A linkage between Cladophora mats and exceedances of
recreational water quality criteria has been suggested, but
not directly studied. Th is study investigates the spatial and
temporal association between Escherichia coli concentrations
within and near Cladophora mats at two northwestern Lake
Michigan beaches in Door County, Wisconsin. Escherichia
coli concentrations in water underlying mats were signifi cantly
greater than surrounding water (p < 0.001). Below mat E. coli
increased as the stranded mats persisted at the beach swash
zone. Water adjacent to Cladophora mats had lower E. coli
concentrations, but surpassed EPA swimming criteria the
majority of sampling days. A signifi cant positive association was
found between E. coli concentrations attached to Cladophora
and in underlying water (p < 0.001). Th e attached E. coli likely
acted as a reservoir for populating water underlying the mat.
Fecal bacterial pathogens, however, could not be detected
by microbiological culture methods either attached to mat
biomass or in underlying water. Removal of Cladophora mats
from beach areas may improve aesthetic and microbial water
quality at aff ected beaches. Th ese associations and potential
natural growth of E. coli in bathing waters call into question the
effi cacy of using E. coli as a recreational water quality indicator
of fecal contaminations.
The Green Alga, Cladophora, Promotes Escherichia coli Growth and Contamination of
Recreational Waters in Lake Michigan
Amy Vanden Heuvel, Colleen McDermott, and Robert Pillsbury University of Wisconsin–Oshkosh
Todd Sandrin Arizona State University
Julie Kinzelman City of Racine Health Department
John Ferguson and Michael Sadowsky University of Minnesota
Muruleedhara Byappanahalli and Richard Whitman USGS
Gregory T. Kleinheinz* University of Wisconsin–Oshkosh
managers and private homeowners on the Great Lakes. Th is green
alga (Chlorophyta: Cladophorales) grows best on fi rm substrates
such as rocks, pilings, piers, and other hard surfaces in shallow
waters (Herbst, 1969), but can break free and form large fl oating
mats that eventually accumulate on beaches. Cladophora mats
communities usually consist of a variety of organisms, including
bacteria, diatoms, cyanobacteria, protozoa, rotifers, and other
multicellular organisms (Chilton et al., 1986; Marks and Powers,
2001; Stevenson et al., 2006). Cladophora may provide a variety
of requirements for growth and survival, such as nutrients and
protection from predation and UV light (Marks and Powers, 2001).
Recently there has been resurgence in the growth of Cladopho-
ra along Lake Michigan shorelines to pre-1970 levels. Eutrophica-
tion of water is associated with increases in Cladophora mat pro-
duction and accumulation (Stevenson et al., 2006; Herbst, 1969)
but water transparency has also been suggested as an important
causation (Barbiero et al., 2006; Bootsma, 2007). As mats age and
ultimately decay, communities of associated organisms change.
High densities of E. coli and enterococci have been recovered in
Cladophora accumulations along Lake Michigan shorelines from
Indiana to Illinois and into southern Wisconsin (Englebert et al.,
2008; Whitman et al., 2003). High densities have been attributed
to in situ growth of these bacteria under favorable conditions (By-
appanahalli et al., 2003). An association between Cladophora mats
and beach water quality has been hypothesized (Englebert et al.,
2008; Whitman et al., 2003), but to our knowledge there has yet
to be a published study that has shown a direct link between these
he occurrence of the nuisance alga, Cladophora, in the form
of mats along shorelines is an increasing problem for beach
Abbreviations: DE, diatomaceous earth; PBW, phosphate-buff ered water.
A. Vanden Heuvel, C. McDermott, R. Pillsbury, and G.T. Kleinheinz, Dep. of Biology and
Microbiology, Univ. of Wisconsin-Oshkosh, 800 Algoma Blvd., Oshkosh, WI 54901.
J. Ferguson and M. Sadowsky, Dep. of Soil, Water, and Climate; and BioTechnology
Institute, Univ. of Minnesota, 1991 Upper Buford Cir., 439 Borlaug Hall, St. Paul, MN
55108. M. Byappanahalli and R. Whitman, U.S. Geological Survey, Lake Michigan
Ecological Research Station, 1100 N. Mineral Springs Rd., Porter, IN 46304. T. Sandrin,
Div. of Mathematical and Natural Sciences, Arizona State University, MC 2352, P.O. Box
37100, Phoenix, AZ 85069. J. Kinzelman, City of Racine Health Dep., 730 Washington
Ave., Racine, WI 53403.
Copyright © 2010 by the American Society of Agronomy, Crop Science
Society of America, and Soil Science Society of America. All rights
reserved. No part of this periodical may be reproduced or transmitted
in any form or by any means, electronic or mechanical, including pho-
tocopying, recording, or any information storage and retrieval system,
without permission in writing from the publisher.
