Journal of Environmental Protection, 2011, 2, 669-674
doi:10.4236/jep.2011.26077 Published Online August 2011 (http://www.SciRP.org/journal/jep)
669
Growth and Toxin Production by Microcystis
Aeruginosa PCC 7806 (Kutzing) Lemmerman at
Elevated Salt Concentrations
Ken Black, Mete Yilmaz, Edward J. Phlips
School of Forest Resources and Conservation, University of Florida, Gainesville, USA.
Received April 27th, 2011; revised May 29th, 2011; accepted July 8th, 2011.
ABSTRACT
One of the most common and widespread bloom-forming cyanobacteria associated with toxin production is Microcystis
aeruginosa (Kutzing) Lemmerman. While norma lly associa ted with fresh water environmen ts, this toxigenic species has
been observed at bloom concentrations in a number of major estuaries worldwide. This study examined the effect of
salinity on growth and toxin production by M. aeruginosa strain PCC 7806 under controlled lab oratory conditions. Salt
concentrations above 12.6 ppt resulted in total cessation of growth. Toxin production was similarly affected, with cul-
tures grown in salt concentrations of 4.6 ppt and above yielding less toxin than the control after 20 days of culture.
Toxin concentratio ns after 20 days of culture were 40% of the control at 4.6 ppt. The relative proportion of extracellu-
lar to intracellular toxin increased over time in cultures with salt concentrations greater than 4.6 ppt. Extracellular
toxins persisted in the med ia long after the cessation of growth. The results sugg est that the influence of M. aeruginosa
and/or its toxins can extend well out into estuarine environments under the influence of significant freshwater inputs.
Keywords: Microcystin, Cyanobacteria, Estuaries
1. Introduction
Cyanobacterial blooms are common across the globe,
affecting both freshwater and marine ecosystems [1-3].
Among these bloom-forming species, there are a number
of toxigenic strains [4-6]. One of the more common and
widespread bloom-forming cyanobacteria associated
with toxin production is Microcystis aeruginosa (Kutzing)
Lemmerman. The toxin most often associated with M.
aeruginosa is microcystin, a hepatotoxin which can
negatively impact aquatic animal and human health on
the cellular and organ level [4,7,8]. Blooms of toxic M.
aeruginosa have been implicated in mass mortalities of
aquatic animals and the destabilization of food webs [3,9,
10]. Consumption of microcystin contaminated drinking
water and tainted food items pose potential human health
risks, especially in third world countries where effective
treatment practices are not uniformly applied [4,11-14].
Because of the potential harmful effects of M. aerugi-
nosa it has become a focus of efforts to control harmful
algae blooms.
Although M. aeruginosa is most commonly associated
with freshwater environments, blooms have been ob-
served in mesohaline regions of estuaries, such as the
Chesapeake Bay [15], the Neuse River in North Carolina
[2], the Neva Estuary in the Gulf of Finland [16], the
Guadina Estuary in Spain [17], the Swan River in Aus-
tralia [18], and the St. Lucie Estuary [19] and St. Johns
River [20] estuaries in Florida. The highest reported salt
concentrations at which M. aeruginosa survives ranges
from 2 to 17 ppt [2,21-24]. Isolates from blooms can
vary in toxicity [25,26]. The appearance of toxic strains
of M. aeruginosa in saline environments has become a
serious issue for the management of affected coastal en-
vironments, particularly those where there is extensive
utilization of marine resources for fishing, recreation or
consumption of potable water after desalinization [27-29].
Previous work indicates that elevated salt concentrations
result in the lysis of cells and the release of microcystins
into the supporting water [23,24].
