Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
Competition between Cyanobacteria
Species and Cyanotoxins: Identifying
the dominant species in a temperate,
hypereutrophic reservoir.
Sarah Bartlett
December 5th, 2012
UW-Milwaukee, School of Freshwater Sciences
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
PROJECT SUMMARY
Cyanobacteria are outcompeting algae in many temperate freshwater reservoirs.
Cyanobacteria, also known as blue green algae, are responsible for harmful algal bloom (HAB)
formation in many eutrophic freshwater systems. HAB’s can create anoxic water conditions and
can also cause harmful effects to ecosystems that need algal species for both food and an energy
source. Warmer water and air temperatures, coupled with an increase in nutrient availability and
stagnant water conditions provide prime breeding grounds for cyanobacteria to flourish. The
harmful algal blooms out-compete not only other cyanobacteria species but also species of algae
that are an important component to a freshwater ecosystem’s food web and productivity.
Cyanobacteria are also responsible for producing toxins, which appear to harm terrestrial
organisms more so than aquatic, and are responsible for producing taste-and-odor compounds. It
is still unclear why cyanobacteria invest the energy to produce toxins and odor compounds.
Finding out the ecological importance and triggers of cyanobacteria toxin production could be
groundbreaking.
Many studies focus on quantifying the presence of one toxin or one strain of
cyanobacteria. This study seeks to understand the composition and community of cyanobacteria
within an ecosystem of a small, temperature, hypereutrophic reservoir throughout the summer
season. Individual cyanobacteria species will be identified, and cyanotoxin production will be
quantified and qualified as will taste-and-odor compounds, specifically looking at the odor
compounds geosmin and 2-methylisoborneol (MIB). Taking into account the total dissolved
phosphorus, weather (lake and air temperatures), and the chemical composition of water
throughout the season, this study hopes to draw conclusions to what environmental factors cause
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
a cyanobacteria strain to be dominant within a temperate freshwater reservoir, and determine
when odor compounds are produced at the greatest quantity.
PROJECT DESCRIPTION
Introduction
The increase in frequency and severity of harmful algal blooms has led many to question
how cyanobacteria are managing to outcompete the algae and become a dominant species in the
freshwater ecosystems during the summer months of temperate lakes. Cyanobacteria, also known
as blue green algae, occurs naturally but many issues surrounding anthropogenic forces has
affected the frequency and duration of blooms and harmful algal blooms are increasing at a rate
greater than what would occur naturally (Paerl, H.W. et al. 2011). This bacteria has the ability to
outcompete other systems in freshwater ecosystems. In the right conditions (warm water and air
temperature, lots of sunlight, little wind, increase of nutrients into a freshwater body possibly due
to runoff from recent rain events), blooms can occur quickly [2].
Blooms can be toxic, food-web altering, and generate hypoxia (Paerl, H.W. et al. 2011),
causing a whole sweep of problems to freshwater ecosystems in addition to outcompeting the
algae that would otherwise naturally grow during the summer season.
Cyanobacteria produce a wide range of toxins that can be harmful to lower and higher
trophic level organisms if exposed via inhalation, ingestion, or dermal contact. Toxins are
typically held intracellularly until cell death and the ecological role and function of these toxins
is still unclear. Some speculation looks at toxins and odor compound production as a means to
ward off predation, although there have been few documented predators of cyanobacteria. It is
also unclear why cyanobacteria of the same genus don’t all produce the same toxin, and why one
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
cyanobacteria of the same genus may not produce toxins at all. Not all cyanobacteria blooms are
harmful because of this discrepancy. It is difficult to draw parallels between cyanobacteria
growth and cyanotoxin production because there doesn’t appear to be a pattern for when to
produce toxins.
