JGLR-00730; No. of pages: 7; 4C: Journal of Great Lakes Research xxx (2014) xxx–xxx
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Notable decomposition products of senescing Lake Michigan Cladophora glomerata Julie R. Peller a,⁎, Muruleedhara N. Byappanahalli b,1, Dawn Shively b,1, Michael J. Sadowsky c,d,2, Chan Lan Chun c,2, Richard L. Whitman b,1 a
Department of Chemistry, Indiana University Northwest, 3400 Broadway, Gary, IN 46408, USA U.S. Geological Survey, Great Lakes Science Center, 1100 N Mineral Springs Road, Lake Michigan Ecological Research Station, Porter, IN 46304, USA BioTechnology Institute, University of Minnesota, St. Paul, MN 55018, USA d Department of Soil, Water and Climate, University of Minnesota, St. Paul, MN 55108, USA b c
a r t i c l e
i n f o
Article history: Received 30 September 2013 Accepted 7 April 2014 Available online xxxx Communicated by John Janssen Keywords: Cladophora Decomposition Persistent organics Algal mats Algal oils
a b s t r a c t Massive accumulations of Cladophora, a ubiquitous, filamentous green alga, have been increasingly reported along Great Lakes shorelines, negatively affecting beach aesthetics, recreational activities, public health and beachfront property values. Previously, the decomposition byproducts of decaying algae have not been thoroughly examined. To better understand the negative consequences and potential merit of the stranded Cladophora, a three month mesocosm study of the dynamic chemical environment of the alga was conducted using fresh samples collected from southern Lake Michigan beaches. Typical fermentation products, such as organic acids, sulfide compounds, and alcohols were detected in the oxygen–deprived algae. Short chain carboxylic acids peaked on day seven, in correspondence with the lowest pH value. Most low molecular mass carbon compounds were eventually consumed, but 4-methylphenol, indole, and 3-methylindole were detected throughout the incubation period. Natural oils were detected in fresh and decomposing algae, indicating the stable nature of these compounds. The mesocosm experiment was validated by directly sampling the fluid within decomposing Cladophora mats in the field; many of the same compounds were found. This study suggests that the problematic Cladophora accumulations may be harvested for useful byproducts, thereby reducing the odiferous and potentially harmful mats stranded along the shorelines. © 2014 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.
Introduction The resurgence of the green macroalga Cladophora in areas of the Great Lakes has been thoroughly reported, and many consequences of the overgrowth have been studied for the past two decades (Auer et al., 2010). The high growth rate of Cladophora, its prominent role in the ecosystem, and its link to human pollution sources have been well established in both fresh and marine waters (Auer and Canale, 1982; Egan et al., 2012; Higgins et al., 2008; Zulkifly et al., 2012). A general hypothesis for the massive growth in the Great Lakes has been linked to the proliferation of invasive mussels (Hecky et al., 2004; Higgins and Vander Zanden, 2010; Higgins et al., 2008). It is thought that the mussels have altered the phosphorous loadings and increased the clarity of the lake water, producing conditions favorable for algal growth. However, the most recent efforts to control Great Lakes Cladophora through phosphorus reduction plans have not been fully successful
⁎ Corresponding author. Tel.: +1 219 980 6744. E-mail addresses:
[email protected] (J.R. Peller),
[email protected] (M.N. Byappanahalli),
[email protected] (D. Shively),
[email protected] (M.J. Sadowsky),
[email protected] (C.L. Chun),
[email protected] (R.L. Whitman). 1 Tel.: +1 219 926 8336. 2 Tel.: +1 612 624 2706.
