Nitrogen limitation, toxin synthesis potential, and toxicity of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida, during the 2016 state of emergency event

Abstract

Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0–25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 μg L⁻¹, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.

Full text

RESEARCH ARTICLE
Nitrogen limitation, toxin synthesis potential,
and toxicity of cyanobacterial populations in
Lake Okeechobee and the St. Lucie River
Estuary, Florida, during the 2016 state of
emergency event
Benjamin J. Kramer
1
, Timothy W. Davis
2
, Kevin A. Meyer
3,4
, Barry H. Rosen
5
, Jennifer
A. Goleski
1
, Gregory J. Dick
4
, Genesok Oh
1
, Christopher J. Gobler
1
*
1School of Marine and Atmospheric Sciences, Stony Brook University, NY, United States of America,
2Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, United States of
America, 3Cooperative Institute for Great Lakes Research (CIGLR), University of Michigan, Ann Arbor, MI,
United States of America, 4Department of Earth and Environmental Sciences, University of Michigan, Ann
Arbor, MI, United States of America, 5USGS, Orlando, FL, United States of America
*christopher.gobler@stonybrook.edu
Abstract
Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that
can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-
induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake
Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a
state of emergency. This study reports on measurements and nutrient amendment experi-
ments performed in this freshwater-estuarine ecosystem (salinity 0–25 PSU) during and
after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-
phosphorus ratios <6, while Microcystis dominated (>95%) phytoplankton inventories from
the lake to the central part of the estuary. Chlorophyll aand microcystin concentrations
peaked (100 and 34 μg L
-1
, respectively) within Lake Okeechobee and decreased east-
wards. Metagenomic analyses indicated that genes associated with the production of micro-
cystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and
multiple diazotrophic genera, respectively. There were highly significant correlations
between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance
across all surveyed sites (p<0.001), suggesting high levels of nitrogen supported the pro-
duction of microcystin during this event. Consistent with this, experiments performed with
low salinity water from the St. Lucie River during the event indicated that algal biomass was
nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were
significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups
displayed nitrogen-limitation. These results indicate that monitoring and regulatory strate-
gies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing
loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 1 / 26
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OPEN ACCESS
Citation: Kramer BJ, Davis TW, Meyer KA, Rosen
BH, Goleski JA, Dick GJ, et al. (2018) Nitrogen
limitation, toxin synthesis potential, and toxicity of
cyanobacterial populations in Lake Okeechobee
and the St. Lucie River Estuary, Florida, during the
2016 state of emergency event. PLoS ONE 13(5):
e0196278. https://doi.org/10.1371/journal.
pone.0196278
Editor: Todd Miller, University of Wisconsin
Milwaukee, UNITED STATES
Received: January 12, 2018
Accepted: April 10, 2018
Published: May 23, 2018
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was funded by Chicago
Community Trust, received by CJG, http://cct.org.
The funders had no role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. This study was also
supported by NOAA Great Lakes Environmental

Introduction
Climate change and eutrophication can promote the dominance of cyanobacteria among
freshwater phytoplankton communities [1–3]. Human activities that enrich freshwater ecosys-
tems with nutrients have been linked to the emergence of dense cyanobacterial blooms that
can attenuate light and create hypoxic (low-oxygen) zones, deleteriously impacting benthic
flora as well as pelagic and benthic fauna, respectively [4]. Shallow, non-stratifying lakes are
especially vulnerable to excessive nutrient levels and the dominance of harmful cyanobacteria
within phytoplankton communities [5,6]. Many of the cyanobacterial genera that bloom
under these conditions, including Aphanizomenon,Dolichospermum (Anabaena), Cylindros-
permopsis,Planktothrix, and Microcystis, can be comprised of strains that produce toxins that
can impact the health of aquatic life and humans [7–9]. Many Microcystis spp., for instance,
can produce the hepatotoxin microcystin [10].
As harmful cyanobacterial blooms recur and/or intensify in freshwater bodies, it is impor-
tant to identify the conditions that promote these events, as well as the causative cyanobacterial
genera. It is traditionally assumed that phosphorus (P)-availability controls primary productiv-
ity in freshwater ecosystems [11,12], and the proportion of cyanobacteria that comprise phy-
toplankton communities can be inversely correlated with total nitrogen-to-phosphorus (N:P)
ratios [13]. The decrease in N:P can lead to the dominance of diazotrophic cyanobacterial gen-
era capable of converting dinitrogen (N
2
) gas to ammonia (NH
3
) [14]. Recent meta-analyses,
however, have demonstrated that the growth and toxin production of some non-diazotrophic
cyanobacterial genera (e.g. Microcystis and Planktothrix) can be controlled by nitrogen (N)
[15,16]. In Lake Taihu, China, for instance, Microcystis spp. outcompete diazotrophs partly
because of the supply of regenerated N and P from resuspended sediments in this shallow (~2
m mean depth) lake [5]. Other factors that may facilitate the dominance of non-N
2
fixing cya-
nobacteria such as Microcystis over N
2
fixers include reductions in water column irradiance
[17] and elevated temperature [3,18,19].
Lake Okeechobee is the largest lake in the southeastern United States and has been sub-
jected to eutrophication since the 1970s [20,21]. The lake exhibits low submerged plant bio-
mass, a benthic invertebrate community dominated by oligochaetes, and a phytoplankton
community mainly comprised of cyanobacteria, all of which significantly limit the lake’s ability
to sequester phosphorus [22]. While at least some regions of Lake Okeechobee were shown to
exhibit N and P co-limitation in the early 1990s [23], Havens et al. (2016) reported that hurri-
canes in the mid-2000s led to some of the largest, most toxic blooms of M.aeruginosa ever
observed in Lake Okeechobee, possibly due in part to the prolonged retention of soluble reac-
tive phosphorus, inorganic nitrogen, and total suspended solids in the water column [24]. Ulti-
mately, however, it is unclear which nutrient or combination of nutrients regulates these
blooms, or whether nutrient limitation characteristics change over time and under different
environmental conditions.
During the 2015–16 winter season, the Florida peninsula was subjected to unusually high
temperatures, storm activity, and rainfall, corresponding to the concurrent El Niño event [25].
Consequently, the lake exhibited a dramatic increase in water level in early 2016 [26], necessi-
tating the release of billions of gallons of water through canals, one of which led to the St. Lucie
River and Estuary in the east. A dramatic, persistent increase in nitrate and decrease in salinity
throughout the estuary made conditions optimal for a Microcystis bloom to develop [27]. This
bloom lasted from May to mid-July and spread throughout this brackish ecosystem [27], lead-
ing Florida to declare a state of emergency due to socioeconomic impacts and human health
concerns. In addition to the dominance of Microcystis, other potential toxin producers were
identified [28]. For this study, transect surveys and experiments were performed from Lake
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 2 / 26
Research Laboratory, received by TWD, https://
www.glerl.noaa.gov. The funders had no role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. This
study was also supported by University of
Michigan Cooperative Institute for Great Lakes
Research, received by TWD, https://ciglr.seas.
umich.edu. The funders had no role in the study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.

Okeechobee through the St. Lucie River and Estuary and the Indian River Lagoon during
and after this bloom event. The objective of the study was to characterize nutrient levels, the
phytoplankton community, their toxins, and toxin synthesis potential via sequencing of genes
associated with microcystin and saxitoxin synthesis. Furthermore, nutrient amendment exper-
iments were conducted to assess limitations on algal and cyanobacterial growth. During and
after the bloom event, it was determined that algal growth and toxicity were tightly coupled to
N availability.
Materials and methods
Area of study and sample collection
Sampling and experiments were performed in July and September 2016, with samples col-
lected from sites in Lake Okeechobee, the canal leading to the St. Lucie River (C-44), the
St. Lucie River Estuary, and the Indian River Lagoon (Fig 1 and Table 1). Water samples from
July (N = 20) and September (N = 22) were collected over the course of 3 and 4 days, respec-
tively. Waterfront areas were accessed from public access points and the collection of small
volumes of water is not a regulated activity in Florida. Temperature (˚C), salinity (PSU) and
dissolved oxygen (O
2
) concentrations (mg L
-1
) were measured at 22 sites using a YSI 556
probe (Table 1). Surface water was collected from seven sites that represented a salinity gradi-
ent across the system to characterize the phytoplankton community, their toxin synthesis
potential, as well as toxin and nutrient concentrations. At four sites in July and three sites in
September, large volumes of water from Lake Okeechobee to the river estuary were collected
and nutrient amendment experiments were performed. For each of the seven full sampling
sites, samples were collected and analyzed according to previously established protocols
described below. For enumeration of phytoplankton, duplicate samples were preserved in
Lugol’s iodine (5% final concentration) and microscopically quantified to at least the genus
level based on morphological traits such as cell dimension, arrangement of cells in colonies or
filaments, and the presence of specialized structures (i.e., aerotopes, heterocytes, akinetes). For
the precise volume of the subsample for counting, a 4-place balance (Ohaus Explorer EX224,
Ohaus Corporation, Parsippany, New Jersey, USA) was used (typically 0.0175 mL or less) and
dispersed under a 22 mm
2
cover slip. This method allows samples to be examined at 400x with
an Olympus BX51 research microscope (Olympus America, Waltham, Massachusetts, USA);
random strip-counts of known width allowed a precise calculation of the volume counted to
obtain an accurate cell count per unit volume of the original sample.
