA post-hoc multiple comparisons were adjusted
using Tukey’s HSD. While F. crotonenis and M. aeruginosa
monoculture and co-culture growth rates are presented
separately in this text, all experiments were performed con-
currently in the same conditions, and thus have been statistically
analyzed using ordinary two-way ANOVAs to compare
both pH and abundance (Supplementary Tables 1,2). All
analyses were performed using GraphPad’s Prism software
(Version 8). For this study, we consider a p-value <0.05 to be
significant but have reported all values so the reader may decide
(Supplementary Tables 1–4).
Role of pH in 2015 Lake Erie Bloom
Succession Trends
Monitoring data from a 2015 M. aeruginosa-dominated bloom
demonstrated that total chlorophyll aconcentration across the
season varied from 1–3 µg/L in June to 20–120 µg/L
in July and August (Chaffin et al., 2018 and Supplementary
Figure 2). The pre-cyanobacterial bloom period (June through
early July) was dominated by diatoms which form 50–80%
of the total chlorophyll aconcentration, whereas cyanobacteria
were less than 10% (Figure 1A). The mean daily pH during
the corresponding diatom bloom period was between 8.08 and
8.56 (Figure 1B). Conversely, during the cyanobacterial bloom
period cyanobacteria dominate, forming 56–84% of the chl a
concentration, whereas diatoms were less than 7% and frequently
not detected (Figure 1A). The mean daily pH during the
Microcystis bloom peaked at 9.27 and remained higher than 9
throughout most of August (Figure 1B).
Alkaline pH Decreases Growth Rate of
F. crotonensis Monocultures
Monoculture experiments demonstrated F. crotonensis growth
was suppressed at high pH. F. crotonensis cultures inoculated
at pH 9.2 attained lower abundances throughout the 30-day
experiment compared to their pH 7.7 counterparts (Figure 2A).
F. crotonensis mean growth rate at pH 7.7 was µ= 0.34,
with pH 9.2 monocultures exhibiting a significantly lower
mean growth rate of µ= 0.22 (p= 0.0002) (Figure 2B).
Overall, F. crotonensis monocultures inoculated at pH 9.2
had a 1.5-fold lower mean growth rate compared to pH
7.7 equivalents.
Effects of pH on M. aeruginosa monocultures were less
pronounced. M. aeruginosa reached higher cell abundances at pH
7.7 compared to pH 9.2 equivalents (Supplementary Figure 6A).
Yet, M. aeruginosa growth rates were unaffected by pH overall
(p= 0.503) (Supplementary Figure 6B).
M. aeruginosa Modulates the Effect of
pH on F. crotonensis in Co-culture
F. crotonensis reached higher abundances at pH 9.2 than
pH 7.7 when co-cultured with non-dominant M. aeruginosa
concentrations of 10:1 and 1:1 (Figures 3A,C). Additionally,
F. crotonensis growth rates at the designated pH treatments were
not significantly different in the 10:1 ratio (p= 0.999) and 1:1
ratio (p= 0.206) (Figures 3B,D). Conversely, at the dominant
M. aeruginosa co-culture ratio of 1:10, F. crotonensis growth was
substantially suppressed at pH 9.2 (Figure 3E). F. crotonensis
mean growth rate at pH 7.7 in the 1:10 co-culture was µ= 0.35,
with pH 9.2 co-cultures exhibiting a significantly lower mean
growth rate of µ=0.23 (p= 0.0002) (Figure 3F). Overall, at the
1:10 ratio and pH 9.2, F. crotonensis has a 1.5 times lower mean
growth rate compared to its pH 7.7 equivalents.
As in the monocultures, the effects of pH on M. aeruginosa
growth in the co-culture replicates were less pronounced.
M. aeruginosa reached higher cell concentrations at pH 7.7
in all co-culture ratios compared to pH 9.2 equivalents
(Supplementary Figures 7A,C,E). Yet, M. aeruginosa growth
FIGURE 2 | (A) In vitro F. crotonensis monoculture growth curves at pH 7.7 (black squares) and pH 9.2 (green squares). (B) F. crotonensis growth rate at pH 7.7
(black squares) and pH 9.2 (green squares). Statistically significant differences between pH treatments are denoted by p-values generated by Two-way ANOVAs.
Standard error of the mean reported by error bars.
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FIGURE 3 | (A) In vitro F. crotonensis co-culture growth curves in a 10:1 ratio (F. crotonensis:M. aeruginosa) at pH 7.7 (black squares) and pH 9.2 (green squares).
(B) F. crotonensis growth rate at 10:1 ratio (C) F. crotonensis growth curves in a 1:1 ratio (D) F. crotonensis growth rate in 1:1 ratio (E) F. crotonensis growth curves
in a 1:10 ratio (F) F. crotonensis growth rate in a 1:10 ratio. Statistically significant differences between pH treatments are denoted by p-values generated by
Two-way ANOVAs. Standard error of the mean reported by error bars.
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rates were unaffected by pH overall (p>0.503) (Supplementary
Figures 7B,D,F).
M. aeruginosa Concentrations
Correspond With pH Increases
While F. crotonensis monocultures inoculated at pH 7.7
remained at this pH throughout the 30-day experiment
(Supplementary Figure 8), M. aeruginosa monocultures
demonstrated a steady climb in pH, reaching 8.0 by day 30
(Supplementary Figure 9). Similarly, all 3 co-culture ratios
inoculated at pH 7.7 demonstrated continual increases in
pH throughout the 30-day experiment, reaching final pH
levels of 8.10 (Supplementary Figure 10). In total, pH
7.7 inoculated M. aeruginosa monocultures and co-cultures
all experienced increases in pH of 0.30, coinciding with
increases in M. aeruginosa concentrations throughout the 30-day
experiment (Supplementary Figures 7,11A). Upon further
analysis, M. aeruginosa concentrations demonstrated a strong
linear relationship with culture pH increases observed in the
mono and co-cultures (Simple linear regression R20.8577)
(Supplementary Figure 12). Collectively, pH was maintained
within a range of approximately +/0.40 pH units throughout
the 30-day experiment (Supplementary Figure 11).