Cyanotoxins appear to be more harm to terrestrial organisms than aquatic. The most
common cyanotoxin is microcystin, the hepatoxins (microcystins and nodularins) which
primarily target the liver, and are mainly produced by Anabaena, Microcystis, and Planktothrix
genera (Backer, L. 202). Other cyanotoxins include neurotoxins (anatoxin-a, anatoxin-a(s) and
saxitoxins), cytotoxins (cylindrospermopsins), and dermatotoxins (aplysiatoxins and
debromoaplysiatoxins) (Sivonen, K. 1999). This study will primarily focus on the hepatoxin,
microcystin and cytoxoins cylindrospermopsis and the ananbaena, microcystis, planktothrix
genera due to prior testing of species in the lake.
Some of the byproducts of the harmful algal bloom toxins are noxious odors that can
create poor air quality and undesirable smells. Cyanobacteria produces these produce taste-andodor compounds, although it is not well understand which strain of cyanobacteria is responsible
for production of these compounds, nor is known when a strain will release these compounds –
either during the growth phase or during cell lysis (Graham, J. 2010). One such study has
indicated that testing for surface water odor is not a reliable toxin indicator and cannot replace
the need for chemical analyses (Watson, S.B. 2003). This proposal does not dispute the need for
more chemical analyses to determine toxin presence, however it may be beneficial to test surface
water for odors as a way to forewarn of a harmful algal bloom, which would be the case if the
odors were produce during the growth phase of cyanobacteria.
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
This study seeks to characterize cyanobacteria production within a temperate reservoir
throughout a season, and determine which strain of cyanobacteria is producing the odor
compounds geosmin and MIB, odor compound that has been more commonly produced in
Midwestern lakes (Graham J. et al 2010).
Objectives
Overall, the objective of this proposal is to identify and classify the cyanobacteria
community in a temperate, hypereutrophic freshwater reservoir. By looking at the change in
environmental factors that occurs throughout a summer season, this study seeks to draw parallels
between environmental factors (water chemical composition, temperature, wind) and the
presence of a dominant cyanobacteria community. With the knowledge of a dominant
community, hopefully future studies will be better equipped to predict the occurrence of a
harmful algal bloom and be able to predict if cyanobacteria will be producing cyanotoxins.
1. What is the diversity index of cyanobacteria in a temperate, hypereutrophic
freshwater reservoir? Several studies have looked at identifying the many different
species of cyanobacteria and a good database with micrographs of each genus has been
created (. Few studies exist looking at the cyanobacteria community or communities that
grow in a temperature reservoir throughout a season. Qualifying and quantifying this
community will be an important step in determining the dominant cyanobacteria strains
that develop and prosper throughout the summer season in the temperate freshwater
reservoir of Tainter Lake.
2. What factors drive cyanotoxin production throughout the summer season and at
what concentration are toxins being produced? The ecological function that induces
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
cyanotoxin production is still unknown. It is possible that toxins are produced by
cyanobacteria a defense mechanism, but little is known why cyanobacteria invest energy
into this production. Current guidelines provided by the World Health Organization
recommend limits for the number of cyanobacteria cells present to ensure deem a water
body at low, moderate, or high risk for human health. Measuring the cyanotoxins that are
produced can help update the WHO guidelines which only look at cell counts of
cyanobacteria species, and no guidelines exist for toxin concentrations. Cyanotoxins will
be quantified from surface water grabs taken once per week and test using the
microcystins measured by enzyme linked immunosorbent assay (ELISA) .
3. When are the odor-producing compounds geosmin and MIB being produced? Is
there a lag time from when toxins are produced to when odor compounds are
produced? Can the production of geosmin be used as an indicator for a forthcoming
algal event? Little is known what causes the cyanobacteria to produce these compounds
and even less is known about which strains of cyanobacteria will produce odor
compounds. This objective seeks to draw correlations between dominant species, toxin
production, and odor production. By understanding which cyanobacteria species respond
to a specific variable such as water temperature or chemical composition of the water, we
will hopefully predict which strain will be present at a given point during the summer
season and thus, predict when air quality will be most impacted by odor compound
production.