For toxin analyses, 10 mL of whole water was stored at -20˚Cprior to quantification. Micro-
cystin and saxitoxin concentrations were quantified using Abraxis enzyme-linked immuno-
sorbent assays (ELISA) following the manufacturer’s protocols. Prior to running a microcystin
ELISA, thawed samples were lysed and filtered using materials provided by Abraxis. ELISA
limits of detection for microcystin and saxitoxin were 0.15 and 0.02 μg L
-1
, respectively. As the
microcystin antibody provided in the Abraxis ELISA kit specifically binds to congeners with
the βamino acid (ADDA) group, toxin concentrations were reported as microcystin–ELISA
equivalents. Duplicate 20 mL, whole water samples were collected and stored frozen for total
nutrient analyses whereas duplicate dissolved nutrient samples were passed through pre-com-
busted (450˚C for 2 h) glass fiber (GFF) filters and stored frozen. GFF filters were also stored
frozen until chlorophyll awas extracted with 5 mL 90% acetone and quantified to approximate
algal biomass on a Turner Designs fluorometer. Concentrations of total N, total P, nitrite and
nitrate (NO
2-
+NO
3-
), ammonia (NH
3
), and soluble reactive phosphorus (SRP)
-
were quanti-
fied on a Lachat Instruments autoanalyzer [29]. During the September transect, a Fluoroprobe
(bbe Moladaenke) was used to quantify the relative abundance of green algae, cyanobacteria,
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 3 / 26

Fig 1. Sampling sites around Lake Okeechobee (A) and on the St. Lucie River Estuary (B) in Florida, USA. Sites
sampled in both July and September 2016 (●) and September 2016 only (×) are represented. Inserts denote the general
region sites were located on the peninsula.
https://doi.org/10.1371/journal.pone.0196278.g001
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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Table 1. GPS coordinates as well as the collection dates (Month / Day) as well as the dissolved oxygen (mg L
-1
), temperature (˚C), and salinity (PSU) levels from the
July and September 2016 transects.
SITES JULY 2016 SEPTEMBER 2016
ID General Location Coordinates (LAT/LONG) Month / Day O
2
(mg L
-1
)
˚C PSU Month / Day O
2
(mg L
-1
)
˚C PSU
LO 1 Eastern
Shore
26.864296,
-80.63255
7/97.5 32.4 0.1 9/265.6 30.9 0.2
LO 2 Eastern
Shore
26.984979,
-80.620918
7/10 6.1 32.4 0.2 9/266.4 30.1 0.2
LO 3 Southwestern Shore 26.760595,
-80.918512
- - - - 9/28 3.6 29.6 0.2
LO 4 Western Shore 26.992225,
-81.067107
- - - - 9/28 1.7 30.1 0.2
LO 5 Northern Shore 27.147202,
-80.869267
- - - - 9/28 3.4 30.2 0.1
LO 6 Northern Shore 27.191742,
-80.76324
- - - - 9/29 2.7 26.9 0.1
Canal 1 C-44
Canal
27.012359,
-80.455056
7/10 6.5 33.9 0.2 9/29 5.8 29.7 0.2
Canal 2 C-44
Canal
27.09528,
-80.296074
7/10 3.6 32.8 0.2 9/29 4.8 29.4 0.2
SLE 1 SF 27.11349,
-80.28313
7/8, 7/96.1 32.6 0.2 9/266.4 30.0 0.2
SLE 2 SF 27.12091,
-80.26969
7/8 6.6 32.2 0.2 9/27 6.1 29.8 0.2
SLE 3 SF 27.13966,
-80.261706
7/8 7.7 33.3 0.2 9/27 5.8 29.9 0.2
SLE 4 SF 27.15646,
-80.25502
7/8 8.5 34.1 0.2 9/27 5.7 29.9 0.2
SLE 5 SF 27.17057,
-80.25821
7/8 6.7 33.1 0.2 9/27 5.4 29.8 0.2
SLE 6 ME 27.18850,
-80.26478
7/8 8.7 32.9 2.2 9/27 5.5 29.8 0.2
SLE 6.5 ME 27.19989,
-80.264114
7/96.9 33.5 3.2 9/265.3 30.2 0.8
SLE 7 ME 27.20684,
-80.26859
7/8 5.9 33.1 3.6 9/27 7.4 31.0 1.8
SLE 8 NF 27.26080,
-80.33047
7/8 6.2 33.9 0.5 9/27 4.8 29.1 0.4
SLE 9 NF 27.22998,
-80.29655
7/8 8.2 34.1 0.7 9/27 5.6 31.3 0.8
SLE 10 ME 27.20792,
-80.25105
7/8, 7/97.8 33.7 7.4 9/26 5.2 30.4 4.2
SLE 11 IRL 27.20509,
-80.21291
7/8 6.8 32.6 12.6 9/27 5.5 30.6 5.7
SLE 12 IRL 27.16769,
-80.19385
7/8 6.8 31.1 27.5 9/27 5.6 30.0 10.3
SLE 13 SLI 27.16516,
-80.16748
7/8 7.0 30.1 32.0 9/27 5.6 29.7 6.0
LO = Lake Okeechobee, Canal = drainage canal connecting the lake to the river estuary, SLE = Saint Lucie Estuary. Sites LO 3–6 were not included until the September
2016 transect.
= dates when water was collected for nutrient amendment experimentation. General locations of sampling sites are also given, emphasizing from which lake shoreline
(LO), canal (C-44), and estuary branch (SF = South Fork, ME = Middle Estuary, NF = North Fork, IRL = Indian River Lagoon, SLI = St. Lucie Inlet) samples were
collected
https://doi.org/10.1371/journal.pone.0196278.t001
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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and diatoms, which were distinguished based on the spectral characteristics of their photosyn-
thetic accessory pigments [30–32], in triplicate.
During the July transect, 20 mL of water was passed through a 2 μm polycarbonate filter
(Millipore) and stored at -80˚C. Frozen sample filters were thawed at room temperature before
being cut into small strips in a sterile petri plate using a flame sterilized blade. Filter strips were
then placed in a 2 mL microcentrifuge tube for DNA extraction using a Qiagen DNeasy1
Blood and Tissue Kit (Qiagen, Hilden, Germany). Briefly, samples were incubated with 100 μL
Qiagen ATL tissue lysis buffer, 300 μL Qiagen AL lysis buffer, and 30 μL proteinase K at
56˚C for 1 h with agitation, followed by vortexing at maximum speed for 10 minutes. Lysates
were homogenized with a QiaShredder™spin-column before purification according to the
DNeasy1protocol. DNA quantity and quality were assessed using a NanoDrop Lite (Thermo
Fisher Scientific, NanoDrop Products, Wilmington, DE, USA).
Analysis of toxin gene presence and abundance
Multiplex qPCR. Genes indicative of the genetic potential to produce microcystin/
nodularin (mcyE), saxitoxin (sxtA) and cylindrospermopsin (cyrA) were enumerated using a
commercially available multiplex qPCR kit (Phytoxigene CyanoDTec™Toxin Genes Test;
Diagnostic Technology, Sydney, Australia) modeled after the multiplex qPCR assay described
in Al-Tebrineh et al. (2012). Briefly, molecular grade H
2
O (80 μL) was added to each tube of a
CyanoDTec™cyanotoxin detection kit and processed following kit directions. A synthetic stan-
dard of known toxin gene copy (Diagnostic Technology) was assayed in serial dilutions to gen-
erate a standard curve spanning five orders of magnitude (100–1,000,000 copies) for each
target toxin gene. Amplification, per the kit protocol, was conducted in 96-well plates on a
7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) in a total volume
of 25 μL. Each sample was run in duplicate. Gene copies in each reaction were calculated using
the Applied Biosystems software and back-calculated to copies mL
-1
[33].
