These authors contributed equally to this paper.
Growth and survival of Escherichia coli and enterococci populations in the macro-alga Cladophora (Chlorophyta)
Article first published online: 5 JAN 2006
FEMS Microbiology Ecology
Volume 46, Issue 2, pages 203–211, November 2003
How to Cite
Byappanahalli, M. N., Shively, D. A., Nevers, M. B., Sadowsky, M. J. and Whitman, R. L. (2003), Growth and survival of Escherichia coli and enterococci populations in the macro-alga Cladophora (Chlorophyta). FEMS Microbiology Ecology, 46: 203–211. doi: 10.1016/S0168-6496(03)00214-9
- Issue published online: 5 JAN 2006
- Article first published online: 5 JAN 2006
- Received 25 June 2003, Revised 12 August 2003, Accepted 15 August 2003
- Environmental condition;
- Escherichia coli;
- Great Lakes
The macro-alga Cladophora glomerata is found in streams and lakes worldwide. High concentrations of Escherichia coli and enterococci have been reported in Cladophora along the Lake Michigan shore. The objective of this study was to determine if Cladophora supported growth of these indicator bacteria. Algal leachate readily supported in vitro multiplication of E. coli and enterococci, suggesting that leachates contain necessary growth-promoting substances. Growth was directly related to the concentration of algal leachate. E. coli survived for over 6 months in dried Cladophora stored at 4°C; residual E. coli grew after mat rehydration, reaching a carrying capacity of 8 log CFU g−1 in 48 h. Results of this study also show that the E. coli strains associated with Cladophora are highly related; in most instances they are genetically different from each other, suggesting that the relationship between E. coli and Cladophora may be casual. These findings indicate that Cladophora provides a suitable environment for indicator bacteria to persist for extended periods and to grow under natural conditions.
Cladophora glomerata (L.) Kütz (Chlorophyta) is a macro-alga that grows as dense strands and mats attached to hard substrates of streams and lakes. In the Great Lakes, massive growths of detached Cladophora have been a common summer problem, creating a shoreline nuisance, affecting recreational activities (fishing, boating, swimming), and potentially influencing water quality . Algal accumulations are especially troublesome in shallow embayments and protected areas near or down current of shoreline hardening and groins. Algae can become detached, drift in, and form large mats along the shoreline or become stranded upon the foreshore, producing malodorous conditions that can detract from visitor enjoyment. Recently, it was determined that Cladophora occurring along the shorelines of Lake Michigan was a significant environmental source of Escherichia coli and enterococci . These findings may have important implications for beach management decisions because Cladophora may increase the level of indicator bacteria in nearshore swimming water and foreshore sand, resulting in higher beach closure rates and lower visitation. Public health implications remain unexplored.
The occurrence of bacteria on algae is common [3–7], but the presence of fecal indicator bacteria –E. coli and enterococci – on algae challenges the discussion of the regulatory basis for using indicator bacteria . The recent findings that Cladophora is a significant environmental source of E. coli and enterococci concur with numerous other reports demonstrating occurrence and perhaps even growth of indicator bacteria in natural habitats (soil, water, plants) [9–19].
The recovery of high concentrations of E. coli and enterococci in Cladophora (median 3.7 and 2.1 log CFU g−1 algal mat, respectively) in numerous Lake Michigan beaches  suggested that Cladophora could sustain these bacteria in ambient conditions. The primary objective of this study was to examine growth potential of E. coli and enterococci in Cladophora. Several laboratory-based experiments were conducted using algae or leachate preparations from Cladophora as the principal growth medium.
2Materials and methods
To demonstrate the growth potential of E. coli and enterococci in Cladophora, two approaches were taken. First, Cladophora leachate (primarily, washings of the alga with unfiltered lake water) containing residual populations of E. coli and enterococci was used as a growth medium to determine the effect of physical and chemical factors on E. coli and/or enterococci growth. In the second approach, small quantities of dried Cladophora mats (containing residual indicator bacteria) were used as the growth medium. Experiments were conducted to determine (1) the growth potential of residual E. coli after rehydration of the algal mat; (2) the effect of nutrient and inoculum loss by sequential mat washings on growth potential and mat carrying capacity of E. coli; and (3) growth patterns of E. coli in rehydrated algal mat suspended in lake water or mixed with beach sand.