Silica Deposition Decreases at Alkaline
pH in F. crotonensis Monocultures
In vitro PDMPO incubations demonstrated a pronounced
effect of alkaline pH on silica deposition. Epifluorescence
microscopy revealed pH 7.7 acclimated cultures deposited
more Si after 48 h PDMPO incubations (Figures 4A,C,E and
Supplementary Figures 13A,C) compared to pH 9.2 acclimated
cultures (Figures 4B,D,F and Supplementary Figures 13B,D).
Quantitative counts of these images also demonstrated pH 9.2
acclimated cultures formed significantly smaller filaments than
pH 7.7 acclimated cultures (p<0.0001; unpaired two-tailed t-test
t= 4.057, df = 197, n= 100) (Supplementary Figure 14A).
In total, 66% of cells in each filament deposited silica after
48 h in the pH 7.7 treatments, while only 30% of the cells in
each filament had deposited Si at pH 9.2 (p<0.0001; unpaired
two-tailed t-test t= 9.457, df = 197, n= 100) (Supplementary
Figure 14B). 100% of F. crotonensis filaments incubated at pH 7.7
exhibited at least one diatom cell depositing Si, while only 66% of
pH 9.2 F. crotonensis filaments demonstrated at least one instance
of Si deposition per filament.
Fluorometric data revealed pH 7.7 acclimated cultures
deposited a mean of 25.28 µmol Si total, while pH 9.2 acclimated
cultures deposited a significantly lower mean of 15.81 µmol Si
total (p<0.0001; unpaired two-tailed t-test t= 8.544, df = 8,
n= 5) (Supplementary Figure 15). Normalization of this data
to abundance (final filament concentration) reflected a similar
trend. F. crotonensis cultures acclimated to pH 7.7 deposited a
mean of 1.17 nmol Si/filament, while cultures acclimated to pH
9.2 deposited a significantly lower mean of 0.59 nmol Si/filament
(p<0.0001; unpaired two-tailed t-test t= 9.446, df = 8, n= 5)
(Figure 5). Overall, diatoms acclimated to pH 9.2 deposited
50% less silica in comparison to their pH 7.7 counterparts.
Silica Deposition Decreases at Alkaline
pH in Lake Erie Diatom Communities
Elevating the pH negatively influenced Si deposition in the
Lake Erie diatom community. Samples incubated at pH 7.7,
control pH (8.6), and pH 9.2 deposited a mean of 219.07 µmol
Si total, 214.52 µmol Si total, and 194.36 µmol Si total,
respectively (Supplementary Figure 16). Total Si deposited in
pH 9.2 treatments was less than pH 7.7 treatments, though not
statistically significant (p= 0.127). Normalization of this data
to chlorophyll aconcentration upheld this observation, with
pH 7.7 treatments depositing a mean of 27.61 µmol Si/Chl
a, control treatments depositing 22.45 µmol Si/Chl a, and pH
9.2 treatments depositing 18.16 µmol Si/Chl a, respectively
(Figure 6). The pH 9.2 treated community deposited significantly
less Si per chlorophyll aconcentration after 48 h compared to the
pH 7.7 treated community (p= 0.0375). Overall, Lake Erie diatom
communities incubated at pH 9.2 deposited 1.5 times less Si per
chlorophyll aconcentration than their pH 7.7 counterparts.
Seasonal succession drivers associated with Microcystis blooms
are complicated. While it remains clear that nutrient-loading
results in the planktonic biomass observed during toxic
cyanobacterial blooms, the environmental conditions that allow
specific organisms to outcompete others are more nuanced
(Wilhelm et al., 2020). Here we build on the idea that pH
serves as a contributing piece to this puzzle. Previous analyses
have suggested a correlation between pH and diatom-Microcystis
succession in Lake Tai, China (Ke et al., 2008) and Lake Erie
(Krausfeldt et al., 2019). In these and other cases, authors have
suggested that the effects of pH on carbon acquisition and
the superior carbon concentrating mechanisms of cyanobacteria
were the major mechanistic drivers of these observations.
Additionally, previous studies have indicated nutrient speciation
at alkaline pH may favor Microcystis, such as the discovery that
urea serves as both a carbon and nitrogen source to M. aeruginosa
at alkaline pH levels (Krausfeldt et al., 2019). While the direct
and indirect effects of pH on freshwater diatom carbon and
nutrient acquisition cannot be discounted or ruled out, our
data demonstrated a previously uncharacterized effect of pH on
freshwater diatoms, which may serve to depress them beyond,
or in addition to, their ability to acquire CO2. We present this
information as a factor that likely enhances the exclusion of
Si depositing phytoplankton observed during heated summer
competition. These observations lead to a take-away message
from this study: sometimes it is not the ability of Microcystis but
the inability of its competitors that results in the taxa succession.
Effect of pH on Freshwater Diatom
We used pH manipulation in mono- and co-culture experiments
to demonstrate that an elevated pH, consistent with Microcystis-
bloom conditions, negatively affected the diatom F. crotonensis.
