Available via license: CC BY 4.0
Content may be subject to copyright.
fmicb-12-598736 February 19, 2021 Time: 14:22 # 1
ORIGINAL RESEARCH
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
*Correspondence:
Steven W. Wilhelm
wilhelm@utk.edu
†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
Citation:
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
INTRODUCTION
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.
Frontiers in Microbiology | www.frontiersin.org 1February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 2
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 Erie’s 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 Erie’s 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 HCO3−acquisition (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.
MATERIALS AND METHODS
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
Frontiers in Microbiology | www.frontiersin.org 2February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 3
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 Na2SiO3•9H2O) (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 26◦C,
with orbital shaking at 70 rpm, and a light intensity of
approximately 55–60 µmol photons m−2s−1on 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 100◦C). 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
Frontiers in Microbiology | www.frontiersin.org 3February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 4
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-
dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole]
(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 Na2SiO3•9H2O. 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 26◦C and approximately 55–60 µmol
photons m−2s−1on 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 (−80◦C). 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 −80◦C 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 = 25.3◦C; 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 100◦C). 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 4◦C
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 −80◦C 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
Frontiers in Microbiology | www.frontiersin.org 4February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 5
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).
RESULTS
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.
Frontiers in Microbiology | www.frontiersin.org 5February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 6
Zepernick et al. Alkaline pH Decreases Diatom Viability
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.
Frontiers in Microbiology | www.frontiersin.org 6February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 7
Zepernick et al. Alkaline pH Decreases Diatom Viability
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 R2≥0.8577)
(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.
DISCUSSION
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
Growth
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
Frontiers in Microbiology | www.frontiersin.org 7February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 8
Zepernick et al. Alkaline pH Decreases Diatom Viability
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.
Frontiers in Microbiology | www.frontiersin.org 8February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 9
Zepernick et al. Alkaline pH Decreases Diatom Viability
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 Na2SiO3•9H2O).
Frontiers in Microbiology | www.frontiersin.org 9February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 10
Zepernick et al. Alkaline pH Decreases Diatom Viability
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.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s.
AUTHOR CONTRIBUTIONS
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.
FUNDING
This work was funded through the Bowling Green State
University Great Lakes Center for Fresh Waters and Human
Frontiers in Microbiology | www.frontiersin.org 10 February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 11
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).
ACKNOWLEDGMENTS
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.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.598736/full#supplementary-material
REFERENCES
Allinger, L. E., and Reavie, E. D. (2013). The ecological history of Lake Erie as
recorded by the phytoplankton community. J. Great Lakes Res. 39, 365–382.
doi: 10.1016/j.jglr.2013.06.014
Amo, Y. D., and Brzezinski, M. A. (1999). The chemical form of dissolved Si taken
up by marine diatoms. J. Phycol. 35, 1162–1170. doi: 10.1046/j.1529- 8817.1999.
3561162.x
Anderson, D. M. (2009). Approaches to monitoring, control and management of
harmful algal blooms (HABs). Ocean Coast. Manag. 52, 342–347. doi: 10.1016/
j.ocecoaman.2009.04.006
Andersson, A., Haecky, P., and Hagström, Å (1994). Effect of temperature and light
on the growth of micro-nano-and pico-plankton: impact on algal succession.
Mar. Biol. 120, 511–520. doi: 10.1007/bf00350071
Azam, F., Hemmingsen, B. B., and Volcani, B. E. (1974). Role of silicon in diatom
metabolism. Arch. Microbiol. 97, 103–114. doi: 10.1007/bf00403050
Beutler, M., Wiltshire, K. H., Meyer, B., Moldaenke, C., Lüring, C., Meyerhöfer, M.,
et al. (2002). A fluorometric method for the differentiation of algal populations
in vivo and in situ.Photosynth. Res. 72, 39–53.
Bramburger, A. J., Reavie, E. D., Sgro, G., Estepp, L., Chraïbi, V. S., and Pillsbury, R.