Cladophora samples were collected from Washington Park Beach in Michigan City, Indiana between June 24 and November 7, 2002. Samples were collected either from the water (floating algae) or from rock pilings (attached algae). All Cladophora samples were collected by hand and placed in plastic bags. Samples were transported to the laboratory at 4°C, and unless otherwise stated, analyzed within 4–6 h of collection. Air and water temperatures were recorded in the field at the time of sampling.
2.3Recovery of bacteria from algae
E. coli and/or enterococci counts were usually determined by using the membrane filtration (MF) technique . Algal sub-samples (1 g) were placed in sterile 15-ml centrifuge tubes, to which aliquots (9 ml) of sterile phosphate-buffered dilution water (PBW, pH 6.8) were added . The sample–PBW mixture was vigorously shaken by hand for 2 min and centrifuged briefly (45 s) at 2000 rpm. When necessary, the supernatant was further diluted in PBW to achieve desirable counts. The filters were aseptically placed on mTEC (E. coli) or mE (enterococci) media (Difco Laboratories, Becton-Dickinson, Sparks, MD, USA) and incubated at 44.5°C (E. coli) or 41°C (enterococci) . Sequential rinsing of algae showed that, on average, 55% of adhered bacteria were recovered using this technique (data not shown). Counts were expressed g−1 dry weight of the sample.
2.4Recovery of bacteria from sand
Pre-washed, then dried, construction-grade (particle size ∼0.5 mm in diameter) silica sand was used in all experiments. Portions (50 g) of sand were weighed into plastic bags, to which aliquots (100 ml) of PBW were added. The sand–PBW mixture was shaken for 2 min and allowed to settle for 30 s. The supernatant was then serially diluted, and appropriate volumes were analyzed for E. coli as described above. Sequential rinsing showed that an average of 86–100% of bacteria were recovered using this technique. Unless otherwise stated, E. coli counts in Cladophora were expressed g−1 dry weight of sand.
If the turbidity of the sand washing (due to the presence of algae) was too high for the MF technique, then the Colilert-18 method (IDEXX, Westbrook, ME, USA), which is based on a defined substrate technology [21,22], was used for E. coli analysis. E. coli was elutriated from sand similarly as described above, and the suspension was serially diluted as necessary. Samples were placed in distribution trays (IDEXX QuantiTray/2000), sealed, and incubated at 35°C for 18 h. A most probable number of E. coli organisms in the sample was determined by counting all fluorescing wells using a long wavelength UV fluorescent lamp.
For quality control purposes, E. coli and enterococci determinations included suitable blanks and reference cultures (E. coli ATCC 25922 and Enterococcus faecalis ATCC 29212). At least 10% of the presumptive colonies for both E. coli and enterococci were confirmed by standard methods of analysis .
Sub-samples (10 g) of representative Cladophora were distributed into 12 sterile, 50-ml centrifuge tubes. To each tube, aliquots (30 ml) of unfiltered lake water were added; tubes were stored upright at 4°C for 48 h, and centrifuged for 30 min at 2000 rpm (653×g). The supernatant was poured through a 40-μm nylon mesh, and the pooled leachate was collected in a sterile dilution bottle and stored at 4°C. The algal leachates were not characterized for their nutrient composition; in this paper, they are referred to as ‘leachates’ instead of ‘nutritional media/broths’ to prevent ambiguity.
Only freshly prepared leachates were used for growth-related experiments throughout this study. On one occasion, the experimental design required five independently prepared leachates; each batch of Cladophora was treated alike to minimize experimental error and to maintain the integrity of the growth media. E. coli and enterococci counts in the leachates ranged from 3 to 1200 and 3 to 162 CFU ml−1, respectively. The leachates were used as is or diluted in PBW or lake water as necessary.
2.6.1Temperature effects on E. coli growth
Aliquots (5 ml) of the leachate were distributed into 12 sterile, 15-ml centrifuge tubes. Sets of four randomly chosen tubes were independently incubated at 25, 30, or 35°C. At 0, 24, 48, and 120 h of incubation, one tube from each temperature treatment was randomly chosen, and the contents were analyzed for E. coli by the MF technique as described above.