F. crotonensis monocultures inoculated at pH 9.2 exhibited lower
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FIGURE 4 | Epifluorescent microscopy images (40x magnification) of F. crotonensis filaments after 48 h PDMPO incubations. Scale bar represents 25 µm.
Chlorophyll aautofluorescence is depicted in red, and PDMPO fluorescence is in blue. (A,C,E) F. crotonensis cultures acclimated to pH 7.7. (B,D,F) F. crotonensis
cultures acclimated to pH 9.2.
growth rates and failed to establish a substantial population,
demonstrating alkaline pH alone decreases the viability of
this model freshwater diatom. Likewise, when co-cultured with
dominant concentrations of M. aeruginosa at the 1:10 ratio, these
alkaline pH effects on abundance were exacerbated. This data
is consistent with freshwater diatom decline at the alkaline pH
levels observed during summer Microcystis blooms. Interestingly,
when F. crotonensis was co-cultured in the 1:10 ratio at pH
7.7, it was able to maintain growth rates resembling those
observed in the pH 7.7 monocultures, suggesting alkaline pH
may have a larger role in diatom viability than previously
thought. Surprisingly, when F. crotonensis was co-cultured with
M. aeruginosa at 10:1 and 1:1 (i.e., where the diatom biomass
dominated) it did not exhibit significant declines in growth rate
at pH 9.2. F. crotonensis abundances in the 10:1 and 1:1 co-
culture were higher at pH 9.2 than their pH 7.7 counterparts,
though statistical significance was lacking. While the underlying
mechanisms of these results remain unelucidated, this data
suggests that while pH is a factor, it alone is likely not the sole
driver of diatom exclusion. Another important observation is that
in all M. aeruginosa mono and co-cultures inoculated at pH 7.7,
pH increases in tandem with M. aeruginosa cell concentration.
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FIGURE 5 | Si deposited per filament after 48 h PDMPO incubations in
F. crotonensis cultures acclimated to pH 7.7 treatments (black squares) and
pH 9.2 (green squares). Statistically significant differences are denoted by
respective p-values generated by unpaired two-tailed t-tests. Standard error
of the mean reported by error bars.
FIGURE 6 | Si deposited per chl aconcentration in pH 7.7 treatments (black
squares), control pH 8.6 (gray squares), and pH 9.2 (green squares) after 48 h
incubations. Statistically significant differences are denoted by respective
p-values generated by One-way ANOVAs. Standard error of the mean
reported by error bars.
This data demonstrates M. aeruginosa is indeed capable of
driving the pH up substantially despite increased buffer use,
and mimics environmental data previously observed during
a 2015 Microcystis bloom. Cumulatively, in vitro co-cultures
suggest diatoms may be able to persist in the water column
during the spring diatom blooms and onset Microcystis blooms
regardless of water column pH. Yet, during peak Microcystis
bloom conditions when the pH is driven to alkaline levels,
diatoms are at a disadvantage. This data further suggests these
persisting alkaline pH levels may prolong the Microcystis bloom
period by preventing diatom fall resurgence as a result of
decreased diatom growth and viability.
Effects of pH on Diatom Silica Deposition
Though previous studies have investigated the effects of pH on
marine diatom biosilicification (Vrieling et al., 1999;Martin-
Jézéquel et al., 2000;Hansen, 2002), this study builds on
these observations through an assessment of pH effects on
freshwater diatoms. We used PDMPO assays to demonstrate that
pH conditions consistent with Microcystis blooms significantly
decrease silica deposition in both cultured and environmental
freshwater diatoms. When interpreting this data it is important
to note, flow cytometry analyses of filamentous microorganisms
such as F. crotonensis count “filaments per volume” rather
than “cells per volume.” As a result, an estimate for average
number of cells per chain is often used to calculate cells/mL
(Bramburger et al., 2017). Our data demonstrate the average
number of cells per filament differs significantly in response
to culture pH (Supplementary Figure 14), which has the
potential to introduce additional error in estimates of cell/mL and
biovolume. Indeed, epifluorescence microscopy revealed pH 9.2-
acclimated F. crotonensis had 1.5 times shorter filaments and
2 times fewer silica depositing cells per filament. Fluorometric
data from the Si deposition assays revealed a similar trend,
with F. crotonensis cultures acclimated to pH 9.2 depositing
50% less silica per filament in comparison to their pH 7.7
counterparts. This trend was further observed in Lake Erie
diatom communities, with communities incubated at pH 9.2
depositing 1.5 times less Si per chlorophyll aconcentration than
their pH 7.7 counterparts. Cumulatively, this data suggests pH-
induced decreases in silica deposition may serve as an important
contributor to the freshwater diatom decline observed during
Microcystis blooms. Furthermore, these results also bring to
light a need to further optimize detection and normalization
techniques in studies concerning filamentous phytoplankton.
While we have demonstrated a decrease in silica deposition
at pH 9.2, the underlying mechanisms remain unclear. Part of
our limitation comes from the lack of knowledge concerning
functions in the organelle responsible for silica deposition, known
as the silica deposition vesicle (SDV). Despite decades of research,
the SDV has yet to be isolated or characterized (Martin-Jézéquel
et al., 2000;Hildebrand et al., 2018). Additionally, intracellular
proteins and pathways associated with diatom biosilicification
remain elusive (Thamatrakoln and Hildebrand, 2008;Vardi et al.,
2009;Otzen, 2012). External alkaline pH may negatively affect
intracellular metabolism in the SDV, which relies on acidic
conditions and an undisturbed pH gradient (Vrieling et al., 1999;
Hervé et al., 2012). Previous studies have also demonstrated high
pH levels may shape intra-cellular diatom silica storage pools
(Werner, 1966;Azam et al., 1974;Sullivan, 1977;Martin-Jézéquel
et al., 2000). Alkaline pH has previously been shown to limit
silica deposition by altering the chemical species of silicic acid or
decreasing diatom-uptake rates of dissolved silica (dSi) (Riedel
and Nelson, 1985;Amo and Brzezinski, 1999). However, this is
unlikely in our study due to the non-limiting concentration of
silicic acid in our media (176 µM Na2SiO39H2O).