(2017). Decreases in diatom cell size during the 20th century in the Laurentian
Great Lakes: a response to warming waters? J. Plankton Res. 39, 199–210.
doi: 10.1093/plankt/fbx009
Brzezinski, M. A., Olson, R. J., and Chisholm, S. W. (1990). Silicon availability
and cell-cycle progression in marine diatoms. Mar. Ecol. Prog. Ser. 67, 83–96.
doi: 10.3354/meps067083
Bullerjahn, G. S., Mckay, R. M., Davis, T. W., Baker, D. B., Boyer, G. L.,
D’anglada, L. V., et al. (2016). Global solutions to regional problems: collecting
global expertise to address the problem of harmful cyanobacterial blooms.
A Lake Erie case study. Harmful Algae 54, 223–238. doi: 10.1016/j.hal.2016.
01.003
Chaffin, J. D., Kane, D. D., Stanislawczyk, K., and Parker, E. M. (2018). Accuracy
of data buoys for measurement of cyanobacteria, chlorophyll, and turbidity
in a large lake (Lake Erie, North America): implications for estimation of
cyanobacterial bloom parameters from water quality sonde measurements.
Environ. Sci. Pollut. Res. 25, 25175–25189. doi: 10.1007/s11356-018-2612-z
Clement, R., Jensen, E., Prioretti, L., Maberly, S. C., and Gontero, B.
(2017). Diversity of CO2-concentrating mechanisms and responses to CO2
concentration in marine and freshwater diatoms. J. Exp. Bot. 68, 3925–3935.
doi: 10.1093/jxb/erx035
Davis, T. W., Stumpf, R., Bullerjahn, G. S., Mckay, R. M. L., Chaffin, J. D.,
Bridgeman, T. B., et al. (2019). Science meets policy: a framework for
determining impairment designation criteria for large waterbodies affected by
cyanobacterial harmful algal blooms. Harmful Algae 81, 59–64. doi: 10.1016/j.
hal.2018.11.016
Flynn, K. J., Clark, D. R., Mitra, A., Fabian, H., Hansen, P. J., Glibert, P. M.,
et al. (2015). Ocean acidification with (de) eutrophication will alter future
phytoplankton growth and succession. Proc. R. Soc. B Biol. Sci. 282:20142604.
doi: 10.1098/rspb.2014.2604
Guillard, R. R., and Lorenzen, C. J. (1972). Yellow-green algae with chlorophyllide
C 1,2. J. Phycol. 8, 10–14. doi: 10.1111/j.0022-3646.1972.00010.x
Hansen, P. J. (2002). Effect of high pH on the growth and survival of marine
phytoplankton: implications for species succession. Aquat. Microb. Ecol. 28,
279–288. doi: 10.3354/ame028279
Hartig, J. H. (1987). Factors contributing to development of Fragilaria crontonensis
Kitton Pulses in Pigeon Bay waters of western Lake Erie. J. Great Lakes Res. 13,
65–77. doi: 10.1016/s0380-1330(87)71628-1
Hartig, J. H., and Wallen, D. G. (1986). The influence of light and temperature on
growth and photosynthesis of Fragilaria crotonensis Kitton. J. Freshw. Ecol. 3,
371–382.
Hervé, V., Derr, J., Douady, S., Quinet, M., Moisan, L., and Lopez, P. J.
(2012). Multiparametric analyses reveal the pH-dependence of silicon
biomineralization in diatoms. PLoS One 7:e46722. doi: 10.1371/journal.pone.
0046722
Hildebrand, M., Lerch, S. J., and Shrestha, R. P. (2018). Understanding diatom cell
wall silicification—moving forward. Front. Mar. Sci. 5:125. doi: 10.3389/fmars.