Metagenomic analysis. Samples were submitted to the University of Michigan DNA
Sequencing Core for sequencing on the Illumina1HiSeq™platform (4000 PE 150, Illumina,
Inc., San Diego, CA, USA). Sequence reads were checked for quality using FASTQC version
0.10.0 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)) and de-replicated (100%
identity over 100% of length) before adapter removal using Scythe and read trimming using
Sickle. Sequences were then assembled de novo with the iterative de Bruijn graph approach for
uneven sequencing depths (IDBA-UD) with the following parameters: minimum kmer size
52, maximum kmer size 92, step size 8, minimum contig 500.
Sequence reads remaining after read trimming as well as assembled scaffolds were searched
for microcystin and saxitoxin production genes using BLASTN against a database complete
target sequence for the mcyE and sxtA standards in the aforementioned multiplex qPCR kit,
provided by Diagnostic Technology (Belrose, Australia). Reads and scaffolds with BLAST hits
with >80% query coverage and >97% ID were then put through a standard nucleotide
BLAST against the NCBI nucleotide collection (nr/nt) for cyanobacteria for taxonomic identi-
fication. The coverage of toxin genes was calculated by mapping trimmed reads against assem-
bled scaffolds using BWA mapper with default parameters and then calculating coverage with
bedtools’ multicov function.
In addition to the aforementioned multiplex qPCR target sequences, the full primer and
probe sequences used in each of the mcyE and sxtA qPCR analyses were searched for in all
samples’ assembled scaffolds using BLASTN. To be considered positive for saxitoxin produc-
tion the forward primer, sequence probe, and reverse primer all needed to hit the same scaffold
with 100% identity for the entire lengths of the primer/probe and be aligned in order. All
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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scaffolds from samples positive for putative saxitoxin production were then binned into puta-
tive taxonomic groups with emergent self-organizing maps (ESOM) of tetranucleotide fre-
quencies (Robust ZT transformation) using Databionics ESOM Tools (http://databionic-
esom.sourceforge.net). Reference genomes were included for ESOM training and binning ref-
erence including two species of Dolichospermum (Anabaena), and one species each of Aphani-
zomenon,Cyanobium,Oscillatoria,Planktothrix,Pseudanabaena, and Synechococccus. Only
contigs longer than 3000 kb were considered for ESOM binning and contigs longer than 5 kb
were cut to fit into the 3–5 kb window. Other training parameters for the ESOM were: K-batch
algorithm (k = 0.15%) for 40 training epochs, standard best match search method with a local
best match search radius of 8, a Gaussian weight initialization, Euclidean data space function,
a starting training radius of 213 with linear cooling to 1, and a starting learning rate of 0.5 with
linear cooling to 0.1. Bins containing scaffolds with BLAST hits to sxtA genes were identified
and all scaffolds in that bin were extracted for taxonomic identification using a combination of
BLASTN of contigs against the Silva SSU Database version 119 and a standard nucleotide
BLAST against the NCBI nucleotide collection (nr/nt).
Nutrient amendment experimental design
During sample collection for the July and September transects, surface water was collected
from Lake Okeechobee (station LO 1), the canal connecting Lake Okeechobee and the
St. Lucie Estuary (SLE 1) and two stations in the main stem of the St. Lucie Estuary (SLE 6.5,
and SLE 10). Sample water was transferred into triplicate Nalgene, polycarbonate bottles (250
mL in July, 1 L in September) and amended with either 20 μM ammonium (NH
4+
), 2 μM
PO
43-
, or NH
4+
and PO
43-
(20 and 2 μM, respectively). Bottles were incubated in the eastern
extent of the St. Lucie River. As Microcystis descends deeper into the water column in response
to elevated irradiance [34], likely to avoid the harmful effects of elevated UV levels [35], bottles
were placed under one layer of neutral density screening, mimicking light levels~ one meter
below the surface. After 24 h, samples were collected for the analysis of chlorophyll aand phy-
toplankton quantification in July, whereas during September, samples for chlorophyll aanaly-
sis were collected after 72 h and were also analyzed via the Fluoroprobe after 48 h. Chlorophyll
avalues from both experiments were used to approximate growth rate (day
-1
) at specific time
points during experimentation using the formula:
ln  ðNt
N0Þ
tð1Þ
where N
t
is the final biomass, N
o
is the initial biomass, and t is time in days.
Nitrogen fixation and statistics
During September transect sampling, N
2
-fixation rates were measured using an acetylene
reduction assay [36]. To quantify rates, 5 mL of sample water was placed in gas-tight 10 mL
glass septum vials, injected with 500 μL of acetylene (C
2
H
2
) via a gas-tight, glass syringe, and
incubated in the field. After 4 h, 90 mM zinc acetate (C
4
H
6
O
4
Z
n
) was added to each vial to pre-
serve samples and cease all biological processes [37]. Samples were stored at room temperature
prior to ethylene (C
2
H
4
) quantification using a Trace 1300 Gas Chromatograph (Thermo
Fisher Scientific). The amount of ethylene produced was quantified using standards and the
amount of N
2
fixed was determined assuming a 4:1 ratio [38].
A one-way analysis of variance (ANOVA) was performed using SigmaPlot (Version 11.0)
to compare differences among transect sites whereas experimental treatments at specific time
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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points for all parameters were compared via a two-way ANOVA where nitrogen and phospho-
rus were the main effects (p<0.05).
Results
Lake-to-ocean transect, July
Across the transect from Lake Okeechobee and the St. Lucie River, salinity levels were 0.2
PSU except for two eastern sites within the St. Lucie River where levels were 3–8 PSU (Fig 2A).
Fig 2. July 2016 transect data of sites that represented a strong salinity gradient. Included are microcystin and salinity values
(A), chlorophyll aconcentrations (B), densities of the five most abundant phytoplankton (C), and total (D) and dissolved (E)
inorganic nitrogen and phosphorus concentrations and ratios. Algal densities are represented on the log scale in C. Error bars
denote standard deviations.
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Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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Chlorophyll alevels were elevated (>20 μg L
-1
), with levels exceeding 100 μg L
-1
at LO 1 (Fig
2B). Cyanobacteria numerically dominated (>95%) the phytoplankton community, with
Microcystis making up the majority (>95%) of cells at all sites (106–10
7
cells mL
-1
), save for
site SLE 10 (Figs 2C and 3). Other cyanobacteria present at lower densities (10
3
–10
5
cells mL
-
1
) at most sites included Dolichospermum (Anabaena) and Aphanocapsa spp. (Figs 2C and 3).
The diatoms Cyclotella spp. and Cymbella spp. were present throughout the transect at densi-
ties comparable to the two non-Microcystis cyanobacteria (10
2
–10
5
cells mL
-1
;Fig 2C).
Across the transect, total nitrogen concentrations were higher (40–160 μM) than total phos-
phorus values (2–7 μM), and the total N:P ratio ranged from 12–25 (Fig 2D). Total nitrogen
and phosphorus levels generally declined from Lake Okeechobee through the St. Lucie River
Estuary (Fig 2D). Dissolved nitrogen (NH
3
) levels (1–6 μM) were much lower than total nitro-
gen levels, though dissolved phosphorus (SRP) levels (1–3 μM) were comparable to total phos-
phorus levels. Furthermore, nitrate levels were largely undetectable, save at site SLE 1 (Fig 2E).
The dissolved nitrogen-to-phosphorus ratio (DIN:DIP) was well below Redfield, ranging from
0.5–5.5 and averaging ~ 2 (Fig 2E).
Microcystin ELISA equivalent levels were highest in Lake Okeechobee (3.6–34 μg L
-1
) but
lower in the St. Lucie River Estuary (0.3–7.8 μg L
-1
) (Fig 2A). Interestingly, regions along the
Fig 3. Cyanobacterial taxa abundant in the 2016 summer transect. Microcystis aeruginosa, bar is 20 μm (A) and 100 μm (B).
Aphanocapsa grevillei, bar is 10 μm (C). Dolichospermum circinale, bar is 10 μm and asterisk () denotes heterocyst (D).
Pseudanabaena spp., bar is 10 μm (E).
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transect that exhibited the highest Microcystis spp. concentrations (LO 2 and SLE 1), did not
correspond to the highest microcystin concentrations (Fig 2A and 2C). Saxitoxin concentra-
tions were at or below the methodological detection limit in all samples.