Published in J. Environ. Qual. 39:333–344 (2010).
Published online 20 Nov. 2009.
Received 24 Apr. 2009.
*Corresponding author (email@example.com).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS: SURFACE WATER QUALITY
334 Journal of Environmental Quality • Volume 39 • January–February 2010
Escherichia coli is used as an indicator of recent fecal contami-
nation of water, however, should it survive for long periods of time
or replicate associated with Cladophora, this application would be
challenged. As a fecal indictor, the presence of E. coli in recreational
water suggests that pathogenic microbes (i.e., Campylobacter, Sal-
monella, Shigella) associated with fecal contamination also may be
present. In a study by Ishii et al. (2006) Campylobacter, Salmonella,
and Shigella were recovered from Cladophora mats in lower Lake
Michigan. Additionally, the pathogenic bacterium, Vibrio, also
has been found to associate with algal accumulations (Islam et al.,
1989). Since E. coli survives (and perhaps replicates) in Cladophora
mats, the same may hold true for pathogenic bacteria; therefore
an increase in Cladophora in the Great Lakes may be a potential
public health risk.
Conversely, should E. coli preferentially survive and repro-
duce for long periods of time in the mats, but the pathogens
do not, then high concentrations of E. coli found at bathing
beaches as a result of effl ux of the bacterium from Cladophora
mats would challenge the paradigm that E. coli is a suitable
indicator of fecal contamination (i.e., does not replicate in the
environment). Likewise, these elevated E. coli concentrations
in beach water would not indicate a signifi cant risk of gastroin-
testinal illness for recreational water users. In this case, beaches
may be closed inappropriately and thus cause an unnecessary
negative impact on tourism.
Door County, Wisconsin, a peninsula located on northern
Lake Michigan, has been plagued by Cladophora accumulations
for several years. In a preliminary study conducted by authors
of this manuscript (Englebert et al., 2008), E. coli concentra-
tions were shown to be elevated in Cladophora mats at beaches
located in the geographical vicinity of our current study area.
At those beaches there was no positive statistical correlation
between presence of stranded Cladophora and beach closures.
In that study, stranded Cladophora mats and the beach water
monitoring sites were often distant from one another. Th ere-
fore, it was hypothesized that E. coli was unable to persist in
open beach water after it was washed from Cladophora mats by
wave and wind action and/or diff use any distance towards the
beach monitoring site. For this study, two beaches (Whitefi sh
Dunes State Park and Lakeside Park) were chosen as study sites
because they historically have had Cladophora accumulations
that have presented a problem for beach managers. In addition,
Cladophora mats at these beaches typically fl oat in close to the
beach water monitoring sites and strand for several days before
disappearing. Escherichia coli concentrations in water at these
beaches have been relatively low compared to concentrations
at beaches on southern Lake Michigan, but occasional beach
closures have occurred due to elevated bacterial concentrations.
Th e overarching objective of this study was to determine if E.
coli concentrations associated with Cladophora mats infl uence E.
coli concentrations in beach water adjacent to the mats. Specifi -
cally, we were interested in determining the spatial and temporal
eff ects that Cladophora accumulations may have on the relative
abundance of E. coli at bathing beaches. In addition, we wanted
to determine if the relative abundance of selected pathogens
mimicked that of E. coli under the same conditions. Th e un-
derlying hypothesis was that Cladophora mats may contribute to
and allow for the persistence of E. coli in recreational beach wa-
ter, but pathogens concentrations would not respond similarly.
Materials and Methods
Th is project was conducted at two popular recreational
beaches on Lake Michigan, located in Door County, Wiscon-
sin, Whitefi sh Dunes State Park beach and Lakeside Park beach
(Fig. 1). Th ese two locations both face east and are separated by
a shoreline distance of 7.1 km. Th ese sites were chosen because
historically they are subject to large accumulation of Cladopho-
ra mats during the recreational bathing season.
Cladophora Mat Selection and Sample Collection
Th e study sites were observed daily for the “arrival” of an
algal mat. Within 24 h, the mat was sampled daily for three
consecutive days. Th ere were three separate sampling events
(16–18 July, 1–3 Aug., 8–10 Aug. 2007) at each beach during
the summer season. Samples collected included the underlying
water of the Cladophora mat proper (within mats), outlying
water on either side of the mat, and Cladophora biomass.