(Higgins et al., 2008). In 2004, an estimated 80% of Lake Michigan's western shoreline was considered impacted by the overgrowth of this alga (Greb et al., 2004). More importantly, the rate of Cladophora growth has not been matched by the rate of decomposition, and the result of this imbalance is a massive amount of algal waste. In the vicinities of heavy Cladophora growth and accumulations along beaches, the water and shorelines are adversely affected, leading to public and environmental health risks, diminished lakeshore recreational and economic values, and water quality concerns (Depew et al., 2011; Verhougstraete et al., 2010). These negative conditions are consequences of decomposing Cladophora mats where the formation of numerous odiferous compounds takes place and further attracts nuisance insects. This immense amount of organic material and/or its natural degradation products, however, may offer economic value in the form of biofuels or other useful chemicals. Consequently, potential applications for this abundant, perennially occurring plant material need to be further evaluated. In decomposing Cladophora mats, the chemical pathways and conditions are influenced largely by the anaerobic conditions created within the mats themselves. Around the Great Lakes, algal decomposition under ambient conditions is mostly confined to 5–6 months, typically between May and October. Several investigations have shown that stranded and attached Cladophora mats support diverse microbial
http://dx.doi.org/10.1016/j.jglr.2014.04.012 0380-1330/© 2014 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Peller, J.R., et al., Notable decomposition products of senescing Lake Michigan Cladophora glomerata, J Great Lakes Res (2014), http://dx.doi.org/10.1016/j.jglr.2014.04.012
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populations, including human pathogens, e.g. Escherichia coli O157:H7, Shigella, Campylobacter, and Salmonella (Byappanahalli et al., 2003; Englebert et al., 2008; Olapade et al., 2006). Aside from these enteric bacteria, a vast array of microbial communities, including sulfatereducing bacteria, which are likely contributing to the malodorous conditions, have been found in decaying Cladophora (Zulkifly et al., 2012). Recent evidence suggests that Cladophora may have a prominent role in the spread of botulism-related illnesses in fish eating birds of the Great Lakes (Byappanahalli et al., 2009; Chun et al., 2013). Within decaying algal mats, an assessment of the evolving chemical constituents, including those that are noxious, has not been reported to our knowledge. In general, chemical toxicities in aqueous environments have been largely focused on synthetic compounds, such as pesticides (Battaglin and Fairchild, 2002) and pharmaceuticals (Cleuvers, 2003), environmental contaminants from industrial (Verma, 2008) or wastewater discharges (Maruya et al., 2012), and naturally toxic compounds formed from harmful algal blooms and unicellular protists (e.g. red tide, cyanobacteria, dinoflagellates) (Gallardo-Rodriguez et al., 2012; Graneli et al., 2012). In addition to the potentially harmful byproducts of fermentation within the Cladophora mats (sulfides, short chain acids, for example), algae contain natural oils, proteins and nutrients. The interest in algal biofuel production has focused mostly on the growth and harvest of microalgae, not macroalgae. However, Cladophora oils were recently esterified and used as a biofuel source in combination with petro diesel. The formulation was shown to perform similar to pure petro diesel, with fewer nitrogen oxide emissions (Grayburn et al., 2013). This study reports on the diversity of chemical compounds formed during the natural decomposition of Cladophora. This knowledge may assist in the development of strategies to minimize the adverse effects of the decomposing algal mats. Additionally, information from this study may provide information for the potential of harvested Cladophora as useful biomass, feedstock, or crop/garden fertilizer. Methods and experiments Location and Cladophora sample collections Fresh, healthy Cladophora samples were collected from rock surfaces of a breakwater at Portage Lakefront and River walk, located on the southern Lake Michigan shoreline in Portage, Indiana (Lat, long: 41.631519°, − 87.179053°). The collection site was chosen because of its juxtaposition to the Burns Ditch waterway and previous extensive research on this waterway and adjacent swimming beaches (Byappanahalli et al., 2010; Nevers et al., 2013). The breakwater collection site is immediately west of the Burns Ditch outfall. The characteristics of the waterway have been described in detail elsewhere (Ishii et al., 2006; Nevers and Whitman, 2005). Briefly, water from the Little Calumet River feeds into Lake Michigan via the Burns Ditch outfall; the river, and hence Burns Ditch, receives a combination of waste, including combined sewage overflows and septic field leachate, industrial discharge, urban