Geosmin is the more dominantly produced odor compound. Previous studies indicate that
depending on the strain of cyanobacteria, geosmin can either be produced during
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
cyanobacteria cell growth or during cell lysis. Determining if there is a lag time between
toxin production and odor production this might tell us that odors are being produced in
the lysis stage of cyanobacteria cells. Maybe odors are produced to outcompete other
cyanobacteria species and producing these compounds during death might be the cell’s
one last attempt to maintain dominancy over an ecosystem.
Current hypotheses include:
1. A dominant cyanobacteria species will develop throughout the summer months, and I
expect to see this same species dominant in the following two years of this study.
However, I also expect to see a dominant species that is different at the northern basin,
than the eastern. This will probably be due to different in lake composition and hopefully
this difference in lake characteristics can be paralleled to the dominant cyanobacteria
species that is apparent.
2. It is expected that Microcystin will be the dominant toxin throughout the entire lake,
although it will be produced by several different strains of cyanobacteria.
3. Toxin and odor compound production will occur simultaneously, with odor more so
occurring during cyanobacteria cell lysis. Odor production will not make a good indicator
to forewarn the general public for the occurrence of a harmful algal bloom.
Tainter Lake Environment
Tainter Lake is located in Dunn County, Wisconsin and has a documented history of
algal blooms accompanied by the production of noxious odors throughout the summer months. It
is 5 km2, with a maximum depth of 11.27 meters and is the formed by the damming of the Red
Cedar River. The lake has experienced high levels of phosphorus inputs for several years due to
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
anthropogenic forces and is considered hypereutrophic by the Wisconsin Department of Natural
Resources using water quality data collected by the Citizen Lake Group for Tainter Lake. The
Citizen Lake Group has been monitoring since 1989, recording the dissolved oxygen,
chlorophyll, total phosphorus and secchi
depth actively at several different areas within
the basin.
Residents of Dunn County have provided
anecdotal accounts of the noxious odors from
Figure 1: Paint-like cyanobacteria sludge covers the surface of
Tainter Lake for several weeks during the summer months.
that lake that seem to occur simultaneously
with the presence of thick algal blooms. Blooms can be described as paint-like, and can persist
and/or reoccur for several weeks of the summer season.
Proposed Research
For the purpose of this study, a season is defined as June 1st to September 1st. While
blooms may occur outside of this time frame, the focus will be on the summer months, when
blooms have been documented to occur more frequently and at greater severity of size and smell.
Sampling will occur three times a week at each of the five locations as determined below [see
Fig. 2] for a total of 13 weeks. The experiment will be duration of 3 seasons in order to gain an
insight on the cyanobacterial community of the lake. Information from the first season will
provide insight on the success or lack of information that sampling 3 times a week will provide.
It is expected that seasons should be similar throughout the course of 3 years, with
obvious changes occurring in the frequency of precipitation, water temperature and air
temperature. These three factors will hopefully help define the triggers that either support or
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
hinder cyanobacterial growth. Identifying triggers such as high precipitation versus low, and
warmer weather versus cooler can then be compared to the dominant cyanobacteria community
that is present. This information will be useful for future studies as the information can be used
to predict the onset of a cyanobacteria bloom based off environmental triggers.
Sampling will occur at five locations throughout Tainter Lake, three that occur along the
shoreline and two that are located at greater depths. These locations were determined due to the
presence of a public pier to allow access to the water, and the middle locations in the lake were
chosen based on the Citizen Lake Group’s sampling in years past. Data from the lake indicated
that blooms have occurred at all five of these locations, more so in the basins areas near the
shoreline.