All sampling occurred before 0900 h to minimize insolation
eff ects and disturbance by visitors. Water samples were collected
using 250-mL sterile bottles attached to long poles; bottles were
capped with a 1 by 1 mm nylon screen to exclude Cladophora
strands. Samples were collected from water with a depth of ap-
proximately 45 cm (approximately 3 m from shore); care was
taken to prevent agitation of the macrophytic mass. Cladophora
biomass was collected and placed into sterile plastic bottles. All
samples were analyzed within 2 to 6 h of collection.
Two diff erent sampling protocols were employed to deter-
mine the impact that accumulated Cladophora mats have on E.
coli concentrations in the surrounding beach water.
First, a random sampling strategy was used to determine
the diff erence in E. coli concentrations in beach water collected
within the mats vs. water collected on either side of the mats,
and to establish any temporal eff ects. Specifi cally, 10 water
samples were collected randomly from underlying water within
the mat and in water on either side of the mat (1–3 m from the
water-mat edge) for a total of 30 samples per mat, per day of
each sampling event (Fig. 2B)
Th e second sampling strategy was used to determine if an
E. coli concentration gradient existed in beach water within or
on either side of the mat. A transect parallel to the shoreline,
in water at a depth of 45 cm, was established across a mat and
extending out to the right and left of the mat edge. Water and
Cladophora biomass sampling took place at 10 evenly spaced
locations within the transect (Fig. 2A). Distance between the
spaced samples depended on the length of the Cladophora mat
(range = 3–6 m between sample sites). For one particularly large
mat at Whitefi sh Dunes beach (16–18 July 2007), more than
Vanden Heuvel et al.: Infl uence of Cladophora Mats on Escherichia coli in Water 335
10 evenly spaced sample locations (17) were designated. Sample
locations were temporarily marked on the beach with fl ags.
Escherichia coli Concentrations from Water
Escherichia coli concentrations from water were measured
using defi ned substrate technology, Colilert with Quantitray
2000 format (IDEXX Corp., Portland, ME) (Clescerl et al.,
2005; Kinzelman et al., 2005). Incubation and E. coli enumer-
ation were conducted following the manufacturer’s recommen-
dations. Analyses were conducted at Th e University of Wiscon-
sin-Oshkosh, Sturgeon Bay Laboratory. Th is facility, which is
State certifi ed for analysis of fecal bacteria, is located within 15
min of the sampling location. All results were reported as most
probable number (MPN) of E. coli per 100 mL of water.
Escherichia coli Attached to Cladophora
For Cladophora samples, an initial bacterial elutriation step
was necessary. One gram of homogenized Cladophora biomass
was placed in sterile 15-mL centrifuge tubes, to which 9 mL of
sterile phosphate-buff ered water diluent, pH 6.8 (PBW) was
added. Th e alga-PBW mixture was shaken vigorously for 2 min
and centrifuged for 45 s at 600 × g to allow the large particles to
settle. Previous studies have shown that this elutriation method
achieves an average of 55% bacterial recovery rate (Whitman et
al., 2003). Th e supernatant was further diluted (1/10 and 1/100)
in PBW, from 1 to 30 mL, and E. coli was enumerated using tra-
ditional membrane fi ltration techniques (USEPA, 2002). Filters
were placed onto modifi ed thermotolerant-E. coli agar medium
(modifi ed mTEC: Becton Dickinson, Chicago, IL), and plates
were incubated at 35°C for 2 h, followed by incubation at 44.5°C
for 22 h (USEPA, 2002). Bacterial concentrations in algae were
expressed as colony forming units per gram (dry weight) of algae
(CFU/g). Approximately 1% of the presumptive colonies for E.
coli were confi rmed using the API 20E rapid identifi cation sys-
tem (bioMérieux, Marcy l’Etoile, France).
Presence of Salmonella, Shigella, or Campylobacter in Water
Water underlying each of the study mats was analyzed for
the presence of pathogens; Salmonella, Shigella, and Campylo-
bacter were identifi ed and enumerated using standard micro-
For Campylobacter, approximately 3 L water samples were
fi ltered through 0.4-μm fi lters (Gelman Scientifi c; Ann Arbor,
MI) and fi lters were placed on 7% Horse Blood Agar plates.
Plates were incubated for up to 48 h in a microaerobic environ-
ment (Gas Pak, Oxoid, Hants, UK) at 42°C. Campylobacter
colonies were identifi ed by gram stain, motility, biochemistry,
and agglutination with specifi c antibodies (Oxoid; Hants, UK).
Salmonella and Shigella were identifi ed by using a modi-
fi ed membrane fi ltration technique (Wisconsin State Labora-
tory of Hygiene, 2003; Clescerl et al., 2005). Ten milliliters,
100 mL, and 1-L aliquots of water were fi ltered through ster-
ile diatomaceous earth (DE). Th e DE plug was suspended in
50 mL Selenite broth (Difco; Sparks, MD), and incubated at
42°C for 24 h. Ten microliters of broth from each water ali-
quot were inoculated onto xylose lysine desoxycholate medium
(XLD) (Difco; Sparks, MD) and incubated at 35°C for 24 h.