2.6.2Effect of leachate concentration on E. coli growth
Two separate sets of media containing different concentrations – 100, 50, 25, 13, 6, and 0% (control) – of original leachate were prepared using PBW and lake water as diluents. The media thus prepared were distributed into a series of 1-ml centrifuge tubes. 50 μl of fresh leachate containing about 500 E. coli cells was added to each sample tube except the uninoculated ones, which received 50 μl of sterile water. Following inoculation, the tubes were put into a cryogenic vial storage container, secured, and placed horizontally on an orbital shaker at 75 rpm. E. coli was enumerated for each set of tubes at 0 and 24 h of incubation at 35°C.
2.6.3Growth of E. coli and enterococci in leachate
The purpose of this experiment was to establish growth curves for E. coli and enterococci using Cladophora leachates as growth media. Five replicate leachates were prepared from independent algal samples. From each replicate, sub-samples (5 ml) were distributed into 15-ml centrifuge tubes. The tubes were maintained horizontally on an orbital shaker at 75 rpm and 35°C. Randomly retrieved tubes were analyzed for E. coli and enterococci at 0, 6, 12, 18, 24, 48,72, 96, and 168 h of incubation.
2.6.4Multiplication of residual E. coli in unwashed and washed algae
Fresh Cladophora was spread on vinyl-coated, 2.5-cm-mesh racks (0.33 m×0.37 m) at a thickness of about 2.5 cm, and placed outdoors in sunlight for four consecutive days: June 24–26, 2002 from 7.00 h to 15.00 h and June 27 from 7.00 h to 12.00 h. Air temperature at the surface of the algal mat ranged from 28.5 to 31°C and averaged 30°C. The dried, sun-bleached algal mat was then stored at 4°C in airtight plastic bags for 6 months. Samples of algal mats were analyzed for E. coli both before and after sun exposure.
Six-gram portions of the dried Cladophora mats were weighed into two 100-ml dilution bottles. Aliquots (100 ml) of PBW were added to each bottle, and the mixture was gently homogenized by shaking. To ensure that the dried mats were sufficiently moistened, the Cladophora and PBW mixtures were incubated together for an additional 20 min. Excess water in one of the bottles was drained and 1-g samples of the moist Cladophora mat (unwashed) were weighed into several 47-mm Petri plates. The contents in the other bottle were shaken for 2 min and filtered through 106-μm nylon netting; fresh PBW was added to wash the algal mat again. This procedure was repeated five more times; at the end of the sixth wash, 1-g portions of the Cladophora mat (washed) were weighed into several 47-mm Petri plates. In addition to these treatments, another set of tubes was prepared that contained 9.9 ml of PBW and 0.1 g of dried, unwashed Cladophora mat. All treatments were incubated at 35°C. Samples of washed and unwashed algae in the Petri plates were analyzed by the Colilert-18 method at 0, 6, 12, 24, 48, 72, and 96 h, and samples in tubes were analyzed by the MF technique at 0, 24, 48, 72, and 96 h.
2.6.5E. coli multiplication in sand and lake water
Preliminary studies showed that there was a positive dose response of E. coli growth with Cladophora content in sand (data not shown). Those experiments yielded a protocol and appropriate concentration ranges of Cladophora in sand for E. coli growth. In this study, to compare the growth patterns of Cladophora-borne E. coli in sand and lake water, 1 g of dried algae and 9 ml of filter-sterilized lake water was shaken in a centrifuge tube for 2 min and poured into a plastic bag containing 50 g of pre-washed, then dried, construction-grade (∼0.5 mm in diameter) E. coli-free silica sand. Remaining algae in the tube were rinsed into the bag with 2 ml of sterile lake water, and the contents were gently mixed. For sand samples not containing algae (control), 11 ml of lake water was added directly to the sand.
For the Cladophora in lake water treatment, sub-samples (1 g) of the previously sun-dried algae were weighed into sterile, 50-ml centrifuge tubes, to which aliquots (40 ml) of lake water were added, and contents were gently mixed. The tubes were maintained horizontally on an orbital shaker at 75 rpm. All samples were incubated at 35°C; samples were analyzed for E. coli at 0, 24, 48, 72, and 96 h by the MF method.