Multiplex qPCR. At all stations sampled during July 2016, both mcyE and sxtA were
above the limit of quantification (Fig 4). mcyE gene copies ranged from 2.8 ±1.8 x 10
3
mL
-1
to
3.8 ±1.0 x 10
5
mL
-1
, whereas sxtA copies ranged from 0.44 ±0.2 x 10
3
mL
-1
to 7.6 ±0.04 x 10
3
mL
-1
(Fig 4). For both mcyE and sxtA, the highest gene copy levels were detected at site LO 1
and decreased throughout the St. Lucie River sites as salinity increased (Figs 2A and 4).
Putative producers of microcystins and saxitoxins. There were positive BLASTN hits
for the mcyE primer, a subunit of the microcystin synthesis gene operon, in the reads and
assembled scaffolds from all samples collected (Fig 4). Scaffold BLASTN hits were limited to a
%ID greater than 98%, which corresponded with a mcyE primer coverage of 55–82%, and run
against the NCBI cyanobacteria database using BLASTN. All scaffolds with a positive mcyE hit
matched to Microcystis, and for all but one sample (SLE 10) the raw coverage of mcyE
was >100X. As Microcystis aeruginosa was the dominant, potentially toxic cyanobacterium in
the Lake Okeechobee-St. Lucie waterway at the time, it was the likely microcystin producer.
Positive BLASTN hits for the sxtA primer, a subunit of the saxitoxin synthesis operon, were
found in the reads of all samples with greater than 98% ID but covering 80% or less of the sxtA
primer. When the same sxtA primer was aligned to assembled scaffolds there were only three
hits: one each to sample SLE 1, SLE 6.5, and Canal 1 with 100% ID and 80% coverage. The
sxtA genes identified in these three samples had raw read coverages of 18, 27, and 9X, respec-
tively. Hits to only these three samples were verified by using the full forward primer, probe,
and reverse primer compliment which again identified the same three samples and scaffolds.
Both primers and the probe were found to align in order along the previously identified scaf-
folds. The scaffolds with these hits were compared to the NCBI cyanobacteria database, which
returned putative taxonomies of Dolichospermum (Anabaena), Aphanizomenon,Lyngbya, or
Cylindrospermopsis.
To increase taxonomic identification, similar scaffolds were binned and extracted by
ESOM and compared to both SSU and nucleotide databases using BLASTN. Within the identi-
fied bin for each sample, putative taxonomy was assigned using the BLASTN bit score (mini-
mum 1000). For all three samples, the highest bit scores were consistently associated with
Dolichopsermum, indicating that this genus was likely the dominant saxitoxin producer in the
Lake Okeechobee-St. Lucie waterway.
Nutrient effects on phytoplankton populations, July
In all estuarine experiments, the addition of NH
4+
yielded the highest levels of phytoplankton
biomass after 24 h (Fig 5). For the experiment using lake water, differences among treatments
were not significant (Fig 5A). However, for the experiments performed at SLE 6.5 and SLE 10,
the addition of NH
4+
with or without PO
43-
yielded biomass levels and/or growth rates that
were significantly greater than the control treatment (p<0.05; Fig 5C and 5D).
Lake-to-ocean transect, September
Salinity levels were near zero throughout most of the September transect, with the site furthest
east reaching ~ 1 PSU (Fig 6A). Chlorophyll alevels across the transect were within the range
found in July, generally between 11–44 μg L
-1
(Fig 6B). Differential algal pigment analyses
revealed that Lake Okeechobee had higher (>20 μg L
-1
) cyanobacterial concentrations than
estuarine sites (Fig 7), suggesting that salinity (Table 1) partially regulated the biomass of this
phytoplankton group. Compared to cyanobacteria, diatom pigment concentrations were low
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Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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(<10 μg L
-1
) throughout the Lake Okeechobee–St. Lucie River waterway except at sites LO 4
and SLE 7 (>20 μg L
-1
;Fig 7). Green algae exhibited a lower variation (9–25 μg L
-1
) in abun-
dance and were the dominant group within the estuarine region (Fig 7).
Lake site LO 1 and the canal site closest to Lake Okeechobee, Canal 1, had phytoplankton
cell densities (10
6
–10
7
cells mL
-1
) and diversity (Microcystis spp. 95% of cell densities) most
similar to sites surveyed during the July transect. Generally, however, fall transect cell densities
were lower (10
5
cells mL
-1
) and cyanobacteria were dominated by Dolichospermum (Ana-
baena) spp. and Aphanocapsa spp. (Fig 6C). While Cymbella spp. abundance was considerably
lower relative to July, Cyclotella spp. abundances were similar (Fig 6C). Total nitrogen concen-
trations were higher (37–81 μM) than total phosphorus concentrations (1–6 μM), and the total
N:P ratio ranged from 12–43 (Fig 6D). Dissolved nutrient concentrations were generally
higher in September (5–20 μM) than in July (2–9 μM), while DIN:DIP was still below Redfield,
ranging from 7–12 except at sites LO 3 and SLE 2 (>16) (Fig 6E). At these two sites, Microcys-
tis spp. concentrations were <10
5
cells mL
-1
and not the dominant cyanobacteria in the algal
community (Fig 6C). Furthermore, NO
3-
concentrations were higher and measurable at all
sites in September. Microcystin concentrations were uniformly low in September, ranging
from 0.15 to 0.35 μg L
-1
, and did not exhibit a spatial trend (Fig 6A). N
2
-fixation was below
detection throughout the transect.
Nutrient effects on phytoplankton, September
During the September experiments, nitrogen additions yielded the highest chlorophyll acon-
centrations in every experiment (Fig 8). At lake site LO 1, ammonium alone yielded signifi-
cantly higher cyanobacterial and green algal pigment concentrations relative to untreated
control bottles (p<0.05; Fig 9A). At the estuary site SLE 1, chlorophyll a, green algae, and dia-
tom pigment concentrations significantly increased in water treated with NH
4+
-only (p<
0.05; Figs 8B and 9B). For the experiment using water from site SLE 6.5, the addition of ammo-
nium with or without phosphate yielded the highest levels of algal biomass and growth rate
with significantly higher growth rates relative to the control group after 72 h (p<0.05; Fig
8C). Cyanobacteria, green algae, and diatoms all exhibited similar responses to nutrient
amendments at SLE 6.5, with samples treated with NH
4+
exhibiting significantly higher pig-
ment concentrations relative to control and PO
43—
only treatments (p<0.05; Fig 9C).
Discussion
While toxic cyanobacterial blooms have become commonplace in freshwater bodies around
the globe, the Lake Okeechobee and the St. Lucie River and Estuary state of emergency bloom
event in 2016 was distinctive in several ways. This event represented a freshwater cyanobacter-
ial bloom spread across an estuary via a man-made canal system. While microscopic, molecu-
lar, and toxin analyses identified Microcystis as the primary human health threat during this
event, cyanobacteria capable of producing saxitoxins were concurrently present. Levels of
microcystin and microcystin synthesis genes paralleled total N levels across the system during
the summer. Furthermore, nutrient concentrations, nutrient ratios, and nutrient amendment
experiments all indicated that N was the element most capable of promoting algal, and specifi-
cally cyanobacterial biomass. Collectively, this study provides important new insights into the
controls and toxicity of cyanobacterial blooms along freshwater-to-estuarine gradients.
Fig 4. mcyE (white) and sxtA (shaded) gene abundances (copies mL
-1
) at sites along the July 2016 transect. Values are log-transformed and represented in
boxplots.
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Fig 5. Chlorophyll a(bar) and specific growth rate (black circle) data from July 2016 24 h nutrient amendment
experiments from samples collected from sites LO 1 (A), SLE 1 (B), SLE 6.5 (C), and SLE 10 (D). Error bars denote
standard deviations. Statistical significance (p<0.05) among sites is denoted by different letter combinations. Top
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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The biomass of phytoplankton communities across Lake Okeechobee and the St. Lucie
River and Estuary was dominated (July 2016) or at least co-dominated (September 2016) by
cyanobacteria that were commonly N-limited during this study. Consistent with these find-
ings, DIN:DIP values were below Redfield at almost all sites, a symptom of N-limitation [39].