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In this study, we observed both a decrease in growth rate and
silica deposition in response to alkaline pH. Though evidence of
a causal link between these two physiological processes is lacking
in this study, prior research has established that diatom silica
uptake and deposition are tightly coupled with the cell cycle, thus
exerting a dependency of silica metabolism on the growth rate
(Martin-Jézéquel et al., 2000;Hildebrand et al., 2018). Yet, while
silica deposition is essential to diatom viability and their ability to
reproduce, diatoms can downregulate silica deposition (i.e., form
thinner frustules) to maintain optimal growth rates (Brzezinski
et al., 1990;Mcnair et al., 2018). Alternatively, previous research
has also demonstrated that as pH increases, growth rates
decrease and intracellular silicic acid increases in marine diatoms,
potentially indicating a decoupling to silica deposition (Hervé
et al., 2012). Hence, pH-associated effects on alternate metabolic
processes such as cellular respiration or photosynthesis (i.e.,
disruptions in normal metabolic regulators) may also contribute
to a decline in silica deposition. Furthermore, thinner-frustules
have been shown to increase the potential for viral infection and
mortality in marine diatoms, exacerbating population declines in
the environment (Kranzler et al., 2019). Ultimately, several of
these underlying mechanisms may contribute to the decreased
silica deposition observed in this study, and further research is
needed before any definitive relationship between growth rate,
silica deposition, and alkaline pH can be established.
A Growing Influence of pH in Future
Phytoplankton Diversity
Climate change continues to pose a threat to freshwater and
marine systems alike. As a result, there is a need to elucidate
its effects on factors constraining the ecological success of
phytoplankton, such as pH. A recent study has demonstrated
ocean acidification has the potential to decrease marine diatom
biosilicification rates (Petrou et al., 2019). Conversely, freshwater
systems are experiencing a basification attributed to increases
in the frequency and duration of HAB events (Wells et al.,
2020), which has the potential to decrease freshwater diatom
biosilicification. In this manner, the effects of projected pH shifts
on phytoplankton succession serve as a critical point of study
for ensuring the integrity of global aquatic systems (Flynn et al.,
2015;Wells et al., 2020). Our results build on these previous
studies, demonstrating pH may play a pivotal role not only in
cyanobacterium-driven diatom decline, but phytoplankton taxa
diversity in general. While previous studies have demonstrated
alkaline bloom-induced pH can serve as a positive feedback
mechanism for M. aeruginosa (Krausfeldt et al., 2019), this
work builds on these efforts by demonstrating these same
conditions can facilitate the exclusion of siliceous algae (diatoms).
Furthermore, while Lake Erie summer cyanobacterial blooms
drive up the western basin pH to an average of 9.2, previous
winter surveys demonstrate the diatom-dominated water column
remains at an average of 7.8–8.2 despite fluctuations in
chlorophyll aand sampling location (Supplementary Figure 17),
though additional surveys are needed concerning winter diatom
blooms. Cumulatively, this data suggests a role of pH on both the
inter and intra-season shifts of phytoplankton taxa within Lake
Erie and demonstrate the need to further assess the role of pH in
phytoplankton succession.
We noted our pH co-cultures of 10:1 and 1:1 yield higher peak
diatom abundance at pH 9.2 in comparison to pH 7.7, despite
the diatom monoculture yielding markedly lower abundances at
the same elevated pH. In this manner, there may be a window
of opportunity for diatoms to persist, and even benefit at low
densities of M. aeruginosa if the cyanobacterial populations do
not become dominant. Many other biological / biogeochemical
processes (e.g., inorganic carbon cycling, nitrogen speciation,
trace metal chemistry) are pH sensitive and likely play a
role in shaping the outcomes of competition for niche space
between phototrophs in fresh waters. Our observations serve
as a salient reminder that competition in aquatic systems is
condition dependent and often complicated by a mix of factors
(Wilhelm et al., 2020).
In this study, we confirmed that pH levels of 9.2 decreased
diatom growth rate in the filamentous diatom F. crotonensis. Our
data further demonstrated that silica deposition in lab cultures
and environmental diatom communities declined at alkaline pH
levels. Cumulatively, these effects reduce diatom viability and
fitness in the competition against Microcystis blooms. While the
pH shift itself may not be sufficient to exclude the diatoms
from this (or any) system, the resulting decrease in competitive
ability for carbon and nutrients, in addition to pressure from
other factors including top-down regulators such as predators
and viruses (Kranzler et al., 2019;Pound et al., 2020) appear
to tip the scale to favor the cyanobacteria. What remains to be
determined beyond this study is how these pressures allow a
specific genus of cyanobacteria to proliferate while in competition
with many others.
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s.
BZ and SW designed the experiments. BZ and LK performed
preliminary culture optimizations and experimental planning.
BZ and EG conducted in vitro co-culture assays and performed
epifluorescence microscopy. BZ conducted in vitro silica
deposition assays and performed statistical analyses. BZ,
HP, and RM conducted in situ Lake Erie silica deposition
assays with logistical support from JC. JC performed data
collection and analyses corresponding to the 2015 M. aeruginosa
bloom in Figure 1. All authors contributed to the drafting
of the manuscript.