Red colonies with black centers (Salmonella) and red colonies
without black centers (Shigella) were confi rmed with API 20E
(bioMérieux, Marcy l’Etoile, France) biochemical analysis. A
MPN technique was used to estimate the number of organisms
in the water samples (Table 1).
Presence of Salmonella, Shigella, or Campylobacter Attached
Four-inch diameter samples of Cladophora were manually
collected from fl oating mats and placed into sterile plastic jars.
Five gram algal subsamples were placed in sterile 50 mL centri-
fuge tubes to which sterile phosphate buff ered water was added
to equal 50 mL total volume. Samples were shaken vigorously
to remove bacteria from the algal mat and briefl y centrifuged to
remove debris (Byappanahalli et al., 2003). Supernatants were
fi ltered onto membrane fi lters as described above for Campy-
lobacter, Salmonella, and Shigella isolation. Identifi cation of
pathogens occurred as described for water samples. Counts are
expressed as CFU/g dry weight
Statistical analysis was performed using SYSTAT 11.0. For
all statistical procedures, unreliable E. coli concentrations were
removed (fi ltration errors, assay maximum surpassed, CFUs
Fig. 1. Map of Door County in relation to the State of Wisconsin and the
location of the two study sites.
336 Journal of Environmental Quality • Volume 39 • January–February 2010
were too numerous to count). Escherichia coli estimates also
were log transformed to conform to normal distribution.
Analysis of variance was used to determine diff erences be-
tween samples, within and between transects. Th e null hy-
pothesis was that there was no diff erence in the mean E. coli
concentrations within and outside the Cladophora mats, and
the alternate hypothesis was that there was a diff erence in these
means. Alpha was set at 0.05 (Zar, 1999).
To examine the relationship between E. coli attached to Cladopho-
ra mats (independent variable) and those in nearby water samples
(dependent variable) a linear regression was applied to E. coli data
pooled from both beaches and all dates. Th en a linear regression was
applied separately to Whitefi sh Dunes Beach and Lakeside Beach to
determine if this relationship diff ered with location.
To determine if the concentration of E. coli was related to
the location of the sample within the alga mat, a new indepen-
dent variable was created (Fig. 2A). To achieve equality with re-
gard to gradient sample location and to off set unequal transect
numbers, a standardized scale was created. A linear regression
was then conducted using the new mat location scale as the
independent variable and the log (CFU E. coli) and log (MPN
E. coli) analyzed separately as the dependent variables.
To determine if diff erences existed in the means of E. coli
concentrations taken on diff erent days of a Cladophora mat
event we ran a two-way ANOVA using Beach (Whitefi sh
Dunes or Lakeside Park) and Day (fi rst, second, or third) of
each sampling event (algal mat moving into shore) as inde-
pendent factors, followed by a Tukey post-hoc test. Th e log
(CFU E. coli) and log (MPN E. coli) were analyzed separately
as dependant variables.
Escherichia coli in Water
Escherichia coli concentrations from the random water sam-
pling within the mat were signifi cantly higher than concen-
trations of the bacterium found outside the mat (p < 0.001)
(Fig. 3 and 4). For Whitefi sh Dunes, mean E. coli concentra-
tions (log MPN/100 mL) for water samples collected within
the Cladophora mats for the three sampling events were 10 to
100 times greater than the mean E. coli concentrations detect-
ed in outlying water. Th e E. coli concentrations on either side
of the mats also were signifi cantly diff erent from one another
(p < 0.001), with concentrations from water samples to the left
(north) of the mat generally greater than those to the right (Fig.
3). Th e prevailing longshore currents at this beach move north
(data not shown) and may transport E. coli and other particu-
lates from the mat into the surrounding water.
For Lakeside Park, mean E. coli concentrations (MPN/100
mL) for water samples collected within the Cladophora mats
Fig. 2. Sampling protocol for staked samples (A) and random water samples from within and outside the Cladophora mat. Oval = Cladophora mat,
numbers and letters represent sample site locations (approximate).
Table 1. Basis for estimation of MPN/100 mL of pathogens in
+ Colonies from:
10 mL, 100 mL, 1 L
100 mL, 1 L
MPN/100 mL water
Vanden Heuvel et al.: Infl uence of Cladophora Mats on Escherichia coli in Water 337
Fig. 3. Log Escherichia coli concentrations from 10 randomly collected water samples from within and surrounding Cladophora mats during three
sampling events at Whitefi sh Dunes State Park Beach. Bars are means of random samples ± standard error.