Though our N and P measurements were limited to our summer and fall transects, our data
support the findings of previous studies of this freshwater-estuarine ecosystem, which have
shown that both Lake Okeechobee [40–42] and the St. Lucie River and Estuary [43] generally
exhibit N-limitation. The absence of nitrogen fixation during our surveys and the dominance
of non-nitrogen fixing cyanobacteria observed in our transects is a common symptom of
N-limitation in freshwater systems [15], and is consistent with earlier reports of infrequent
blooms of diazotrophs in Lake Okeechobee [44]. While we cannot discount the importance of
P in affecting algal populations as a co-limiting nutrient at other times, the preponderance of
evidence accumulated during this and some prior studies [23,24,43] indicates that excessive
N loading can promote cyanobacterial and algal populations across Lake Okeechobee and the
St. Lucie River and Estuary.
N-limitation in the estuary that subsequently promotes cyanobacterial blooms can also be
due to excessive phosphorus loading originating from the North Fork of the St. Lucie River
[45], which receives fertilizer inputs from agricultural activities and golf courses [46]. Such
inputs may have contributed for the lowest DIN:DIP values of the July and September tran-
sects being in the mid-estuary sites SLE 6.5 and SLE 10, and subsequently promoted algal
responses to NH
4+
during experiments. Furthermore, LaPointe et al. (2017) emphasized that
N emanating from on-site sewage treatment and disposal systems along the St. Lucie River was
likely the primary source of excessive nitrogen supporting the 2016 bloom event [47]. As such,
the remediation of these systems via upgrading to denitrifying systems or connection to sew-
age treatment plants would be a logical managerial action to mitigate these events in the
future.
Beyond the effects of N on biomass, N seems to have played an important role in control-
ling the toxicity of the July 2016 event across the Lake Okeechobee—St. Lucie River Estuary
gradient, as levels of total nitrogen, microcystin, and mcyE gene copies were all highly and sig-
nificantly correlated with each other (R
2
>0.91; p<0.0001 for all; Fig 10). In contrast, micro-
cystin concentrations were not correlated with total Microcystis densities, total phosphorus, or
inorganic nutrient pools. These findings reinforce several aspects of Microcystis eco-toxicology.
Firstly, as a N-rich compound, it has been shown in culture studies [48,49], transcriptomic
studies [50,51], field surveys [10,15,52] and field experiments [53] that higher levels of N
yield higher levels of microcystin per cell. Furthermore, field and lab studies have shown that
higher levels of N favor the dominance of toxic Microcystis strains that possess the microcystin
synthetase cassette over non-toxic strains lacking these genes [54,55]. Hence, the tight correla-
tions among microcystin, toxic cells, and total N suggest that this element played a central role
in controlling the toxicity of the Lake Okeechobee—St. Lucie River Estuary Microcystis bloom.
These data also highlight the importance of using molecular methods (e.g. qPCR) to monitor
potentially-toxic cell abundances, as these populations have been shown in several studies to
better track with toxin concentrations than total cell abundances ([15] and references therein).
Given the role that genes such as ntcA play in both microcystin synthesis and nitrogen regula-
tion [56–58], other such targets and their co-regulators such as carbon levels and C:N ratios
could be quantified to generate a more holistic understanding of N–microcystin interactions.
and bottom letters above bar graphs correspond to statistical differences in chlorophyll aand specific growth rate,
respectively.
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Fig 6. September 2016 transect data of sites that represented a strong salinity gradient. Included are microcystin and salinity values
(A), chlorophyll aconcentrations (B), densities of the five most abundant phytoplankton (C), and total (D) and dissolved (E) inorganic
nitrogen and phosphorus concentrations and ratios. Algal densities are represented on the log scale in C. Error bars denote standard
deviations.
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Oehrle et al. (2017) also collected samples along the St. Lucie River Estuary in early July and
reported that NO
3-
levels in the estuary in the months prior to July were elevated relative to
non-bloom years [59], suggesting that these high nitrate levels contributed toward the intensi-
fication of the bloom during June. The same study used LC-MS/MS to determine that micro-
cystin-LR was the dominant (~ 85%) congener and that levels were within the range reported
here for open waters of the SLE (160 - <1μg L
-1
) but also found exceedingly higher levels in
scums accumulated along shorelines (~ 4000 and ~ 1000 μg L
-1
in early and mid–July, respec-
tively) in the vicinity of our site SLE 10, where we found 0.5 μg L
-1
microcystin-ELISA equiva-
lents at this open water site. Shoreline scum samples are known to exhibit significantly higher
concentrations of microcystins relative to water samples [60] and the presence of this scum
emphasizes the highly serious nature of the human health risk this bloom event posed.
By September, the cyanobacterial bloom along the Lake Okeechobee—St. Lucie River sys-
tem had diminished with cyanobacteria co-dominating the phytoplankton community at most
sites along with green algae and diatoms. Levels of microcystin were lower at this time and
Fig 7. September 2016 diatom, cyanobacteria, and green algal pigment concentrations from all transect sites. Error bars represent standard deviations.
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correlations between this toxin and other biological and environment variables were not
detected. Despite these substantial changes in algal community abundance and composition,
N-limitation persisted in the system. Nutrient amendment experiments on samples collected
from other freshwater systems indicate that several freshwater phytoplankton groups, includ-
ing cyanobacteria, green algae, and diatoms, can exhibit N and P co-limitation seasonally [61].
Prior studies have shown freshwater diatoms such as Stephanodiscus minutulus and Asterio-
nella formosa exhibit N-limitation with respect to biomass and biovolume [61,62]. Freshwater
green algae can also exhibit species-specific responses to nutrient limitation. Yang and Gao
(2003), for instance, reported that at elevated CO
2
levels Chlorella pyrenoidosa growth rates are
N-limited [63]. Moreover, culture and field-based experiments with Mougeotia spp. indicated
that this green alga exhibits N and P co-limitation [64]. Ultimately, our results indicate that
freshwater cyanobacteria, green algae, and diatoms exhibited N, rather than P, limitation, at
several sites along the September transect, an outcome consistent with the low N:P ratios pres-
ent at that time.
For almost 30 years, cyanobacteria have comprised the majority of phytoplankton biovo-
lume (50–80%) in Lake Okeechobee, likely due in part to excessive P enrichment [11] in the
late 20
th
century that progressively lowered the total N:P from 30:1 to below 15:1 from the
mid-1970s to the late 1990s [41]. Historically, cyanobacterial communities in the lake have
been dominated by blooms of Oscillatoria and Planktothrix [65], with infrequent blooms of
diazotrophs [44]. The abundance of non-N
2
fixers relative to N
2
-fixers may be due to the high
turbidity and subsequent low irradiance conditions that can be further exacerbated by storm
and hurricane activity [66]. Previous work has also shown that Planktothrix thrives in low-
light environments [67–69]. In addition, Planktothrix has also been shown to dominate the
phytoplankton community in Sandusky Bay, Lake Erie where turbidity is high and N:P ratios
are low [70]. The absence of Oscillatoria and minor amounts of Planktothrix and the domi-
nance of Microcystis, another non-diazotrophic cyanobacteria, in 2016 and during the past
decade [24] might be partly explained by the dissolved N:P in the Lake Okeechobee–St. Lucie
River and Estuary system, which at nearly all sites was <15. N-enrichment in this case may
have favored the dominance of those cyanobacterial strains that do not fix nitrogen. As Lake
Okeechobee is a shallow freshwater system, it is likely that Microcystis spp. outcompeted other
cyanobacterial taxa due to the availability of regenerated N and P from the sediment, enabling
them to dominate the summer algal bloom’s community composition [5,15].
In addition to nutrients, salinity was likely an important factor in controlling the diversity
and toxicity of phytoplankton communities across the Lake Okeechobee and the St. Lucie
River Estuary system. During July, site SLE 6.5 and sites further east were brackish (salinity >
3) and coincided with sharp declines in mcyE gene copies and Microcystis spp. abundance.
Chen et al. (2015) reported a negative correlation between salinity and growth rate and pig-
ment concentration in cultures of Microcystis aeruginosa. While this is consistent with our
findings, the same study found that production of microcystin increased significantly in
response to elevated salinity [71], an outcome not observed during this study. However,
another strain of M.aeruginosa was shown to produce lower amounts of microcystin in
response to elevated salinity [72], suggesting that production of microcystin in response to
changes in salinity is strain-specific.
Fig 8. Chlorophyll a(bar) and specific growth rate (black circle) data at 24 and 72 h from the September 2016 nutrient amendment experiment. Samples were
collected from sites LO 1 (A), SLE 1 (B), and SLE 6.5 (C). Error barsdenote standard deviations. Statistical significance (p<0.05) among sites is denoted by different
letter combinations. Top and bottom letters above bar graphs correspond to statistical differences in chlorophyll aand specific growth rate. For B, statistical differences
in chlorophyll aand growth rate were only present at 24 and 72 h, respectively.