and rural runoff, sedimentation, and nutrient loads. Cladophora samples were collected from the boulders along the breakaway, where the algae are continually washed by the waves. The green algae composition was largely Cladophora (N 95%), with a small amount of other filamentous green algae, such as Spirogyra, Mougeotia, Zygnematales, and Pithophora. Additionally, other microbes and eukaryotic organisms constituted even smaller parts of the algal biomass. For the oxygen depletion experiments, samples were periodically collected from the boulders during the spring and summer 2012. On September 17, 2012, a large amount of Cladophora was collected specifically for use in the mesocosm experiment. Field collections of decomposing Cladophora took place at Jeorse Park Beach on August 29, 2013. Jeorse Park Beach is located on the southwest shore of Lake Michigan in East Chicago, Indiana (41.651110°/− 87.433422°) and decomposing Cladophora mats are
commonly observed along this lakefront area. These Cladophora samples were likely contaminated with other biomass, such as bird feces and assortment of invertebrates and microbes. Three areas of decaying Cladophora mats were sampled: near the far west area among the rocks and frequently washed by lake waves, on the beach approximately 1.2 m from the lake water, and on the beach approximately 2.0 m from the lake water. For this component of the study, liquid from the stranded algal mats was collected using a 10 mL syringe, fitted with a 9 cm, gauge 22 stainless steel needle. Mesocosm experiments Oxygen decline in Cladophora mats Independent experiments were conducted to determine the rate of change in dissolved oxygen within a simulated algal mat environment. For this purpose, a glass column (22 cm × 25 mm) with three side arms was used to probe the oxygen levels within the algae mesocosm at different depths. Fresh Cladophora samples were lightly packed into the column and an oxygen microelectrode (MI-730, Microelectrodes, Inc., Bedford, NH) was used to monitor the dissolved oxygen levels in the simulated algal mat environment. A one point calibration of nitrogen-saturated water was used to determine the zero oxygen reading, as per the manufacturer's instructions. Briefly, after packing the algae gently in the column, it was covered either completely with foil to avoid light exposure, or the lower portion of the column was covered with foil to simulate limited light exposures of stranded algal mats along shorelines. The microelectrode was used to monitor the oxygen levels at the three different depths over time. Cladophora decomposition in vitro Fresh algal samples were manually homogenized in the lab by mixing all of the collected algae in a large vat. Sub-samples of approximately 20 mL were transferred into a series of sterile 30 mL plastic centrifuge tubes. All tubes, with the exception of time 0, were incubated at 27 °C in the dark, and covered with loose fitting lids to prevent evaporation. Duplicate samples were randomly chosen for chemical analyses after the designated times of incubation, representing 15 different stages of algal decomposition: at 0, 3, 6, 12, 24, and 48 h, and after 3, 4, 5, 6, 7, 14, 28, 61, and 91 days of incubation. At each designated time, a 3 mL aliquot of the liquid surrounding the Cladophora was aseptically removed and transferred to a 5 mL vial for chemical analyses. A solid phase microextraction (SPME) fiber (Supelco, 50/30 μm DVB/CAR/PDMS Stableflex, purchased from Sigma-Aldrich Co.) was exposed to the water for 1 h and then stored in a refrigerator in a stainless steel container for up to 2 days, until the gas chromatography–mass spectrometry (GC–MS) analysis was performed. Using solid phase microextraction (SPME) procedures, semi-quantitative data were collected and analyzed. For quantitative analyses, this method of extraction assumes that the compounds in solution have been completely extracted, which is often not the case (Zhang et al., 2007). Since the extractions were performed in a consistent manner, the patterns that emerge from the data sets are useful, while the actual quantitative values are likely low for the organic compounds evaluated in this study. SPME extraction success was checked using standard liquid–liquid extraction methods, with methylene chloride (CH2Cl2) as the extracting solvent. Larger volumes of water solutions were utilized (N 250 mL), and the pH was adjusted to less than 5 to ensure solubility of the organic acids in the organic solvent. All the compounds extracted by the traditional liquid–liquid extraction methodology were successfully extracted using the microextraction fibers. The remaining algal liquid (~ 15 mL) was transferred into a small sterile beaker and tested for pH; the liquid was then filtered through a 0.22 μm membrane into a sterile side-armed flask. The filtrate was transferred into an amber glass vial, and kept frozen until ion chromatography was performed as described below. All steps were repeated for each duplicate sample.