338 Journal of Environmental Quality • Volume 39 • January–February 2010
Fig. 4. Log Escherichia coli concentrations from 10 randomly collected water samples from within and surrounding Cladophora mats during three
sampling events at Lakeside Park Beach. Bars are means ± standard error.
Vanden Heuvel et al.: Infl uence of Cladophora Mats on Escherichia coli in Water 339
for the three sampling events also were signifi cantly higher
than surrounding water (p < 0.001) (Fig. 4). Escherichia coli
concentrations in randomly collected water samples to the left
and right of the mats were not signifi cantly diff erent from one
another (p = 0.401). Th ere is little longshore current at this
beach (data not shown) and E. coli may move by simple diff u-
sion out of the mat proper.
Th ese random samples not only establish that E. coli con-
centrations in water are greatest within Cladophora mats, but
also allow for day-to-day comparisons of E. coli concentrations.
For both beaches (Fig. 3 and 4) two of the three sampling
events (Events 1 and 3) showed an increase in E. coli concen-
trations for consecutive days of the event (Day 1 < Day 2 < Day
3). Event 2 at both beaches (1–3 Aug. 2007) did not fi t the
same pattern as Events 1 and 3. For Event 2, E. coli concentra-
tions decreased over consecutive days of the event at Whitefi sh
Dunes and showed no particular pattern at Lakeside Park.
At Whitefi sh Dunes State Park beach E. coli concentrations
increased from 4.0 log10 on Day 1, to 4.3 log10 on Day 2, to 4.4
log10 on Day 3. A two-way ANOVA was signifi cant for Beach
(p < 0.001), and Day (p = 0.001), with no signifi cant interac-
tion between these two factors. A one-way ANOVA showed
that E. coli concentrations in water over the three sampling
days at Whitefi sh Dunes State Park beach were signifi cantly
diff erent (p = 0.036), with Day 3 being signifi cantly higher
than Day 1. Similar results were seen for Lakeside Beach with
E. coli concentrations ranging from 3.6 log10 on Day 1 and Day
2 and increasing to 4.0 on Day 3. Concentrations from Day 3
were signifi cantly higher than both Days 1 and 2 (p = 0.003).
Results of the random sampling established that E. coli concen-
trations in water were greatest within mats, and generally increased
over time, but could not help determine if a spatial gradient of E.
coli concentrations existed within the mat and radiated outward.
To answer this question water samples also were collected from
evenly spaced positions within and outside of the Cladophora mat
for each of the three (3 d) sampling events (Fig. 2A).
For Whitefi sh Dunes, mean E. coli concentrations
(MPN/100mL) from evenly spaced water samples collected
within the Cladophora mats for the three sampling events were
signifi cantly higher than surrounding water (p < 0.001). Th e E.
coli concentrations on either side of the mats also were signifi -
cantly diff erent from one another at p < 0.001, as was seen with
the randomly collected water samples (Fig. 5).
For Lakeside Park, mean E. coli concentrations (MPN/100mL)
from evenly spaced water samples collected within the Cladopho-
ra mats for the three sampling events were signifi cantly higher
than in surrounding water (p < 0.001). In contrast to Whitefi sh
Dunes, the E. coli concentrations in water collected to the left
and right side of the mats at Lakeside Park were not signifi cantly
diff erent from one another (p = 0.151) (Fig. 6).
When E. coli concentrations in water from the edges of the
Cladophora mat were compared with E. coli in water from the
center of the mat, there was no consistent pattern in E. coli con-
centrations (p = 0.447, r2 = 0.003). A signifi cant (p = 0.013,
r2 = 0.036, N = 171) positive relation was found, however, using
log (MPN E. coli) as the dependent variable and mat location.
Th is suggests that there is more E. coli in water near the center of
the Cladophora mat compared to the edges (Fig. 5 and 6).
Escherichia coli Attached to Cladophora Mats
Stranded mats were comprised primarily of Cladophora
glomerata (L.) Kuetzing. Escherichia coli concentrations ex-
ceeded 5.40 log10 E. coli/g dry weight of mat material during
two sampling events (Events 2 and 3) at Whitefi sh Dunes State
Park when E. coli was detached from mat biomass (Fig. 7), and
concentrations reached 5.42 log10 CFU/g dry wt during the
fi rst sampling event. Th is was the highest concentration of E.
coli found in all samples. Th e second sampling event contained
the highest average concentrations of E. coli with a mean of
5.03 log10 CFU/g dry wt. Lakeside Park demonstrated a similar
relationship, with the second event having the highest mean
concentration of E. coli at 4.04 log10 CFU/g dry wt (Fig. 8).