2018.00125
Ke, Z., Xie, P., and Guo, L. (2008). Controlling factors of spring–summer
phytoplankton succession in Lake Taihu (Meiliang Bay. China). Hydrobiologia
607, 41–49. doi: 10.1007/s10750-008-9365-5
Kranzler, C. F., Krause, J. W., Brzezinski, M. A., Edwards, B. R., Biggs, W. P.,
Maniscalco, M., et al. (2019). Silicon limitation facilitates virus infection and
mortality of marine diatoms. Nat. Microbiol. 4, 1790–1797. doi: 10.1038/
s41564-019-0502-x
Krausfeldt, L. E., Farmer, A. T., Castro Gonzalez, H., Zepernick, B. N., Campagna,
S. R., and Wilhelm, S. W. (2019). Urea is both a carbon and nitrogen source
for Microcystis aeruginosa: tracking 13C incorporation at bloom pH conditions.
Front. Microbiol. 10:1064. doi: 10.3389/fmicb.2019.01064
Leblanc, K., and Hutchins, D. A. (2005). New applications of a biogenic silica
deposition fluorophore in the study of oceanic diatoms. Limnol. Oceanogr.
Methods 3, 462–476. doi: 10.4319/lom.2005.3.462
Martin-Jézéquel, V., Hildebrand, M., and Brzezinski, M. A. (2000). Silicon
metabolism in diatoms: implications for growth. J. Phycol. 36, 821–840. doi:
10.1046/j.1529-8817.2000.00019.x
Mcnair, H. M., Brzezinski, M. A., and Krause, J. W. (2018). Diatom populations in
an upwelling environment decrease silica content to avoid growth limitation.
Environ. Microbiol. 20, 4184–4193. doi: 10.1111/1462-2920.14431
Michalak, A. M., Anderson, E. J., Beletsky, D., Boland, S., Bosch, N. S.,
Bridgeman, T. B., et al. (2013). Record-setting algal bloom in Lake Erie caused
by agricultural and meteorological trends consistent with expected future
conditions. Proc. Natl. Acad. Sci. U.S.A. 110, 6448–6452. doi: 10.1073/pnas.
1216006110
Otzen, D. (2012). The role of proteins in biosilicification. Scientifica 2012:867562.
Paerl, H. W., Scott, J. T., Mccarthy, M. J., Newell, S. E., Gardner, W. S., Havens,
K. E., et al. (2016). It takes two to tango: when and where dual nutrient (N &
P) reductions are needed to protect lakes and downstream ecosystems. Environ.
Sci. Technol. 50, 10805–10813. doi: 10.1021/acs.est.6b02575
Peng, G., Martin, R. M., Dearth, S. P., Sun, X., Boyer, G. L., Campagna, S. R.,
et al. (2018). Seasonally relevant cool temperatures interact with n chemistry
to increase microcystins produced in lab cultures of Microcystis aeruginosa
NIES-843. Environ. Sci. Technol. 52, 4127–4136. doi: 10.1021/acs.est.7b0
6532
Petrou, K., Baker, K. G., Nielsen, D. A., Hancock, A. M., Schulz, K. G., and
Davidson, A. T. (2019). Acidification diminishes diatom silica production in
the Southern Ocean. Nat. Clim. Chang. 9, 781–786. doi: 10.1038/s41558-019-
0557-y
Pound, H. L., Gann, E. R., Tang, X., Krausfeldt, L. E., Huff, M., Staton, M.,
et al. (2020). The “neglected viruses” of Taihu: abundant transcripts for viruses
Frontiers in Microbiology | www.frontiersin.org 11 February 2021 | Volume 12 | Article 598736
fmicb-12-598736 February 19, 2021 Time: 14:22 # 12
Zepernick et al. Alkaline pH Decreases Diatom Viability
infecting eukaryotes and their potenital role in phytoplankton succession.
Front. Microbiol. 11:338. doi: 10.3389/fmicb.2020.00338
Raven, J. (1981). Nutrient transport in microalgae. Advances Microb. Physiol. 21,
47–226. doi: 10.1016/s0065-2911(08)60356-2
Raven, J. A. (2010). Inorganic carbon acquisition by eukaryotic algae: four current
questions. Photosynth. Res. 106, 123–134. doi: 10.1007/s11120-010- 9563-7
Reavie, E. D., Barbiero, R. P., Allinger, L. E., and Warren, G. J. (2014).