This study examined the response of a toxic strain of
M. aeruginosa to a range of salt concentrations, in terms
of survival, growth, toxin production and the fate of the
toxins produced over the growth cycle. Most previous
studies have focused on cells from discrete portions of

Growth and Toxin Production by Microcystis Aeruginosa PCC 7806 (Kutzing) Lemmerman 670
at Elevated Salt Concentrations
the growth cycle or from natural bloom samples. The
objectives of this study were: 1) To determine changes in
cell abundance and microcystin content of M. aeruginosa
grown over a range of salinities, 2) To evaluate the fate
of microcystin in terms of its relative distribution within
cells and the surrounding media over the growth cycle,
and 3) To examine the longevity of microcystin in saline
media.
2. Methods
Microcystis aeruginosa PCC 7806 cells were grown in
Hoagland’s medium buffered to 8.0 with HEPES [30],
yielding a baseline salt concentration of 0.6 ppt. Cul-
tures were grown at 25˚C, and light was provided by
cool-white fluorescent bulbs at approximately 60
μmolphotonsm–2s–1 PAR irradiance, on a 12:12 light:
dark (L:D) photoperiod.
Treatment groups were based on culture media (salin-
ity of 0.6 ppt), to which NaCl was added to reach addi-
tional final salinities of 2.6, 4.6, 6.6, 8.6, 10.6, 12.6, 14.6,
20.6, 25.6, 30.6, and 35.6 ppt. Treatment groups were set
up in triplicate in 500 ml Erlenmeyer flasks. Flasks were
inoculated with M. aeruginosa cells in the exponential
growth phase. Inoculums were added at a ratio of 1:10,
culture to media, yielding a starting concentrations of
approximately 40 μgL–1 chlorophyll a, 106 cellsml–1,
and 35 μgL–1 of microcystin.
Two methods were used to quantify changes in cell
biomass, chlorophyll concentrations and cell counts. In
vivo chlorophyll a was determined fluorometrically using
a Turner fluorometer [31] at two day intervals from t = 0
to t = 20 days. Fluorescence values were converted to
chlorophyll a concentrations using standard relationships
obtained from replicate samples analyzed for chlorophyll
a using spectrophotometric analysis [32] after methanol
extraction [33] using a Hitachi dual beam spectropho-
tometer.
Samples for cell counts were collected at t = 0, 2, 8, 14
and 20 days. Samples were preserved with Lugol’s solu-
tion [32]. Cell counts were carried out microscopically
using the Utermöhl sedimentation method [32,34]. Sub-
samples were allowed to settle in an Utermöhl chamber
for 24 hours. Cells were counted on a Nikon inverted
light microscope at 400x magnification.
Both intracellular and extracellular microcystin con-
centrations were determined for samples collected from
the 0.6, 4.6, 8.6, 12.6 and 20.6 ppt salt treatment groups
at t = 0, 2, 8, 14 and 20 days of culture. Two separate
five ml aliquots were collected during each sampling. To
obtain the extracellular fraction, one of the samples was
filtered through a 0.7 μm pore size glass fiber filter. Fil-
trates were frozen and stored until toxin analysis. The
other whole water sample was separately frozen and
analyzed for toxin concentration.
Samples were thawed and boiled in a 100˚C water bath
for 60 seconds [35]. Cell debris was pelleted by cen-
trifugation and discarded. Toxin concentrations were
determined via Enzyme Linked Immuno-Sorbent Assay
(ELISA). Envirologix© Competitive ELISA kits for the
quantification of Microcystin-LR and congeners were
utilized, following the methods outlined by the manufac-
turer. Toxin concentrations were measured using a Stat
Fax 3200 microplate reader. If necessary, samples were
diluted in order to accommodate the ELISA’s assay
range of 0.16 to 2.5 μgL–1 Microcystin-LR. The concen-
tration of the intracellular microcystin was calculated by
subtracting the filtered sample values from the corre-
sponding unfiltered sample.
ANOVA and Tukey’s (HSD) post-hoc tests were util-
ized to determine the significance of the salinity effects.
Statistical analyses were done with a SPSS Version 17.0
statistics package.