While the trends in E. coli concentrations were similar from
event to event at both locations, the concentrations of E. coli
were much lower in the Lakeside Park mats. Th e highest con-
centration of E. coli was found to be 4.19 log10 CFU/g dry wt.
during the third event. At Lakeside Park only two mean E. coli
concentrations from sampling event days averaged over 4.30
log10 CFU/g dry wt. In contrast, Whitefi sh Dunes State Park
had 9 samples where E. coli concentrations exceeded 4.70 log10
CFU/g dry wt.
Association between Escherichia coli in Water and Cladophora
For both beaches combined, there was a signifi cantly posi-
tive association between the concentration of E. coli attached
to Cladophora and E. coli concentrations from water samples
collected within the mats (p < 0.001, r2 = 0.42) (Fig. 9). A
similar pattern was revealed when the study beaches were ana-
lyzed separately. For Whitefi sh Dunes State Park beach there
remained a signifi cant relationship between the measurements
(log (CFU) vs. log (MPN)) (N = 76, r2 = 0.365, p < .001).
Likewise, for Lakeside Park beach there was also a signifi cant
relationship between the measurements [log (CFU) vs. log
(MPN)] (N = 82, r2 = 0.438, p < 0.001).
Fecal Pathogens Associated with Cladophora mats
Despite high E. coli concentrations in water and the algal
mats at all sampling events, no Salmonella, Shigella, or Campy-
lobacter were detected in these samples.
Escherichia coli concentrations in water underlying
Cladophora mats were routinely higher than in water away
from mats. Th e concentration of E. coli in outlying water to the
left or right of the mat seemed to be dependant on the prevail-
ing water current direction. Determination of current direc-
tion by beach managers (a simple process) could provide a clue
to the possible presence of elevated of E. coli concentration in
adjacent beach water and thus aid in protecting public health.
Random sampling of water underlying and surrounding per-
sistent Cladophora mats for consecutive days allowed us to iden-
340 Journal of Environmental Quality • Volume 39 • January–February 2010
Fig. 5. Log Escherichia coli concentrations from 10 evenly spaced water samples from within and surrounding Cladophora mats during three
sampling events at Whitefi sh Dunes State Park Beach. Bars are means of three consecutive sampling days ± standard error.
Vanden Heuvel et al.: Infl uence of Cladophora Mats on Escherichia coli in Water 341
Fig. 6. Log Escherichia coli concentrations from 10 evenly spaced water samples from within and surrounding Cladophora mats during three
sampling events at Lakeside Park Beach. Bars are the means of three consecutive sampling days ± standard error.
342 Journal of Environmental Quality • Volume 39 • January–February 2010
Fig. 7. Log Escherichia coli concentrations attached to the Cladophora mat at Whitefi sh Dunes State Park Beach. Each bar represents the mean of
three consecutive sampling days ± standard error.
Fig. 8. Log Escherichia coli concentrations attached to the Cladophora mat at Lakeside Park Beach. Each bar represents the mean of three consecutive
sampling days ± standard error.
Vanden Heuvel et al.: Infl uence of Cladophora Mats on Escherichia coli in Water 343
tify changes in E. coli concentrations with time. As the algae mat
persisted on the beach, E. coli concentrations increased. Th is sug-
gests that the algae mats provide a suitable habitat not only for
the persistence, but also the growth of E. coli. A genetic analysis
of the E. coli isolates recovered as part of this study would go
farther in answering questions concerning the ability of E. coli
to replicate in the Cladophora mat, and is part of another study
submitted elsewhere. Th is study alone, however, supports the
fi ndings of others (Byappanahalli et al., 2003; Englebert et al.,
2008; Whitman et al., 2003) that Cladophora mats can infl uence
E. coli survival in the beach environment.
Generally, as E. coli concentrations within the Cladophora
mats increased, E. coli in the surrounding water increased. While
E. coli concentrations of the surrounding water were low relative
to the concentrations within the mats, mean concentrations were
greater than the allowable criteria for recreational freshwaters; 235
CFU/100 mL (USEPA, 1986) for three of six (50%) random
sampling events at Whitefi sh Dunes State Park (Fig. 3). Likewise,
at Lakeside Park the mean E. coli concentrations outside the mats
exceeded the allowable criteria for four of six (66.7%) random
sampling events (Fig. 4). Th ese fi ndings suggest that the presence
of stranded Cladophora mats in nearshore water or on beach sand
may elevate E. coli concentrations in beach water used to deter-
mine beach openings and closures. Removal of these algal mats by
beach managers may positively impact recreational water quality
at beaches with Cladophora accumulations.