2.7Genetic analysis of E. coli
2.7.1E. coli isolates
Initially, E. coli from Cladophora or its leachates was isolated by the MF technique using mTEC agar . Following substrate test for urease activity, yellow, yellow-green, or yellow-brown colonies were further streaked onto MacConkey agar (Difco Laboratories) to obtain pure colonies; after secondary isolation on the same medium, presumptive E. coli colonies were stored in 2-ml cryogenic vials containing 1 ml tryptic soy broth and 10% glycerol at −45°C, until used. To confirm presumptive isolates for E. coli, isolates were streaked onto mFC agar (Difco Laboratories) plates and incubated at 44.5°C for 24 h. Characteristic blue colonies from the mFC plates were picked and evaluated using selective and differential media as previously described . A total of 44 isolates were used for repetitive polymerase chain reaction (rep-PCR) analysis.
2.7.2Rep-PCR DNA fingerprinting
E. coli isolates were streaked onto Plate Count Agar (Difco Laboratories) and grown overnight at 37°C. Colonies were picked with a 1-μl sterile inoculating loop and suspended in 100 μl of sterile water. Fluorophore-enhanced rep-PCR (FERP) analyses were performed using slight modifications to previously described procedures [23,24]. FERP images were acquired using a Typhoon 8600 Variable Mode Imager (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA, USA) operating in the fluorescence acquisition mode using the following settings: green (532 nm) excitation laser; 610 BP 30 and 526 SP emission filters in the autolink mode with 580 nm beam splitter; normal sensitivity; 200 μm/pixel scan resolution; and +3 mm focal plane. The separated gel images were processed using ImageQuant image analysis software (Molecular Dynamics) and converted to 256 gray scale TIF images for import into the BioNumerics software.
Gel images were normalized and analyzed using BioNumerics v.3.0 software (Applied Maths, Sint-Martens-Latem, Belgium). FERP gel lanes were normalized using Genescan-2500 ROX internal lane standards. DNA fingerprint similarities were calculated using the curve-based Pearson product moment correlation coefficient with 1% optimization. Dendrograms were generated using the unweighted pair-group method using arithmetic means (UPGMA).
Statistical analyses and graphical presentations were performed using SPSS version 11.5. Statistical procedures were performed on log10-transformed data to meet parametric assumptions. Earlier studies demonstrated normality and equality of variances with log transformation in area sands and water , but there remain insufficient data to test distribution of E. coli in algae. Unless otherwise stated, statistical analyses were done using P=0.05.
From initial numbers at the time of leachate production, the growth of indigenous E. coli markedly increased with incubation temperature of algal leachate. At 120 h, mean concentration of E. coli at 25, 30 and 35°C was 3.2, 3.7, and 4.4 log CFU ml−1, respectively. At all temperatures, E. coli counts declined marginally between 24 and 48 h and then increased slightly between 48 and 120 h. Even though E. coli growth in algal leachate was best at 35°C, the nominal growth at 25°C is environmentally significant because this temperature corresponds to the nearshore lake temperature during warm summer months.
3.2Leachate concentration and E. coli growth
A paired t-test indicated that PBW and lake water mean E. coli concentrations did not differ significantly (P=0.275) and the two media responses were correlated (P=0.025). Therefore, the corresponding data were pooled for further analysis, and paired observations were treated as duplicates. E. coli growth was directly proportional to percent algal leachate in the medium (Fig. 1). An exponential growth model likely described the relationship, although, statistically, a linear best-fit line yielded a higher coefficient of determination (R2=0.97 vs. R2=0.83, respectively). Undefined, growth-promoting substances leached from Cladophora supported the growth of E. coli.
The initial concentration of E. coli and enterococci in the undiluted leachate was 1.3 log CFU ml−1. E. coli numbers increased by approximately 1000-fold after 18 h, while enterococci counts increased by about 100-fold during the same time; counts of these bacteria declined gradually during the following 44 h and then leveled off to 2.3 and 1.1 log CFU ml−1, respectively (Fig. 2). At the end of 168 h, E. coli counts were still higher than initial counts, while enterococci numbers had dropped to their initial density. These results clearly indicated that Cladophora leachates contained substances that can readily support the growth of E. coli and enterococci. Further, the manner in which E. coli and enterococci grew in Cladophora leachates was generally comparable to their growth patterns in standard microbiological media (e.g., TSB/BHI broth).