This work was funded through the Bowling Green State
University Great Lakes Center for Fresh Waters and Human
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Zepernick et al. Alkaline pH Decreases Diatom Viability
Health through the NIH (1P01ES028939-01) and NSF (OCE-
1840715) to SW and JC, an NSF GRFP to BZ (DGE-1938092).
This work was also supported by the Kenneth & Blaire Mossman
Endowment to the University of Tennessee (SW).
We thank Dr. Gary LeCleir, Dr. Matthew Saxton, Dr. Robert
McKay, Dr. George Bullerjahn, and Naomi Gilbert for comments
and suggestions. We also thank Keara Stanislawczyk for
facilitating field work at OSU Stone Lab.
The Supplementary Material for this article can be found
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Zepernick, Gann, Martin, Pound, Krausfeldt, Chaffin and
Wilhelm. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original author(s) and the copyright owner(s)
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Frontiers in Microbiology | 12 February 2021 | Volume 12 | Article 598736
... However, the development of microalgae has always been a problem for the management of the duckweed-based treatment systems, which greatly affects the growth of duckweed and the performance of the system. Many previous studies have reported that the thriving of microalgae is always accompanied by dissolved oxygen depletion and biodiversity loss in the water bodies (Lu et al., 2013;Nolan & Cardinale, 2019;Zepernick et al., 2021). Among the observed microalgae, Microcystis is one of the most dominant and widespread cyanobacteria species. ...
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Microcystis is commonly found in duckweed-based treatment systems, which greatly affects the duckweed growth and performance of the system. In this study, the effects of nitrogen and population density on duckweed and microalgae competition were investigated in terms of their growth sensitivity response to mutual competition under laboratory conditions. A comparatively strong mutual competition between S. polyrhiza and Microcystis sp. was observed at nitrogen concentrations of 5 and 10 mg N/L. At high nitrogen concentrations (40 mg N/L), the growth of Microcystis sp. was remarkably inhibited, which facilitated the S. polyrhiza growth. The magnitude of competitive activity between S. polyrhiza and Microcystis sp. displayed a density-dependent trend. Under 100% duckweed coverage, the intraspecific competition was stronger than the interspecific competition. This study will greatly facilitate the biocontrol of Microcystis sp. development in the duckweed-based water treatment system and improve the system’s efficiency.
... The unique cell structure of diatoms is a natural pH buffer, but diatom species have distinct optimal pH requirements. Elevated pH (> 9.2) negatively affected the growth and silica deposition of diatoms (Zepernick et al., 2021). The growth rate of Chaetoceros muelleri decreased at a pH below 7.9 (Iwasaki et al., 2021). ...
... As for the pH, it is known that an increase in this parameter is connected with the consumption of CO 2 during photosynthesis. Many authors suggest that alkaline pH is considered advantageous to cyanobacteria ( Wilson, et al., 2010 ;Shruthi & Rajashekhar, 2014 ;Zepernick, et al., 2021 ), due to their unique carbon concentrating mechanisms (CCMs) which confer a competi- JID: ECOHYD [m3Gdc;September 21, 2023;14:23 ] tive advantage during growth at low CO 2 / high pH conditions. The other important phytoplankton groups were Bacillariophyta. ...
... More broadly, eukaryotic algae often dominate in the winter/spring but subsequently give way to cyanobacterial dominance as the summer approaches or progresses, reflecting, at least in part, differences in thermal optima among various taxa [104,183,184]. This eukaryote-to-prokaryote succession may also be partly due to the relatively large increase in pH (basification) that occurs during intense cyanobacterial blooms in the summer coupled with the high tolerance of cyanobacteria to basic water conditions [185][186][187]. Cyanobacterial blooms in U.S. inland waters can last up to several months in the summer [188]. ...
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Cyanobacteria are the most common toxigenic algae in inland waters. Their toxins can affect the health of aquatic and terrestrial organisms, including humans. Other algal groups, such as haptophytes (e.g., Prymnesium parvum) and euglenoids (e.g., Euglena sanguinea), can also form harmful algal blooms (HABs) whose toxins cause injury to aquatic biota but currently have no known effects on human health. Prymnesium parvum, however, is responsible for some of the worst HAB-related ecological disasters recorded in inland waters. Here, we provide an overview of the primary toxigenic algae found in U.S. inland waters: cyanobacteria (planktonic forms), P. parvum, and E. sanguinea with the objective of describing their similarities and differences in the areas of HAB ecology, algal toxins, and the potential for future range expansion of HABs. A detailed account of bloom habitats and their known associations with land cover and use is provided from the perspective of water quality. This review revealed that salinity may have an influence on inland cyanobacterial blooms and cyanotoxins that had not been fully recognized previously.
... The shift of DO, pH, and chlorophyll-a in the water is closely related to photosynthesis and respiration (Huang and Chen, 2013). During bloom-forming, the photosynthesis of cyanobacteria is greater than respiration, resulting in the consumption of carbon dioxide and the rise of DO, pH, and chlorophyll-a (Paerl and Huisman, 2008;Verspagen et al., 2014;Zepernick et al., 2021). Conversely, if photosynthesis is weaker than respiration, DO, pH, and chlorophyll-a will reduce. ...