Phytoplankton trends in the Great Lakes, 2001–2011. J. Great Lakes Res. 40,
618–639. doi: 10.1016/j.jglr.2014.04.013
Reavie, E. D., Cai, M., Twiss, M. R., Carrick, H. J., Davis, T. W., Johengen, T. H.,
et al. (2016). Winter–spring diatom production in Lake Erie is an important
driver of summer hypoxia. J. Great Lakes Res. 42, 608–618. doi: 10.1016/j.jglr.
2016.02.013
Riedel, G. F., and Nelson, D. M. (1985). Silicon uptake by algae with no kown
Si requirement. II. Strong pH dependence of uptake kinetic parameters in
Phaeodactylum tricornutum (Bacillariophyceae) 1. J. Phycol. 21, 168–171. doi:
10.1111/j.0022-3646.1985.00168.x
Sandrini, G., Tann, R. P., Schuurmans, J. M., Van Beusekom, S. A., Matthijs,
H. C., and Huisman, J. (2016). Diel variation in gene expression of the
CO2-concentrating mechanism during a harmful cyanobacterial bloom. Front.
Microbiol. 7:551. doi: 10.3389/fmicb.2016.00551
Saxton, M. A., D’souza, N. A., Bourbonniere, R. A., Mckay, R. M. L., and Wilhelm,
S. W. (2012). Seasonal Si: C ratios in Lake Erie diatoms—evidence of an active
winter diatom community. J. Great Lakes Res. 38, 206–211. doi: 10.1016/j.jglr.
2012.02.009
Shapiro, J. (1990). Current beliefs regarding dominance by blue-greens: the case
for the importance of CO2 and pH. Internationale Vereinigung für Theoretische
und Angewandte Limnologie Verhandlungen 24, 38–54. doi: 10.1080/03680770.
1989.11898689
Shruthi, M., and Rajashekhar, M. (2014). Effect of salinity and pH on the growth
and biomass production in the four species of estuarine cyanobacteria. J. Algal
Biomass Utln. 5, 29–36.
Steffen, M. M., Belisle, B. S., Watson, S. B., Boyer, G. L., Bourbonniere, R. A.,
and Wilhelm, S. W. (2015). Metatranscriptomic evidence for co-occurring
top-down and bottom-up controls on toxic cyanobacterial communities. Appl.
Environ. Microbiol. 81, 3268–3276. doi: 10.1128/aem.04101-14
Steffen, M. M., Belisle, B. S., Watson, S. B., Boyer, G. L., and Wilhelm, S. W. (2014).
Status, causes and controls of cyanobacterial blooms in Lake Erie. J. Great Lakes
Res. 40, 215–225. doi: 10.1016/j.jglr.2013.12.012
Sullivan, C. (1977). Diatom mineralization of silicic acid. II. Regulation of Si (OH)
4 transport rates during the cell cycle of Navicula pelliculosa 1. J. Phycol. 13,
86–91. doi: 10.1111/j.1529-8817.1977.tb02892.x
Suttle, C. A., and Fuhrman, J. A. (2010). Aquatic Viral Ecology. Manual Aquat.
Viral Ecol. 145. doi: 10.4319/mave.2010.978-0-9845591-0-7.145
Taraldsvik, M., and Myklestad, S. (2000). The effect of pH on growth rate,
biochemical composition and extracellular carbohydrate production of the
marine diatom Skeletonema costatum.Eur. J. Phycol. 35, 189–194. doi: 10.1080/
09670260010001735781
Thamatrakoln, K., and Hildebrand, M. (2008). Silicon uptake in diatoms revisited:
a model for saturable and nonsaturable uptake kinetics and the role of silicon
transporters. Plant Physiol. 146, 1397–1407. doi: 10.1104/pp.107.107094
Twiss, M., Mckay, R., Bourbonniere, R., Bullerjahn, G., Carrick, H., Smith, R., et al.