Collection of evenly spaced water samples underlying
Cladophora mats allowed us to identify patterns of E. coli dis-
tribution within a mat. In general, E. coli concentrations were
greatest in underlying water located near the center of the mat.
Th e center of the mat is protected from wind and wave action
and E. coli from this area may not be moved into surrounding
water as easily as E. coli located at the perimeter of the mat.
Escherichia coli attached to Cladophora biomass in mats
was removed by vigorous washing and was enumerated and
expressed as CFU E. coli/g dry wt Cladophora to determine if
the algal strands off ered attachment points for the bacterium.
At both beaches, extremely elevated concentrations of E. coli
were removed from Cladophora biomass (4–5 log10/g dry wt).
Wind and wave action on the stranded Cladophora biomass
should contribute to detachment of E. coli from the algae (as
did mechanical shaking during the wash procedure), seeding
underlying water with the indicator organism and potentially
allowing weather conditions to move E. coli into contact with
bathers. Th e signifi cant, positive correlation found between
concentrations of E. coli attached to Cladophora and in free
in adjacent water (Fig. 9) supports this. Since E. coli appears
to persist in Cladophora mats for extended time periods and
perhaps replicates, Cladophora mats on swimming beaches rep-
resent a challenge to maintaining beach water quality.
Surprisingly, common fecal pathogens (Salmonella, Shigel-
la, and Campylobacter) were not detected in water underlying
nor attached to Cladophora mats in this study. Th ese bacte-
rial pathogens have been detected in Cladophora mats, albeit in
low densities, in other Lake Michigan beachsheds (Ishii et al.,
2006; Byappanahalli et al., 2008). We propose two hypotheses
for their nondetection in the current study. First, the counts
of these bacteria were too low to be enumerated by the cul-
tural methods used, due to environmental conditions, such as
dilution, temperature (Rhodes and Kator, 1988), solar radia-
Fig. 9. Relationship between Escherichia coli concentrations found in water log (MPN/100 mL) and E. coli concentrations attached to Cladophora
biomass log (CFU/g dry wt).
344 Journal of Environmental Quality • Volume 39 • January–February 2010
tion (Stevenson and Stoermer, 1982; McCambridge and Mc-
Meekin, 1981), predation (Rhodes and Kator, 1988), and via-
ble-but nonculturable (VBNC) state of the organisms (Roszak
et al., 1984). We plan to use molecular techniques to detect
these pathogens in future years of the study. While Cladopho-
ra attached to its hard substrate in southern Lake Michigan
was previously shown to contain these pathogens (Ishii et al.,
2006), both culture and molecular methods have been unable
to detect Salmonella from northern Lake Michigan, including
Door County (Byappanahalli et al., 2008)). Our second hy-
pothesis centers on the paucity of occurrence of these patho-
gens in northern Lake Michigan. Th e presence of these patho-
gens in southern Lake Michigan may be a factor of input from
particular sources (wastewater) not found commonly in the
northern areas of this lake.
If E. coli, the indicator of fecal contamination, is able to
persist and perhaps replicate in Cladophora mats while fecal
pathogens are not present, the usefulness of E. coli as an indica-
tor organism is called into question. Further studies to answer
these questions have been planned.
In summary, this study has measured some of the greatest
concentrations of E. coli found in water within Cladophora mats
(> 5.4 log10 MPN/100 mL), as well as concentrations of E. coli
attached to strands of Cladophora material recovered from mats
(> 5.4 log10 CFU/g dry wt.). Th is study further demonstrated a
direct relationship at both sites between attached E. coli concen-
trations within the mat and E. coli concentrations in water with-
in the mat. Additionally, at both study sites the E. coli concentra-
tions increased from Day 1 through Day 3 of the 3-d sampling
events. Th is, coupled with our molecular analysis (data not pre-
sented here) suggests that E. coli may be replicating within the
Cladophora mats. A lack of detection of fecal pathogens within
Cladophora mats calls into question the ability of E. coli to serve
as an appropriate indicator of recent fecal contamination.
Th is project was funded by the University of Wisconsin Sea
Grant Research Program. Special thanks to Rhonda Daily for
her assistance in sample collection and laboratory analysis.
Barbiero, R.P., M.C. Tuchman, and E.S. Millard. 2006. Post-dreissenid
increases in transparency during summer stratifi cation in the off shore
waters of Lake Ontario: Is a reduction in whiting events the cause? J.
Great Lakes Res. 32:131–141.
Bootsma, H. 2007. Linking Cladophora growth to mussel metabolism and
nearshore hydrodynamics. p. 2 In Proc. of Cladophora and Great Lakes,
Oshkosh, WI. 17 Jan. 2007.