Microcystis, one of the common cyanobacteria, often causes blooms in reservoirs, which has seriously threatened the safety of drinking water worldwide. To identify the growth characteristic of total and microcystin-producing Microcystis in large deep reservoirs, we used Quantitative PCR (qPCR) to measure the cell density of total and microcystin-producing Microcystis and monitored water quality in the water samples collected in Dongzhang Reservoir once a month. Microcystis blooms occurred in Dongzhang Reservoir in April 2017, which was composed of microcystin-producing and non-microcystin-producing Microcystis. Water temperature, dissolved oxygen, pH, and chlorophyll-a showed significant vertical stratification during Microcystis blooms. Total and microcystin-producing Microcystis grew rapidly under the high concentration of total phosphorus and rising water temperatures. Nitrate-nitrogen had a significant linear correlation with the abundance of microcystin-producing Microcystis. Our results indicated that nutrients and water temperature could be key triggers of Microcystis blooms and nitrate-nitrogen potentially regulates the competition between microcystin-producing and non-microcystin-producing Microcystis. This study improves our understanding of the characteristics of Microcystis blooms and the competition between microcystin-producing and non-microcystin-producing Microcystis in large deep reservoirs.
... The blooms of cyanobacteria increase pH (Hinners et al., 2015). This was noted, for example, for Microcystis spp (Zepernick et al., 2021), which caused a lower abundance of other species in the phytoplankton community, e.g., diatoms. This means that testing of the pH effect on NSAID toxicity would be problematic due to high modification of pH by growing cyanobacteria. ...
Aquatic species are continuously exposed to pharmaceuticals and changeable water conditions simultaneously, which can induce changes in the toxicity of pollutants. Cyanobacterium are an organism for which less ecotoxicological tests have been performed compared to green algae. In this study, we decided to check how selected non-steroidal anti-inflammatory drugs (NSAID) affect the grow of Synechocystis salina, picocyanobacterium isolated from the Baltic Sea, with salinity as potential modulator of toxicity. S. salina was exposed to diclofenac (DCF), ibuprofen (IBF) and naproxen (NPX) (nominal 100 mg L-1) in BG11 medium and sea salt supplemented BG11 medium (38 PSU) over 96 h in continuous light at 23 °C. No acute toxicity was found in both tested salinity levels. The comparable grow rate in exposed culture compared to control culture over 4 days indicate lack of stress for several generations which need to be overcome with substantial energy consumption. S. salina was found to be halotolerant and can be species for ecotoxicology test where salinity in an additional stressor. Furthermore, resistant of S. salina to target NSAIDs provide a competitive advantage over other phytoplankton species.
... This negative association is in accordance with the findings from Horn and Uhlmann (1995), finding high yields of Fragilaria caused low yields of Cyanobacteria and vice versa. More recent work has proven that elevated pH associated with cyanobacterial productivity negatively affected the growth rate of Fragilaria and diatom silica deposition (Zepernick et al., 2021). Similarly, Ulnaria also experienced a negative cooccurrence with Gloeotrichia PYH6, yet there is no supporting literature to explain reasons behind this negative relationship. ...
... This internally sourced P can be assimilated into the food web and contribute to eutrophication. In-lake mechanisms of P mobilization may be enhanced by rising water temperature (Boström et al., 1982;Gomez et al., 1998;Jeppesen et al., 1997), photosynthetically driven pH increases (Sabur et al., 2021;Søndergaard et al., 2003;Welch and Cooke, 1995) in shallow environments characterized by dense cyanobacterial blooms (Chen et al., 2014(Chen et al., , 2018Zepernick et al., 2021), hypolimnetic hypoxia and anoxia (Nürnberg et al., 2019;Orihel et al., 2017;Welch and Cooke, 1995), salinization (Radosavljevic et al., 2022), and coastal sediment resuspension and shoreline erosion (Eadie et al., 1990(Eadie et al., , 2008McKinney et al., 2019;Scavia et al., 2019aScavia et al., , 2019bScavia et al., , 2020. Despite growing evidence that these mechanisms can mobilize significant amounts of P (e.g., Nürnberg and LaZerte, 2016;Scavia et al., 2020), quantifying internal P loads in large lakes is difficult because the underlying mechanisms are generally highly dynamic in time and space. ...
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Since the discovery of the first “giant virus,” particular attention has been paid toward isolating and culturing these large DNA viruses through Acanthamoeba spp. bait systems. While this method has allowed for the discovery of plenty novel viruses in the Nucleocytoviricota , environmental -omics-based analyses have shown that there is a wealth of diversity among this phylum, particularly in marine datasets. The prevalence of these viruses in metatranscriptomes points toward their ecological importance in nutrient turnover in our oceans and as such, in depth study into non-amoebal Nucleocytoviricota should be considered a focal point in viral ecology. In this review, we report on Kratosvirus quantuckense (née Aureococcus anophagefferens Virus), an algae-infecting virus of the Imitervirales . Current systems for study in the Nucleocytoviricota differ significantly from this virus and its relatives, and a litany of trade-offs within physiology, coding potential, and ecology compared to these other viruses reveal the importance of K. quantuckense . Herein, we review the research that has been performed on this virus as well as its potential as a model system for algal-virus interactions.
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Blooms of the toxin-producing cyanobacterium Microcystis are increasing globally, leading to the loss of ecosystem services, threats to human health, as well as the deaths of pets and husbandry animals. While nutrient availability is a well-known driver of algal biomass, the factors controlling “who” is present in fresh waters are more complicated. Microcystis possesses multiple strategies to adapt to temperature, light, changes in nutrient chemistry, herbivory, and parasitism that provide a selective advantage over its competitors. Moreover, its ability to alter ecosystem pH provides it a further advantage that helps exclude many of its planktonic competitors. While decades of nutrient monitoring have provided us with the tools to predict the accumulation of phytoplankton biomass, here, we point to factors on the horizon that may inform us why Microcystis is presently the dominant bloom former in freshwaters around the world.