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Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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Fig 9. Diatom, cyanobacteria, and green algal pigment concentrations at 48 h from the September 2016 nutrient amendment
experiment. Samples were collected from sites LO 1 (A), SLE 1 (B), and SLE 6.5 (C). Error bars represent standard deviations. Statistical
significance (p<0.05) of specific pigments among sites is denoted by different letter combinations. Letters are ordered relative to the order of
parameters listed in the legend. For A, only cyanobacterial and green algal pigment concentrations exhibited statistical differences among
treatments.
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While cyanobacteria across the Lake Okeechobee—St. Lucie River system possessed a gene
important for the synthesis of saxitoxin [73], this toxin was not detected during this study, a
finding that could have been related to multiple factors. First, given toxin synthesis is depen-
dent on the full translation of the entire biosynthetic pathway, the presence, but not necessarily
transcription and translation, of a single gene in the pathway may not result in the production
of the toxin. Furthermore, the ELISA kits used to quantify saxitoxins here have somewhat ele-
vated detection limits and only detect some of the more than 25 congeners of the toxin [74]. A
study utilizing high-performance liquid chromatography (HPLC), which is a more robust and
sensitive method to detect saxitoxins than the ELISA used in this study (limit of detection = 2
x 10
−5
μg mL
-1
), found that samples from blooms in several Australian lakes with sxtA gene
abundances comparable to values reported in this study had very low, but detectable, saxitoxin
concentrations 8.6 x 10
−6
μg mL
-1
[75]. This suggests that saxitoxin might have been present
in the Lake Okeechobee–St. Lucie River Estuary waterway, albeit below the limit of detection
for our method. Furthermore, these results highlight the importance of incorporating molecu-
lar techniques into routine monitoring programs, as these methods are sensitive enough to
Fig 10. Regression of total nitrogen concentrations, total microcystin concentrations (black circles, dashed regression) and mcyE copies (white circles, dotted
regression) during the July transect.
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detect the genetic potential to produce toxins, even if toxin concentrations are too low to quan-
tify. This is important as future changes to several established environmental drivers of bloom
growth and toxicity including temperature, land use, invasive species, and rainfall patterns [2]
may facilitate shifts in bloom dominance. Therefore, monitoring the genetic potential for
toxin production is critically needed, especially during blooms where more than one potential
toxin producer is present and in systems where the full suite of toxin producers is unknown.
In conclusion, the cyanobacterial bloom found in Lake Okeechobee and throughout the
St. Lucie River and Estuary in the summer of 2016 was dominated by Microcystis and exhibited
N-limitation. In addition, the levels of microcystin and toxic Microcystis cells (possessing
mcyE) were highly correlated to the levels of total N in this system. Though the bloom had
diminished by September, multiple phytoplankton groups including cyanobacteria consis-
tently exhibited N-limitation. Collectively, evidence indicates that reductions in N-loading
associated with on-site sewage treatment and disposal systems [47] are likely an important
managerial step that could minimize the intensity of future toxic Microcystis-dominated cya-
nobacterial blooms across the Lake Okeechobee—St. Lucie River system.
Supporting information
S1 File. Microcystin concentrations (μg L
-1
).
(XLSX)
S2 File. Chlorophyll aconcentrations (μg L
-1
).
(XLSX)
S3 File. Cell density (cells mL
-1
).
(XLSX)
S4 File. Total nutrient concentration (μM) and ratio.
(XLSX)
S5 File. Dissolved nutrient concentration (μM) and ratio.
(XLSX)
S6 File. McyE and sxtA gene abundances (copies mL
-1
) at sites along the July 2016 transect.
(XLSX)
S7 File. Chlorophyll adata (μg L
-1
) from July 2016 24 h nutrient amendment experiments
from samples collected from sites LO 1, SLE 1, SLE 6.5, and SLE 10.
(XLSX)
S8 File. Specific growth rate data (day
-1
) from July 2016 24 h nutrient amendment experi-
ments from samples collected from sites LO 1, SLE 1, SLE 6.5, and SLE 10.
(XLSX)
S9 File. Microcystin concentrations (μg L
-1
).
(XLSX)
S10 File. Chlorophyll aconcentrations (μg L
-1
).
(XLSX)
S11 File. Cell density (cells mL
-1
).
(XLSX)
S12 File. Total nutrient concentration (μM) and ratio.
(XLSX)
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 21 / 26

S13 File. Dissolved nutrient concentration (μM) and ratio.
(XLSX)
S14 File. September 2016 diatom, cyanobacteria, and green algal pigment concentrations
(μg L
-1
) from all transect sites.
(XLSX)
S15 File. Chlorophyll adata (μg L
-1
) at 24 and 72 h from the September 2016 nutrient
amendment experiment.
(XLSX)
S16 File. Specific growth rate data (day
-1
) at 24 and 72 h from the September 2016 nutrient
amendment experiment.
(XLSX)
S17 File. Diatom, cyanobacteria, and green algal pigment concentrations (μg L
-1
) at 48 h
from the September 2016 nutrient amendment experiment.
(XLSX)
S18 File. Regression of total nitrogen concentrations (μM), total microcystin concentra-
tions (μg L
-1
) and mcyE gene abundances (copies mL
-1
) during the July transect.
(XLSX)
Acknowledgments
We deeply appreciate the employees of Hutchinson Island’s Marriott Beach Resort & Marina
and TC Paddle & Watersports, Inc. of Stuart, FL, USA 34996 for allowing us to use their facili-
ties and dock to run our experiments. We also thank Craig Young of Stony Brook University,
NY, USA 11968 for his assistance in preparing figures for publication.
Author Contributions
Conceptualization: Benjamin J. Kramer, Timothy W. Davis, Gregory J. Dick, Genesok Oh,
Christopher J. Gobler.
Data curation: Benjamin J. Kramer, Timothy W. Davis, Gregory J. Dick, Genesok Oh.
Formal analysis: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen,
Jennifer A. Goleski, Gregory J. Dick.
Funding acquisition: Timothy W. Davis, Christopher J. Gobler.
Investigation: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen,
Christopher J. Gobler.
Methodology: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jen-
nifer A. Goleski, Gregory J. Dick, Genesok Oh, Christopher J. Gobler.
Project administration: Benjamin J. Kramer, Timothy W. Davis, Christopher J. Gobler.
Resources: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jennifer
A. Goleski, Gregory J. Dick, Genesok Oh, Christopher J. Gobler.
Software: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jennifer
A. Goleski, Gregory J. Dick, Genesok Oh, Christopher J. Gobler.
Supervision: Benjamin J. Kramer, Timothy W. Davis, Christopher J. Gobler.
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 22 / 26

Validation: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jennifer
A. Goleski, Gregory J. Dick, Christopher J. Gobler.
Visualization: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jen-
nifer A. Goleski, Gregory J. Dick, Christopher J. Gobler.
Writing – original draft: Benjamin J. Kramer, Timothy W. Davis, Christopher J. Gobler.
Writing – review & editing: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry
H. Rosen, Genesok Oh, Christopher J. Gobler.
References
1. Carey C, Ibelings B, Hoffmann E, Hamilton D, Brookes J. Eco-physiological adaptations that favour
freshwater cyanobacteria in a changing climate. Water Res. 2012; 46(5):1394–1407. https://doi.org/10.
1016/j.watres.2011.12.016 PMID: 22217430
2. O’Neil J, Davis T, Burford M, Gobler C. The rise of harmful cyanobacteria blooms: The potential roles of
eutrophication and climate change. Harmful Algae. 2012; 14:313–334.
3. Paerl H, Paul V. Climate change: links to global expansion of harmful cyanobacteria. Water Res. 2012;
46(5):1349–1363. https://doi.org/10.1016/j.watres.2011.08.002 PMID: 21893330
4. Jeppesen E, Søndergaard M, Meerhoff M, Lauridsen T, Jensen J. Shallow lake restoration by
nutrient loading reduction—some recent findings and challenges ahead. Hydrobiologia. 2007; 584
(1):239–252.
5. Paerl H, Xu H, Hall N, Zhu G, Qin B, Wu Y, et al. Controlling cyanobacterial blooms in hypertrophic Lake
Taihu, China: will nitrogen reductions cause replacement of non-N
2
fixing by N
2
fixing taxa? PLoS One.
2014; 9(11):1–13.