Byappanahalli, M.N., R. Sawdey, S. Ishii, DA. Shively, J.A. Ferguson, R.L.
Whitman, and M.J. Sadowsky. 2008. Seasonal stability of Cladophora-
associated Salmonella in Lake Michigan watersheds. Water Res.
Byappanahalli, M.N., D.A. Shively, M.B. Nevers, M.J. Sadowsky, and R.L.
Whitman. 2003. Growth and survival of Escherichia coli and enterococci
populations in the maco-alga Cladophora (Chlorophyta). FEMS
Microbiol. Ecol. 46:203–211.
Chilton, E.W., R.L. Lowe, and K.M. Schurr. 1986. Invertebrate communities
associated with Bangia atropurpurea and Cladophora glomerata in western
Lake Erie. J. Great Lakes Res. 12:149–153.
Clescerl, L.S., A.E. Greenberg, and A.D. Eaton (ed.). 2005. Standard methods
for examination of water and wastewater. 20th ed. American Public
Health Assoc., Washington, DC.
Englebert, E.T., C. McDermott, and G.T. Kleinheinz. 2008. Eff ects of the
nuisance algae, Cladophora, on Escherichia coli at recreational beaches in
Wisconsin. Sci. Total Environ. 404:10–17.
Herbst, R.P. 1969. Ecological factors and the distribution of Cladophora
glomerata in the Great Lakes. Am. Midl. Nat. 82:90–98.
Ishii, S., T. Yan, D.A. Shively, M.N. Byappanahalli, R.L. Whitman, and
M.J. Sadowsky. 2006. Cladophora (Chlorophyta) spp. harbor human
bacterial pathogens in nearshore water of Lake Michigan. Appl. Environ.
Islam, M.S., B.S. Drasar, and D.J. Bradley. 1989. Attachment of toxigenic Vibrio
cholerae O1 to various freshwater plants and survival with fi lamentous
green alga Rhizoclonium fontanum. J. Trop. Med. Hyg. 92:396–401.
Kinzelman, J., A. Singh, C. Ng, K. Pond, R. Bagley, and S. Gradus. 2005. Use
of IDEXX Colilert-18 and Quanti-Tray/2000 as a more rapid and simple
enumeration method for detection of E. coli in recreational freshwater.
Lake Reservoir Manage. 21:73–77.
Marks, J.C., and M.E. Powers. 2001. Nutrient induced changes in the species
composition of epiphytes on Cladophora glomerata Kutz (Chlorophyta).
McCambridge, J., and T.A. McMeekin. 1981. Eff ect of solar radiation and
predacious microorganisms on survival of fecal and other bacteria. Appl.
Environ. Microbiol. 41:1083–1087.
Rhodes, M.W., and H. Kator. 1988. Survival of Escherichia coli and Salmonella
spp. in estuarine environments. Appl. Environ. Microbiol. 54:2902–2907.
Roszak, D.B., D.J. Grimes, and R.R. Colwell. 1984. Viable but nonrecoverable
stage of Salmonella enteritidis in aquatic systems. Can. J. Microbiol.
Stevenson, R.J., S.T. Rier, C.M. Riseng, R.E. Shultz, and M.J. Wiley. 2006.
Comparing eff ects of nutrients on algal blooms in streams in two regions
with diff erent disturbance regimes and with application for developing
nutrient criteria. Hydrobiologia 561:149–165.
Stevenson, R.J., and E.F. Stoermer. 1982. Seasonal abundance patterns of
diatoms on Cladophora in Lake Huron. J. Great Lakes Res. 8:169–183.
USEPA. 1986. Ambient water quality criteria for bacteria 1986. USEPA,
Offi ce of Water Regulations and Standards, Washington, DC.
USEPA. 2002, Method 1603—Escherichia coli in water by membrane
fi ltration using modifi ed membrane-thermotolerant Escherichia coli agar.
EPA 821-R-02–23. USEPA, Washington, DC.
Whitman, R.L., D.A. Shively, H. Pawlik, M.B. Nevers, and N. Byappanahalli.
2003. Occurrence of Escherichia coli and enterococci in Cladophora
(Chlorophyta) in nearshore water and beach sand of Lake Michigan.
Appl. Environ. Microbiol. 69:4714–4719.
Wisconsin State Laboratory of Hygiene. 2003. ESS Micro Methods, SOP 348,
Detection of Salmonella in water samples, Rev 2.0. 12 Dec. 2003.
Zar, J.H. 1999. Biostatistical analysis, 4th ed. Prentice Hall, Englewood Cliff s, NJ.