(2012). Diatoms abound in ice-covered Lake Erie: an investigation of offshore
winter limnology in Lake Erie over the period 2007 to 2010. J. Great Lakes Res.
38, 18–30. doi: 10.1016/j.jglr.2011.12.008
Vanderploeg, H. A., Liebig, J. R., Carmichael, W. W., Agy, M. A., Johengen,
T. H., Fahnenstiel, G. L., et al. (2001). Zebra mussel (Dreissena polymorpha)
selective filtration promoted toxic Microcystis blooms in Saginaw Bay (Lake
Huron) and Lake Erie. Can. J. Fish. Aquat. Sci. 58, 1208–1221. doi: 10.1139/f01-
066
Vardi, A., Thamatrakoln, K., Bidle, K. D., and Falkowski, P. G. (2009).
Diatom genomes come of age. Genome Biol. 9:245. doi: 10.1186/gb-2008-9-
12-245
Verspagen, J. M., Van De Waal, D. B., Finke, J. F., Visser, P. M., Van Donk, E., and
Huisman, J. (2014). Rising CO2levels will intensify phytoplankton blooms in
eutrophic and hypertrophic lakes. PLoS One 9:e104325. doi: 10.1371/journal.
pone.0104325
Vrieling, E. G., Gieskes, W., and Beelen, T. P. (1999). Silicon deposition in diatoms:
control by the pH inside the silicon deposition vesicle. J. Phycol. 35, 548–559.
doi: 10.1046/j.1529-8817.1999.3530548.x
Watanabe, M., Kawachi, M., Hiroki, M., and Kasai, F. (2000). NIES-Collection
List of Strains. Microalgae and Protozoa. National Institute for Environmental
Studies. Tsukuba: Environment Agency, 159.
Wells, M. L., Karlson, B., Wulff, A., Kudela, R., Trick, C., Asnaghi, V., et al. (2020).
Future HAB science: directions and challenges in a changing climate. Harmful
Algae 91:101632. doi: 10.1016/j.hal.2019.101632
Welschmeyer, N. A. (1994). Fluorometric analysis of chlorophyll a in the presence
of chlorophyll b and pheopigments. Limnol. Oceanogr. 39, 1985–1992. doi:
10.4319/lo.1994.39.8.1985
Werner, D. (1966). Die kieselsäure im stoffwechsel von Cyclotella cryptica
Reimann. Lewin und Guillard. Archiv für Mikrobiologie 55, 278–308. doi:
10.1007/bf00410249
Wilhelm, S. W., Bullerjahn, G. S., and McKay, R. M. L. (2020). The complicated
and confusing ecology of Microcystis blooms. mBio 11:e00529-20. doi: 10.1128/
mBio.00529-20
Wilhelm, S. (2017). CT Media. Berkeley, CA: protocols.io. doi: 10.17504/protocols.
io.iizccf6 (accessed Jan 29, 2021).
Wilson, A., Kinney, J. N., Zwart, P. H., Punginelli, C., D’haene, S., Perreau, F., et al.
(2010). Structural determinants underlying photoprotection in the photoactive
orange carotenoid protein of cyanobacteria. J. Biol. Chem. 285, 18364–18375.
doi: 10.1074/jbc.m110.115709
Zepernick, B. N., Krausfeldt, L. E., and Wilhelm, S. W. (2020). Flaming as
part of aseptic technique increases CO2(g) and decreases pH in freshwater
culture media. Limnol. Oceanogr. Methods bioRxiv [Prepring] doi: 10.1101/78
0353
Zepernick, B. N., Saxton, M., and Wilhelm, S. W. (2019). Quantifying Biogenic
Silica (bSi) Deposition Rates Adapted Method. Berkeley, CA: Protocols.io. doi:
10.17504/protocols.io.72vhqe6 (accessed Jan 29, 2021).
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)
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Microbiology | www.frontiersin.org 12 February 2021 | Volume 12 | Article 598736