3.4E. coli in washed and unwashed algal mats
E. coli in Cladophora following sun-drying steadily declined in number (from 6.0 to 5.0 log CFU g−1) during storage (June 2002 to February 2003) at 4°C. In June 2002, E. coli counts in rewetted mats (immediately following drying) maintained an equilibrium density of 8.5 log CFU g −1 (data not shown). While the initial nominal concentrations of E. coli in the same refrigerated mats dropped when the experiment was repeated after 6 months (in December 2002) and after 8 months (in February 2003), the mat carrying capacity of E. coli remained the same, approximately 8 log E. coli CFU g−1 (Fig. 3); both growth curves fit similar logarithmic models: R2=0.94 and R2=0.95, respectively. After 8 months of storage, E. coli growth in washed Cladophora also fit a logarithmic curve (R2=0.98) but displayed relatively lower initial counts and a diminished carrying capacity: 7 log CFU g−1. Beyond these parameters, the three growth curves were remarkably similar, with realized growth rate (μ) of 0.61 after 6 months (unwashed algal mats), 0.62 after 8 months (unwashed algal mats), and 0.68 after 8 months (washed algal mats) (Fig. 3). The lower equilibrium phase coupled with the lack of convergence with washed algal mats suggests that nutrients were lost during rinsing. Both treatments were inspected microscopically and had similar potential foraging communities (e.g., ciliates, sarcodines, rotifers, gastrotriches).
3.5Growth in sand and lake water
Compared to lake water, E. coli populations were sustained longer and at higher density in sand. E. coli counts in Cladophora suspended in lake water declined exponentially during the first 48 h (Fig. 4A); counts were relatively stable between 48 and 72 h and again declined between 72 and 96 h. Over the entire incubation period, the reduction in E. coli counts was almost in excess of 3 log. When Cladophora was added to moist sand (Fig. 4B), E. coli counts increased by as much as 1 log during the first 24 h; however, the numbers declined over the next 72 h. At 96 h of incubation, the sand had higher E. coli counts than lake water (3 log CFU g−1 vs. 1 log CFU ml−1).
3.6Genetic relatedness of E. coli isolates from Cladophora
In order to determine if the E. coli associated with Cladophora constituted a unique group of isolates, we examined 44 isolates obtained from different Cladophora mats, located on rocks within a 15–20-m transect, for genetic relatedness using FERP. The isolates could be divided into two major clusters (I and II) based on their genomic fingerprints (Fig. 5). Cluster I isolates consisted of three sub-groups, A, B, and C, while those in cluster II comprised a single group, D, with one outlier. Although the cluster I isolates were very related to each other, with similarity values of about 90% or greater, the strains were not identical. Strains in sub-group D were only related to each other at an 80% or greater level. Generally, isolates from a specific mat did not group with each other, suggesting a high degree of genetic diversity within and between Cladophora filaments obtained from different rocks within the transect. The non-correspondence of isolates between tufts (rocks bearing Cladophora) was verified using the principal component and MANOVA analyses (data not shown).
Historically, a close link between algae and bacteria has consistently been observed in aquatic environments [3–7], although the ecological significance of these associations is not fully understood. Generally, it is known that bacterial survival and growth in aquatic habitats increases when they are attached to particulate matter or other solid surfaces. Algal surfaces provide sites for attachment, protection against harmful radiation and predation, and nourishment to associated epiphytes, which can vary from relatively simple to multicellular organisms [26–29]. Algal exudates are perhaps one of the most important nutrient sources for heterotrophic bacteria in aquatic environments [30–35]. Although the nature of compounds released by algae under natural conditions is not well characterized, limited research suggests that the exudates may contain a variety of growth-promoting organic compounds, such as carbohydrates and proteins [36,37].
The primary objective of this study was to determine the growth potential of E. coli and enterococci on Cladophora under laboratory conditions and to further explain previous field observations related to indicator bacteria and Cladophora association . Our hypothesis was that unidentified substances released by Cladophora and perhaps other material attached to or associated with this alga were sufficient to promote the growth of indicator bacteria found in the algal matrix. Results showed that E. coli growth in Cladophora leachate was directly proportional to leachate concentration, indicating that these undetermined substances were responsible for E. coli growth. Washing experiments suggested that growth was primarily due to leachates rather than easily rinsed epi- or periphytic material. Residual populations of E. coli in the sun-dried Cladophora mats (that had been stored at 4°C for over 6 months) were induced to grow just by rehydration, reaching a surprisingly high carrying capacity of 8 log CFU g−1 within 48 h at 35°C. Collectively, these experiments suggest that Cladophora provides a suitable environment for indicator bacteria not only to persist for extended periods but also to grow in natural environments, particularly during warm summer months.