6. Lee T, Rollwagen-Bollens G, Bollens S, Faber-Hammond J. Environmental influence on cyanobacteria
abundance and microcystin toxin production in a shallow temperate lake. Ecotox Environ Safe. 2015;
114:318–325.
7. Dawson R. The Toxicology of Microcystins. Toxicon. 1998; 36(7):953–962. PMID: 9690788
8. Osswald J, Rellan S, Gago A, Vasconcelos V. Toxicology and detection methods of the alkaloid neuro-
toxin produced by cyanobacteria, anatoxin-a. Environ Int. 2007; 33(8):1070–1089. https://doi.org/10.
1016/j.envint.2007.06.003 PMID: 17673293
9. Agnihotri V. Anabaena flos-aquae. Crit Rev Environ Sci Technol. 2014; 44(18):1995–2037.
10. Harke M, Steffen M, Gobler C, Otten T, Wilhelm S, Wood S, et al. A review ofthe global ecology, geno-
mics, and biogeography of the toxic cyanobacterium, Microcystis spp. Harmful Algae. 2016; 54:4–20.
https://doi.org/10.1016/j.hal.2015.12.007 PMID: 28073480
11. Smith V. Low nitrogen to phosphorus ratios favor dominance by blue-green algae in lake phytoplankton.
Science. 1983; 221:669–671. https://doi.org/10.1126/science.221.4611.669 PMID: 17787737
12. Smith V. Effects of eutrophication on maximum algal biomass in lake and river ecosystems. Inland
Waters. 2016; 6(2):147–154.
13. Filstrup C, Heathcote A, Kendall D, Downing J. Phytoplankton taxonomic compositional shifts across
nutrient and light gradients in temperate lakes. Inland Waters. 2016; 6(2):234–249.
14. Fogg G. Nitrogen fixation. In: WDP S, editor. Algal physiology and biochemistry. Oxford: Blackwell;
1974. p. 560–583.
15. Gobler C, Burkholder J, Davis T, Harke M, Johengen T, Stow C, et al. The dual role of nitrogen supply in
controlling the growth and toxicity of cyanobacterial blooms. Harmful Algae. 2016; 54:87–97. https://doi.
org/10.1016/j.hal.2016.01.010 PMID: 28073483
16. Paerl H, Scott J, McCarthy M, Newell S, Gardner W, Havens K, et al. It Takes Two to Tango: When and
Where Dual Nutrient (N & P) Reductions Are Needed to Protect Lakes and Downstream Ecosystems.
Environ Sci Technol. 2016; 50:10805–10813.
17. de Tezanos Pinto P, Litchman E. Interactive effects of N:P ratios and light on nitrogen-fixer abundance.
Oikos. 2010; 119(3):567–575.
18. Davis TW, Berry DL, Boyer GL, Gobler CJ. The effects of temperature and nutrients on the growth and
dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae.
2009; 8(5):715–725.
19. Sharma R, Chahar O, Bhatnagar M, Bhatnagar A. Impact of osmotic stress and temperature on pig-
ments and proteins of Anabaena strains. J Environ Biol. 2013; 34(5):941–943. PMID: 24558809
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 23 / 26

20. James R, Jones B, Smith V. Historical trends in the Lake Okeechobee ecosystem II. Nutrient budgets.
Arch Hydrobiol Suppl. 1995; 107:25–47.
21. Havens K, Aumen N, James R, Smith V. Rapid ecological changes in a large subtropical lake undero-
ging cultural eutrophication. AMBIO. 1996; 25:150–155.
22. Havens K, Schelske C. The importance of considering biological process when setting total maximum
daily loads (TMDL) for phosphorus in shallow lakes and reservoirs. Environ Pollut. 2001; 113(1):1–9.
PMID: 11351756
23. Aldridge F, Phlips E, Schelske C. The use of nutrient enrichment bioassays to test for spatial and tem-
poral distribution of limiting factors affecting phytoplankton dynamics in Lake Okeechobee, Florida.
Arch Hydrobiol, Advances in Limnol. 1995; 45:177–190.
24. Havens K, Paerl H, Phlips E, Zhu M, Beaver J, Srifa A. Extreme weather events and climate variability
provide a lens to how shallow lakes may respond to climate change. Water. 2016; 8(229):1–18.
25. Stefanova L, Zierden D. Climate Summary for Florida Florida Climate Center: FSU (Florida State Uni-
versity); 2016 [Available from: https://climatecenter.fsu.edu/products-services/summaries/climate-
summary-for-florida-january-2016.
26. USGS Surface-Water Daily Data for Florida [Internet]. U.S. Department of the Interior. 2015 and 2016
[cited 2.24.18]. Available from: https://waterdata.usgs.gov/fl/nwis/dv?
27. Chlorophyll, nitrate, phosphate, salinity, and temperature levels at SLE-SF, SF2, NF, and ME from
December 2015 to November 2016 [Internet]. 2015 and 2016 [cited 2.23.18]. Available from: http://fau.
loboviz.com/loboviz/.
28. Rosen B, Davis T, Gobler C, Kramer B, Loftin K. Cyanobacteria of the 2016 Lake Okeechobee Water-
way harmful algal bloom: U.S. Geological Survey Open-File Report 2017–1054. 2017. p. 34.
29. Parsons T, Maita Y, Lalli C. A manual of chemical and biological methods for seawater analysis.
Oxford: Pergamon Press; 1984.
30. Harke M, Davis T, Watson S, Gobler C. Nutrient-Controlled Niche Differentiation of Western Lake Erie
Cyanobacterial Populations Revealed via Metatranscriptomic Surveys. Environ Sci Technol. 2016; 50
(2):604–615. https://doi.org/10.1021/acs.est.5b03931 PMID: 26654276
31. Harke MJ, Davis TW, Watson SB, Gobler CJ. Nutrient-Controlled Niche Differentiation of Western Lake
Erie Cyanobacterial Populations Revealed via Metatranscriptomic Surveys. Environ Sci Technol. 2016;
50(2):604–615. https://doi.org/10.1021/acs.est.5b03931 PMID: 26654276
32. Beutler M, Wiltshire K, Meyer B, Moldaenke C, Lu¨ring C, Meyerho
¨M, et al. A fluorometric method for
the differentiation of algal populations in vivo and in situ. Photosynth Res. 2002; 72(1):39–53. https://
doi.org/10.1023/A:1016026607048 PMID: 16228533
33. Al-Tebrineh J, Pearson L, Yasar S, Neilan B. A multiplex qPCR targeting hepato- and neurotoxigenic
cyanobacteria of global significance. Harmful Algae. 2012; 15:19–25.
34. Yao B, Liu Q, Gao Y, Cao Z. Characterizing vertical migration of Microcystis aeruginosa and conditions
for algal bloom development based on a light-driven migration model. Ecol Res. 2017; 32:961–969.
35. Babele P, Singh G, Singh A, Kumar A, Tyagi M, Sinha R. UV-B radiation and temperature stress-
induced alterations in metabolic events and defense mechanisms in a bloom-forming cyanobacterium
Microcystis aeruginosa. Acta Physiol Plant. 2017; 39(248):1–11.
36. Flett R, Hamilton R, Campbell N. Aquatic acetylene-reduction techniques: solutions to several prob-
lems. Can J Microbiol. 1976; 22:43–51. PMID: 814983
37. Sim M, Ono S, Bosak T. Effects of iron and nitrogen limitation on sulfur isotope fractionation during
microbial sulfate reduction. Appl Environ Microb. 2012; 78(23):8368–8376.
38. Capone D. Determination of nitrogenase activity in aquatic samples using the acetylene reduction pro-
cedure. In: Kemp P, Cole J, Sherr B, Sherr E, editors. Handbook in methods in aquatic microbial ecol-
ogy. Organic matter decomposition and nutrient regeneration. 1 ed. Boca Raton: Lewis Publishers;
1993. p. 621–631.
39. Reynolds C. The ecology of freshwater phytoplankton. Cambridge: Cambridge University Press; 2006.
40. Havens K. Secondary nitrogen limitation in a subtropical lake impacted by non-point source agricultural
pollution. Environ Pollut. 1995; 89:241–246. PMID: 15091513
41. Havens K, James R, East T, Smith V. N:P ratios, light limitation, and cyanobacterial dominance in a sub-
tropical lake impacted by non-point source nutrient pollution. Environ Pollut. 2003; 122(3):379–390.
PMID: 12547527
42. McCarthy M, James R, Chen Y, East T, Gardner W. Nutrient ratios and phytoplankton community struc-
ture in the large, shallow, eutrophic, subtropical Lakes Okeechobee (Florida, USA) and Taihu (China).