3. Results and Discussion
Rates of increase in chlorophyll a concentration and cell
numbers of M. aeruginosa decreased with increased salt
concentrations (Figure 1). After two days growth, cul-
tures grown at salt concentrations of 8.6 ppt or higher
varied significantly from the control in terms of chloro-
phyll a concentration (ANOVA, F = 11.84, p <0.05, n =
39) and cell density (ANOVA, F = 10.41, p < 0.05, n =
39). After twenty days exposure, cultures grown at 4.6
ppt or higher exhibited significantly lower chlorophyll a
concentrations compared to the 0.6 ppt and 2.6 ppt
groups (ANOVA, F = 64.64, p < 0.05, n = 39). In terms
of cell numbers at 20 days, cultures grown at 2.6 ppt or
higher had significantly lower cell densities than cultures
grown at 0.6 ppt (ANOVA, F = 85.30, p < 0.05, n = 39).
Cultures grown at or above 16.6 ppt showed no signifi-
cant increases in chlorophyll a or cell numbers over the
incubation period, similar to the observations of Ver-
spagen et al. [29]
At two days of culture, no significant differences were
observed in total microcystin content of cultures grown
at different salt concentrations (ANOVA, F = 2.467, p =
0.113, n = 15) (Figure 2). After twenty days, total toxin
content of cultures grown at 4.6 ppt salt or greater were
significantly lower than in the 0.6 ppt group (ANOVA,
F = 123.977, p < 0.05, n = 12). A strong increase in total
microcystin concentration was observed in the 0.6 ppt
and 4.6 ppt treatment groups between 8 and 14 days of
culture growth, reaching mean concentrations of up to
1500 μgL–1 after 20 days. At salinities of 0.6, 4.6 and 8.6
ppt the mean percentage increases in cell numbers and

Growth and Toxin Production by Microcystis Aeruginosa PCC 7806 (Kutzing) Lemmerman 671
at Elevated Salt Concentrations
0
5
10
15
20
25
30
0510 15 20
Cell density (millions of cells mL-1)
0.6 ppt
2.6 ppt
4.6 ppt
8.6 ppt
12.6 ppt
20.6 ppt
(a)
0
5
10
15
20
25
30
051015 20
Cell density (millions of cells mL-1)
0.6 ppt
2.6 ppt
4.6 ppt
8.6 ppt
12.6 ppt
20.6 ppt
(b)
Figure 1. Changes in abundance of M. aeruginosa over time
at a range of salt concentrations in terms of mean chloro-
phyll a concentrations (a) and cell numbers (b). Standard
deviations are shown as vertical bars. All treatment groups
at or above 16.6 ppt salt showed no increase in chlorophyll
a or cell numbers.
Figure 2. Changes in total microcystin concentration over
time, under a range of salt concentrations. Standard devia-
tions are shown as vertical bars.
total microcystin concentrations over twenty days of
culture were similar, i.e. within 10% of each other.
Differences were observed over the culture period and
between treatment groups in the distribution of toxins
within (intracellular) and outside (extracellular) of the M.
aeruginosa cells. The percentage of intracellular micro-
cystin increased over time in cultures grown at salt con-
centrations up to 12.6 ppt, reflecting increases in cell
density (Figures 3). At twenty days, cultures grown at
12.6 ppt or greater showed a greater proportion of ex-
tracellular than intracellular microcystin (ANOVA, F =
5.865, p <0.05, n = 15), as observed for M. aeruginosa
blooms entering San Francisco Bay in California [15].
Extracellular microcystin concentrations in the ambi-
ent media persisted for the entire 20-day culture period.
Over 80% of the initial extracellular toxin concentration
remained after 20 days of culture in the 20.6 ppt treat-
ment group, despite a lack of cell growth and rapid deg-
radation of cells (i.e. initial concentration = 15.81 μgL–1,
SD = 1.00, N = 3; final concentration = 13.21 μgL–1,
SD = 3.21, N = 3).