Results of this study also show that the E. coli strains associated with Cladophora are highly related yet in most instances are genetically distinct from each other. This suggests that the relationship between E. coli and Cladophora may not be interdependent; perhaps water-borne E. coli can readily populate newly emerging algal filaments. In this sense, the E. coli can be viewed as zymogenous microorganisms, taking advantage of newly found resources for their growth and reproduction.
Although both E. coli and enterococci are heterotrophic, their nutritional requirements are vastly different. E. coli nutritional requirements are relatively simple, and it has the capacity to synthesize its cellular components from glucose and minerals . Enterococci are rather fastidious in their nutritional requirements (in laboratories, they are usually grown on complex media, such as tryptic soy agar plus sheep blood and brain heart infusion broth/agar). In this study, growth of enterococci in the Cladophora leachate is an indication that the leachate has all the necessary ingredients to support their growth. The idea that algal exudates and their cellular products are important nutrient sources for heterotrophic bacteria in aquatic environments is not new [6,39]; however, the findings that such products can promote the growth of indicator bacteria are significant because they suggest potential growth of common ‘fecal bacteria’ under natural conditions. This situation does not appear to be localized, since high concentrations of these indicator bacteria were consistently recovered from Cladophora samples from numerous Lake Michigan beaches, from Wisconsin through northern Michigan . In our studies there was a significant loss of E. coli in lake water, perhaps due to grazing organisms (ciliates, rotifers, bryozoans, sarcodines), depletion of nutrients, or competition from other organisms. Microscopic examination of the algal thalli revealed the presence of protozoan and metazoan grazers. While interspecific pressure was relatively uninhibited in the lake water infusion, sand may have provided a better refuge for E. coli and perhaps some competitive advantage.
The long-term survival of E. coli and enterococci in dried Cladophora mats has environmental and potential public health implications. Accumulated masses of Cladophora are a common sight along the shorelines of Lake Michigan. The mats that wash onto foreshore sand may remain there or move back and forth between nearshore water and foreshore sand, often getting buried in the sand by wave action and beach grooming. Algal mats containing indicator bacteria can serve as an inoculum to the moist foreshore sand. Once the mats are buried in sand, algal-borne indicator bacteria are protected from harmful effects of radiation and may multiply in the warm, moist environment, with abundant available nutrients from the decomposing mats . Thus, it is likely that indicator bacteria may survive for extended periods in both floating and stranded Cladophora mats. The algal mats can serve as a continuous source of indicator bacteria to the foreshore sand, which in turn can impact the perceived quality of nearshore bathing water.
Although the experiments in this study were conducted at 35°C to optimize growth of the indicator bacteria, the ambient temperature of southern Lake Michigan during late summer when Cladophora is most abundant is around 25°C. Consistently high concentrations of indicator bacteria in Cladophora (often exceeding 104–106 g−1) during warm summer months indicate that these bacteria can grow at reasonable rates under these conditions. Compared to nearshore water of the study area, foreshore sand is usually several degrees warmer (22–24°C in lake water vs. 31–36°C in foreshore sand, at 4–6 cm depth). Warm conditions can enhance the rate of Cladophora decomposition, which releases more nutrients to the surrounding algal matrix and can potentially increase counts of indicator bacteria.
These findings concur with previous observations that populations of indicator bacteria can naturally occur and may even grow in habitats such as soil, water, and plants [9,11,14–16,19,41]. If Cladophora can support the growth of resident indicator bacteria, then there is potential for pathogenic bacteria to persist and grow on this alga. The efficacy of using E. coli and enterococci as indicator bacteria relies on a reasonable link to fecal pollution from animals and humans. Even if environmental E. coli and enterococci are found not to originate in Cladophora, evidence that these bacteria persist and multiply in Cladophora may compromise their use as fecal indicators in areas rich in macro-algae.
We thank Eric Garza and Douglas Wilcox, USGS, for their critical review of the manuscript, and John Ferguson for rep-PCR analyses. Special thanks to Heather Pawlik, USGS, who helped in the initial stages of this research. This article is Contribution 1256 of the USGS Great Lakes Science Center.
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