Limnology. 2009; 10(3):215–227.
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 24 / 26

43. Phlips E, Badylak S, Hart J, Haunert D, Lockwood J, O’Donnell K, et al. Climatic influences on autoch-
thonous and allochthonous phytoplankton blooms in a subtropical estuary, St. Lucie Estuary, Florida,
USA. Estuar Coast. 2012; 35(1):335–352.
44. Jones B. Lake Okeechobee eutrophication research and management. Aquatics. 1987; 9:21–26.
45. Lapointe B, Herren L, Bedford B. Effects of Hurricanes, Land Use, and Water Management on Nutrient
and Mirobial Pollution: St. Lucie Estuary, Southeast Florida. J Coast Res. 2012; 28(6):1345–1361.
46. Graves G, Wan Y, Fike D. Water quality characteristics of storm water from major land uses in south
Florida. J Am Water Resour Assoc. 2004; 40(6):1405–1419.
47. Lapointe B, Herren L, Paule A. Septic systems contribute to nutrient pollution and harmful algal blooms
in the St. Lucie Estuary, Southeast Florida, USA. Harmful Algae. 2017; 70:1–22. https://doi.org/10.
1016/j.hal.2017.09.005 PMID: 29169565
48. Codd G, Poon G. Cyanobacterial Toxins. Rogers L, Gallon J, editors. Oxford, England: Clarendon
Press; 1988. 283–296 p.
49. Orr P, Jones G. Relationship between microcystin production and cell division rates in nitrogen-limited
Microcystis aeruginosa cultures. Limnol Oceanogr. 1998; 43(7):1604–1614.
50. Harke M, Gobler C. Global transcriptional responses of the toxic cyanobacterium, Microcystis aerugi-
nosa, to nitrogen stress, phosphorus stress, and growth on organic matter. PLoS One. 2013; 8(7):1–15.
51. Harke M, Gobler C. Daily transcriptome changes reveal the role of nitrogen in controlling microcystin
synthesis and nutrient transport in the toxic cyanobacterium, Microcystis aeruginosa. BMC Genomics.
2015; 16(1068):1–18.
52. Chaffin J, Bridgeman T, Bade D. Nitrogen constrains the growth of late summer cyanobacterial blooms
in Lake Erie. Adv Microbiol. 2013; 3:16–26.
53. Gobler C, Davis T, Coyne K, Boyer G. Interactive influences of nutrient loading, zooplankton grazing,
and microcystin synthetase gene expression on cyanobacterial bloom dynamics in a eutrophic New
York lake. Harmful Algae. 2007; 6(1):119–133.
54. Vezie C, Rapala J, Vaitomaa J, Seitsonen J, Sivonen K. Effect of nitrogen andphosphorus on growth of
toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations. Microb Ecol.
2002; 43:443–454. https://doi.org/10.1007/s00248-001-0041-9 PMID: 11953809
55. Davis T, Harke M, Marcoval M, Goleski J, Orano-Dawson C, Berry D, et al. Effects of nitrogenous com-
pounds and phosphorus on the growth of toxic and non-toxic strains of Microcystis during cyanobacter-
ial blooms. Aquat Microb Ecol. 2010; 61(2):149–162.
56. Alexova R, Haynes P, Ferrari B, Neilan B. Comparative protein expression in different strains of the
bloom-forming cyanobacterium Microcystis aeruginosa. Mol Cell Proteomics. 2011; 10:1–16.
57. Gehringer MM, Wannicke N. Climate change and regulation of hepatotoxin production in Cyanobacte-
ria. FEMS Microbiol Ecol. 2014; 88:1–25. https://doi.org/10.1111/1574-6941.12291 PMID: 24490596
58. Ginn H, Pearson L, Neilan B. NtcA from Microcystis aeruginosa PCC 7806 is autoregulatory and binds
to the microcystin promoter. Appl Environ Microbiol. 2010; 76:4362–4368. https://doi.org/10.1128/AEM.
01862-09 PMID: 20453121
59. Oehrle S, Rodriguez-Matos M, Cartamil M, Zavala C, Rein K. Toxin composition of the 2016 Microcystis
aeruginosa bloom in the St. Lucie Estuary, Florida. Toxicon. 2017; 138:169–172. https://doi.org/10.
1016/j.toxicon.2017.09.005 PMID: 28899665
60. S
ˇulčius S, PilkaitytėR, Mazur-Marzec H, KasperovičienėJ, Ezhova E, Blaszczyk A, et al. Increased risk
of exposure to microcystins in the scum of the filamentous cyanobacterium Aphanizomenon flos-aquae
accumulated on the western shoreline of the Curonian Lagoon. Mar Pollut Bull. 2015; 99(1–2):264–270.
https://doi.org/10.1016/j.marpolbul.2015.07.057 PMID: 26234611
61. Mu¨ller S, Mitrovic S. Phytoplankton co-limitation bynitrogen and phosphorus in a shallow reservoir: pro-
gressing from the phosphorus limitation paradigm. Hydrobiologia. 2014; 744(1):255–269.
62. Lynn S, Kilham S, Kreeger D, Interlandi S. Effect of nutrient availability on the biochemical and elemen-
tal stoichiometry in the freshwater diatom Stephanodiscus minutulus (Bacillariophyceae). J Phycol.
2000; 36(3):510–522. https://doi.org/10.1046/j.1529-8817.2000.98251.x PMID: 29544004
63. Yang Y, Gao K. Effects of CO
2
concentrations on the freshwater microalgae, Chlamydomonas reinhard-
tii,Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta). J Appl Phycol. 2003; 15(5):379–
389.
64. Middleton C, Frost P. Stoichiometric and growth responses of a freshwater filamentous green alga to
varying nutrient supplies: slow and steady wins the race. Freshwater Biology. 2014; 59(11):2225–2234.
65. Havens K, Phlips E, Cichra M, Li B. Light availability as a possible regualtor of cyanobacteria species
composition in a shallow subtropical lake. Freshwater Biol. 1998; 39:547–556.
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
PLOS ONE | https://doi.org/10.1371/journal.pone.0196278 May 23, 2018 25 / 26

66. Beaver J, Casamatta D, East T, Havens K, Rodusky A, James R, et al. Extreme weather events influ-
ence the phytoplankton community structure in a large lowland subtropical lake (Lake Okeechobee,
Florida, USA). Hydrobiologia. 2013; 709(1):213–226.
67. Mur L, Schreurs H. Light as a selective factor in the distribution of phytoplankton species. Water Sci
Technol. 1995; 32(4):25–34.
68. Scheffer M. Ecology of shallow lakes. London: Chapman and Hall; 1998.
69. Nixdorf B, Mischke U, Rucker J. Phytoplankton assemblages and steady state in deep and shallow
eutrophic lakes—an approach to differentiate the habitat properties of Oscillatoriales. Hydrobiologia.
2003; 502:111–121.
70. Davis T, Bullerjahn G, Tuttle T, McKay R, Watson S. Effects of increasing nitrogen and phosphorus con-
centrations on phytoplankton community growth and toxicity during Planktothrix blooms in Sandusky
Bay, Lake Erie. Environ Sci Technol. 2015; 49(12):7197–7207. https://doi.org/10.1021/acs.est.
5b00799 PMID: 25992592
71. Chen L, Mao F, Kirumba G, Jiang C, Manefield M, He Y. Changes in metabolites, antioxidant system,
and gene expression in Microcystis aeruginosa under sodium chloride stress. Ecotox Environ Safe.
2015; 122:126–135.
72. Martin-Luna B, Sevilla E, Bes M, Fillat M, Peleato M. Variation in the synthesis of microcystin in
response to saline and osmotic stress in Microcystis aeruginosa PCC7806. Limnetica. 2015; 34
(1):205–214.
73. Kellmann R, Mihali T, Jeon Y, Pickford R, Pomati F, Neilan B. Biosynthetic intermediate analysis and
functional homology reveal a saxitoxin gene cluster in cyanobacteria. Appl Environ Microb. 2008; 74
(13):4044–4053.
74. Van Dolah F. Marine algal toxins: origins, health effects, and their increased occurrence. Environ Health
Persp. 2000; 108(Supplemental 1):133–141.
75. Al-Tebrineh J, Mihali T, Pomati F, Neilan B. Detection of saxitoxin-producing cyanobacteria and Ana-
baena circinalis in environmental water blooms by quantitative PCR. Appl Environ Microb. 2010; 76
(23):7836–7842.
Nitrogen limitation of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida
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