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Drivers of algal bloom dynamics remain poorly understood, but viruses have been implicated as important players. Research addressing bloom dynamics has generally been restricted to the virus-infection of the numerically dominant (i.e. bloom forming) taxa. Yet this approach neglects a broad diversity of viral groups, limiting our knowledge of viral interactions and constraints within these systems. We examined hallmark virus marker genes in metatranscriptomic libraries from a seasonal and spatial survey of a Microcystis aeruginosa bloom in Lake Tai (Taihu) China to identify active infections by nucleocytoplasmic large DNA viruses (NCLDVs), RNA viruses, ssDNA viruses, bacteriophage, and virophage. Phylogenetic analyses revealed a diverse virus population with seasonal and spatial variability. We observed disproportionately high expression of markers associated with NCLDVs and ssRNA viruses (consistent with viruses that infect photosynthetic protists) relative to bacteriophage infecting heterotrophic bacteria or cyanobacteria during the height of the Microcystis bloom event. Under a modified kill-the-winner scheme, we hypothesize viruses infecting protists help suppress the photosynthetic eukaryotic community and allow for the proliferation of cyanobacteria such as Microcystis. Our observations provide a foundation for a little considered factor promoting algal blooms.
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Diatoms, large bloom-forming marine microorganisms, build frustules out of silicate, which ballasts the cells and aids their export to the deep ocean. This unique physiology forges an important link between the marine silicon and carbon cycles. However, the effect of ocean acidification on the silicification of diatoms is unclear. Here we show that diatom silicification strongly diminishes with increased acidity in a natural Antarctic community. Analyses of single cells from within the community reveal that the effect of reduced pH on silicification differs among taxa, with several species having significantly reduced silica incorporation at CO2 levels equivalent to those projected for 2100. These findings suggest that, before the end of this century, ocean acidification may influence the carbon and silicon cycle by both altering the composition of the diatom assemblages and reducing cell ballasting, which will probably alter vertical flux of these elements to the deep ocean.
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Diatoms are among the most globally distributed and ecologically successful organisms in the modern ocean, contributing upwards of 40% of total marine primary productivity1,2. By converting dissolved silicon into biogenic silica, and photosynthetically fixing carbon dioxide into particulate organic carbon, diatoms effectively couple the silicon (Si) and carbon cycles and ballast substantial vertical flux of carbon out of the euphotic zone into the mesopelagic and deep ocean3–5. Viruses are key players in ocean biogeochemical cycles6,7, yet little is known about how viral infection specifically impacts diatom populations. Here, we show that Si limitation facilitates virus infection and mortality in diatoms in the highly productive coastal waters of the California Current Ecosystem. Using metatranscriptomic analysis of cell-associated diatom viruses and targeted quantification of extracellular viruses, we found a link between Si stress and the early, active and lytic stages of viral infection. This relationship was also observed in cultures of the bloom-forming diatom Chaetoceros tenuissimus, where Si stress accelerated virus-induced mortality. Together, these findings contextualize viruses within the ecophysiological framework of Si availability and diatom-mediated biogeochemical cycling.
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The use of urea as a nitrogenous fertilizer has increased over the past two decades, with urea itself being readily detected at high concentrations in many lakes. Urea has been linked to cyanobacterial blooms as it is a readily assimilated nitrogen (N) - source for cyanobacteria that possess the enzyme urease. We tested the hypothesis that urea may also act as a carbon (C) source to supplemental growth requirements during the alkaline conditions created by dense cyanobacterial blooms, when concentrations of dissolved CO2 are vanishingly low. High rates of photosynthesis markedly reduce dissolved CO2 concentrations and drive up pH. This was observed in Lake Erie during the largest bloom on record (2015) over long periods (months) and short periods (days) of time, suggesting blooms experience periods of CO2-limitation on a seasonal and daily basis. We used 13C-urea to demonstrate that axenic cultures of the model toxic cyanobacterium, Microcystis aeruginosa NIES843, assimilated C at varying environmentally relevant pH conditions directly into a spectrum of metabolic pools during urea hydrolysis. Primarily, 13C from urea was assimilated into central C metabolism and amino acid biosynthesis pathways, including those important for the production of the hepatotoxin, microcystin, and incorporation into these pathways was at a higher percentage during growth at higher pH. This corresponded to increased growth rates on urea as the sole N source with increasing pH. We propose this ability to incorporate C from urea represents yet another competitive advantage for this cyanobacterium during dense algal blooms.
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Microcystin (MCY)-producing harmful cyanobacterial blooms (cHABs) are an annual occurrence in Lake Erie, and buoys equipped with water quality sondes have been deployed to help researchers and resource managers track cHABs. The objective of this study was to determine how well water quality sondes attached to buoys measure total algae and cyanobacterial biomass and water turbidity. Water samples were collected next to two data buoys in western Lake Erie (near Gibraltar Island and in the Sandusky subbasin) throughout summers 2015, 2016, and 2017 to determine correlations between buoy sonde data and water sample data. MCY and nutrient concentrations were also measured. Significant (P < 0.001) linear relationships (R2 > 0.75) occurred between cyanobacteria buoy and water sample data at the Gibraltar buoy, but not at the Sandusky buoy; however, the coefficients at the Gibraltar buoy differed significantly across years. There was a significant correlation between buoy and water sample total chlorophyll data at both buoys, but the coefficient varied considerably between buoys and among years. Total MCY concentrations at the Gibraltar buoy followed similar temporal patterns as buoy and water sample cyanobacterial biomass data, and the ratio of MCY to cyanobacteria-chlorophyll decreased with decreased ambient nitrate concentrations. These results suggest that buoy data are difficult to compare across time and space. Additionally, the inclusion of nitrate concentration data can lead to more robust predictions on the relative toxicity of blooms. Overall, deployed buoys with sondes that are routinely cleaned and calibrated can track relative cyanobacteria abundance and be used as an early warning system for potentially toxic blooms.