The relatively high tolerance of toxic M. aeruginosa to
elevated salt concentrations highlights the potential im-
portance of this species in terms of the ecology of estu-
aries, including the health of aquatic animals [11]. Toxic
cells consumed through the gastrointestinal tract can en-
ter the blood stream and effect internal organs [36]. Mi-
crocystin has been shown to bioaccumulate in the tissues
0
10
20
30
40
50
60
70
80
90
00000
Mi crocyst i n concentrat i on (µg L
-1
)
Me di a sa l t content ( ppt )
Intracellular
microcystin
fractio n
Extracellular
microcystin
fractio n
0
200
400
600
800
1000
1200
1400
1600
1800
00000
Mi crocyst i n concentrat i on (µg L
-1
)
Me di a sa l t content ( ppt )
Intracellular
microcystin
fraction
Extracellular
microcystin
fraction
0.6 4.6 8.6 12.6 20.60.6 4.6 8.6 12.6
20.60.6 4.6 8.6 12.6
Salt concentration (ppt)
Microc ystin concentration (µg L
-1
)
2 days
20 days
Figure 3. Total microcystin concentrations divided into
intracellular and extracellular fractions, after 2 (top) and 20
(bottom) days of growth at salt concentrations of 0.6, 4.6,
8.6, 12.6, and 20.6 ppt. Standard deviations are shown as
vertical bars.

Growth and Toxin Production by Microcystis Aeruginosa PCC 7806 (Kutzing) Lemmerman 672
at Elevated Salt Concentrations
of a wide range of organisms [4,8], including zooplank-
ton [37] ,shellfish [37-40], and fish [36,41], thereby po-
tentially exposing all trophic levels of the food web to
microcystin [42]. Soluble microcystins in the water have
been shown to affect the gills of fish, reducing the capac-
ity for gas and ion exchange [43].
The presence of intracellular and extracellular micro-
cystins may also impact human health. One of the major
concerns is microcystin contamination of potable water
[4,13]. In estuaries, concerns center on the increasing use
of desalinated water for human consumption [44]. Man-
agement options depend on whether the toxins are prin-
cipally intracellular or extracellular [24,27,29]. If the
toxin is primarily intracellular, removal of cells can sub-
stantially reduce the toxin threat, but if the toxin is pri-
marily extracellular chemical treatments may be neces-
sary.
Another human health concern is the consumption of
shellfish and fish containing microcystin, however, con-
siderable uncertainty remains over the potential risks.
Several researchers have observed significant levels of
microcystin in the tissues of commercially important fish,
such as tilapia [36], and shellfish, such as the blue mussel
[45]. The highest concentrations tend to be localized in
gastrointestinal organs [36].
In addition to consumptive issues, recreational use of
estuarine waters might also be affected by the presence
of toxic M. aeruginosa blooms [7]. Irritation due to con-
tact of microcystin with epithelial tissues has been ob-
served in humans, including blistering of affected tissues
and hepatoenteritis [4,46]. Toxin exposure can occur
through direct exposure to water via inhalation of aero-
solized cells and contaminated water particles. Anecdotal
evidence has linked several cases of pneumonia to rec-
reational usage of waters during a M. aeruginosa bloom
[47]. A microcystin LR concentration of 20 μgL–1 has
been suggested as a threshold level of concern for recrea-
tional exposure [4], but more definitive guidelines re-
main an issue of debate and continued research [48].
The longevity of the effects of toxic M. aeruginosa
blooms in different ecosystems depend on factors that
accelerate the degradation or dilution of the toxin, such
as ultraviolet radiation, strong oxidizers, naturally occur-
ring bacteria which deactivate or otherwise eliminate
microcystins [37], and hydrologic considerations, such as
tidal flushing and water residence time, which define the
rates of dilution of both toxic cells and extracellular toxin.
Greater rates of exchange with coastal marine water also
decrease the spatial and temporal window of salinities
favorable for survival. The results suggest that the influ-
ence of M. aeruginosa and/or its toxins can extend well
out into estuaries, particularly those with restricted water
exchange with coastal waters where mesohaline condi-
tions can persist for extended periods of time.
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