Figure 2: Sampling locations on Tainter Lake
What is the diversity index of cyanobacteria in a temperate, hypereutrophic freshwater
reservoir? To create a diversity index, we will catalogue the cyanobacteria that occur throughout
the summer season as collected from water samples. Sampling will be surface grabs, and
collected in 1-L glass amber bottles. Water samples will be analyzed for cyanobacteria
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
composition and the number of cells present will be quantified, using techniques as developed by
the United States Geological Survey (USGS) (Graham, J. 2010). Cyanobacteria will be analyzed
under a microscope and micrographs will be created to develop this diversity index. Additional
environmental parameters will also be recorded such as air and water temperature, dissolved
oxygen, and wind speeds. These environmental factors will help create triggers that will later be
used to determine what causes a cyanobacteria genus to become dominant over other species and
algae.
Which cyanotoxins are produced throughout the summer season and at what
concentrations are they producing? A single grab sample from the surface will be taken at
each of the five sites for cyanotoxin analysis. Samples will be stored in 125-ml glass amber
bottles and will be immediately processed back in the lab. Samples will be inverted several times
to ensure homogeneity and then put through a freeze-thaw process three times to lyse the
cyanobacteria cells and release the toxins and odor compounds. Quantifying and identifying the
toxins will occur using two methods - LC/MS/MS analysis and enzymelinked immunosorbent
assay (ELISA). One drawback to ELISA is the analysis doesn’t distinguish between multiple
variants. In the case of microcystin, with at least 60-known variants (Srivastava, A. 2012), this
analysis could potentially be limiting. ELISA is however, a fast method for toxin identification
and is well-utilized in many studies of cyanotoxins .
Using LC/MS/MS allows the flexibility to have a sample spiked with a known toxin to be
entered into the machine, and other water samples can then be loaded, and the spikes that occur
in the machine can be matched to the known toxins, which allow us to determine the specific
toxin variant.
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
When are the odor-producing compounds geosmin and MIB being produced? Is there a lag
time from when toxins are produced to when odor compounds are produced? Can the
production of geosmin be used as an indicator for a forthcoming algal event?
Determining when a cyanobacteria species produces an odor – during cell growth or
during cell lysis – may be an indicator of a harmful algal bloom being formed, even if it isn’t yet
visible on the surface of the lake. To determine the production of gesomin and MIB, a single
grab samples from the water surface will be taken at each of the five sites, three times a week for
odor compound analysis. Laboratory analysis will follow a protocol determined by the USGS
(Graham, J. 2010). Information required from this analysis will be to determine which compound
is being formed, geosmin or MIB, and when is it being formed. It will also be beneficial to
compare the production of an odor compound on the same time scale as the production of
cyanotoxins and cyanobacteria growth.
An overlap in cyanobacteria growth coupled with a peak in odor production could be an
indication that odors are being produced during the cellular growth stage. Alternatively, when
cyanobacteria cell counts are low and odor production is high could be telling that odors are
being produced during cellular lysis.
Environmental Factors
The following factors will be taken during each sample at each of the five sites: dissolved
oxygen, air temperature, water temperature, wind speed. An additional surface water sample will
be collected in an amber bottle and taken back to the lab for total dissolved phosphorus analysis.
Protocols for TDP will be used as determined by the Miller Lab, School of Public Health, and
UW-Milwaukee.
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
These factors will be used to identify possible triggers that may be responsible for the
presence of a dominant cyanobacteria species or dominant cyanobacteria communities.
Budget
Work will be conducted over the course of 3 seasons (2 months). To account for staff needed on
the proposal, a PI and two graduate students, $63,000 from the budget will pay the staff. Indirect
costs including overhead, use of facilities, fringe benefits, and travel will cost $18,000 each
season. Expendable lab items (amber bottles, filters, 10-ml tubes, chemicals) will cost $20,000
for the duration of the experience and upkeep on lab items (DO2 sensors, Mass spectrometry, gas
chromotograph) will be $1200 per season. Grand total needed for this proposal: $140,600.
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Bartlett, S. Competition between cyanobacteria: Identifying Dominant Species
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