There is increasing concern that accelerating environmental change attributed to human-induced warming of the planet may substantially alter the patterns, distribution and intensity of Harmful Algal Blooms (HABs). Changes in temperature, ocean acidification, precipitation, nutrient stress or availability, and the physical structure of the water column all influence the productivity, composition, and global range of phytoplankton assemblages, but large uncertainty remains about how integration of these climate drivers might shape future HABs. Presented here are the collective deliberations from a symposium on HABs and climate change where the research challenges to understanding potential linkages between HABs and climate were considered, along with new research directions to better define these linkages. In addition to the likely effects of physical (temperature, salinity, stratification, light, changing storm intensity), chemical (nutrients, ocean acidification), and biological (grazer) drivers on microalgae (senso lato), symposium participants explored more broadly the subjects of cyanobacterial HABs, benthic HABs, HAB effects on fisheries, HAB modelling challenges, and the contributions that molecular approaches can bring to HAB studies. There was consensus that alongside traditional research, HAB scientists must set new courses of research and practices to deliver the conceptual and quantitative advances required to forecast future HAB trends. These different practices encompass laboratory and field studies, long-term observational programs, retrospectives, as well as the study of socioeconomic drivers and linkages with aquaculture and fisheries. In anticipation of growing HAB problems, research on potential mitigation strategies should be a priority. It is recommended that a substantial portion of HAB research among laboratories be directed collectively at a small sub-set of HAB species and questions in order to fast-track advances in our understanding. Climate-driven changes in coastal oceanographic and ecological systems are becoming substantial, in some cases exacerbated by localized human activities. That, combined with the slow pace of decreasing global carbon emissions, signals the urgency for HAB scientists to accelerate efforts across disciplines to provide society with the necessary insights regarding future HAB trends.
Aseptic technique has historically served as a fundamental practice in microbiology, helping maintain culture purity and integrity. This technique has been widely encouraged and employed for use with cultures of heterotrophic bacteria as well as freshwater and marine algae. Yet, recent observations have suggested these approaches may bring their own influences. We observed variations in growth amongst replicate experimental cyanobacterial cultures upon flaming of the culture tube opening during sample transfer and collection. Investigation revealed the pH of culture media had decreased from the initial pH established during media preparation. Flaming of sterile culture media alone confirmed a significant decrease, by as much as 1.7 pH units, and correlated with increased flaming events over time. We hypothesized that the causative factor was the introduction of carbon dioxide into the media. To test this hypothesis, qualitative and quantitative analyses were used to identify the primary driver of pH decline. We further assessed the direct effects of flaming and subsequent pH changes on Microcystis aeruginosa cultures, showing flame-driven pH changes and/or the introduction of carbon dioxide influenced experimental results. Our observations provide a cautionary tale of how classic and well-accepted approaches may not always provide the results promised, suggesting new approaches may be necessary in research areas assessing pH or carbon related-effects on microbial communities.
Toxic cyanobacterial harmful algal blooms (cyanoHABs) are one of the most significant threats to the security of Earth's surface freshwaters. In the United States, the Federal Water Pollution Control Act of 1972 (i.e., the Clean Water Act) requires that states report any waterbody that fails to meet applicable water quality standards. The problem is that for fresh waters impacted by cyanoHABs, no scientifically-based framework exists for making this designation. This study describes the development of a data-based framework using the Ohio waters of western Lake Erie as an exemplar for large lakes impacted by cyanoHABs. To address this designation for Ohio's open waters, the Ohio Environmental Protection Agency (EPA) assembled a group of academic, state and federal scientists to develop a framework that would determine the criteria for Ohio EPA to consider in deciding on a recreation use impairment designation due to cyanoHAB presence. Typically, the metrics are derived from on-lake monitoring programs, but for large, dynamic lakes such as Lake Erie, using criteria based on discrete samples is problematic. However, significant advances in remote sensing allows for the estimation of cyanoHAB biomass of an entire lake. Through multiple years of validation, we developed a framework to determine lake-specific criteria for designating a waterbody as impaired by cyanoHABs on an annual basis. While the criteria reported in this manuscript are specific to Ohio's open waters, the framework used to determine them can be applied to any large lake where long-term monitoring data and satellite imagery are available.
A mix of adaptive strategies enable diatoms to sustain rapid growth in dynamic ocean regions, making diatoms one of the most productive primary producers in the world. We illustrate one such strategy off coastal California that facilitates continued, high, cell division rates despite silicic acid stress. Using a fluorescent dye to measure single‐cell diatom silica production rates, silicification (silica per unit area) and growth rates we show diatoms decrease silicification and maintain growth rate when silicon concentration limits silica production rates. While this physiological response to silicon stress was similar across taxa, in situ silicic acid concentration limited silica production rates by varying degrees for taxa within the same community. Despite this variability among taxa, silicon stress did not alter the contribution of specific taxa to total community silica production or to community composition. Maintenance of division rate at the expense of frustule thickness decreases cell density which could affect regional biogeochemical cycles. The reduction in frustule silicification also creates an ecological tradeoff: thinner frustules increase susceptibility to predation but reducing Si quotas maximizes cell abundance for a given pulse of silicic acid, thereby favoring a larger eventual population size which facilitates diatom persistence in habitats with pulsed resource supplies. This article is protected by copyright. All rights reserved.