Please cite this article as: Peller, J.R., et al., Notable decomposition products of senescing Lake Michigan Cladophora glomerata, J Great Lakes Res (2014), http://dx.doi.org/10.1016/j.jglr.2014.04.012
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A Thermo Scientific (Thermo Fisher Scientific Inc.) gas chromatography–mass spectrometry (GC–MS) ISQ single quadrupole instrument, equipped with a nonpolar DB-5MS column (Agilent Technologies, Santa Clara, CA) was used to separate and identify the volatile, ionizable compounds adsorbed on the solid phase microextraction fibers. The experimentally exposed fiber was inserted into the heated GC injection port, set at 230 °C, where the extracted compounds were released into the column. The GC temperature was set at 35 °C, where it was held for 5 min, and then increased to 290 °C at 8 °C/min. The column remained at 290 °C for an additional 3 min to ensure elution of all compounds. All detected compounds were baseline separated with this method. The mass transfer line and the ion source temperature were each set at 250 °C. Since a wide variety of compounds were expected, the mass spectrometer single quadrupole ISQ's scan range was set at 10–500, with a scan time of 0.2 s. Compounds were identified by comparison of their mass spectra and retention times with those of standards, or by comparison of mass spectra with those in the NIST Mass Spectral Search Program (version 2.0), where b80% match was used as a cut-off, in addition to triplicate replicates. Ion analyses were performed using a Waters (Waters Corporation, Milford, MA) high performance liquid chromatography (HPLC) system, equipped with a conductivity detector. For anion analyses (SO2− 4 ), the IC-Pak™ anion column (Waters Corp.) was used. One liter of the aqueous mobile phase was prepared using 20 mL of concentrated sodium borate gluconate, 20 mL of n-butanol, and 120 mL of acetonitrile. For the detection and quantification of ammonium, a Waters ICPak™ cation column was fitted to the HPLC and conductivity detector. The mobile phase consisted of 0.1 mM EDTA and 0.019% nitric acid. Standards solutions ranging from 1 to 20 ppm were prepared for NH+ 4 , and used to verify and quantify the ions in the algal solutions. Results and discussion Oxygen depletion in Cladophora mats When Cladophora accumulates along the shorelines, the environment within the mats becomes anoxic over time, providing favorable conditions for the sustenance and growth of facultative anaerobes and strict anaerobes. Rapid loss of oxygen was measured in the lower side arm of the vertical column of algae (Fig. 1). This experiment was repeated with small variations in light exposure and temperature, and similar oxygen depletion rates were determined in all three side arms, or depths, of the column. The decrease in dissolved oxygen in the glass column of algae was accompanied by the production of other gas pressures. Within 24 h, the pressure created was great enough to float the algae, sometimes expelling the mesocosm's lid. The liquid below the algae was observed to change to a dark purple color, suggesting changes in the bacterial communities and chemical changes. This displacement of the algae occurred whether the experimental set-up was open or closed to the atmosphere, and demonstrated the inability of atmospheric oxygen to penetrate the algae. As anticipated, these experiments suggest that gasses created by chemical reactions within Cladophora mats along the shorelines, develop pressures greater than atmospheric pressure and impose anaerobic conditions in the mats.
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Time (min) Fig. 1. Oxygen concentrations (mg/L) in the column of green algae, measured in the lowest side arm of the 22 cm vertical glass tube, after loose packing of algae in the column.
Cladophora mesocosm study The decomposition chemistry within the Cladophora mats was tracked throughout the mesocosm over a 3 month period. The initial pH of the algae water medium (t = 0) was 6.6, and dropped to a low value of 5.8 after 7 days of incubation. Chemical analyses indicated that this was likely due to the formation of carboxylic acids. A small rise in pH was noted at weeks 2 and 3, followed by an increase in pH to 8.4 and 8.7 at t = 61 and 91 days, respectively (Fig. 2). In the later stages of decomposition, the carboxylic acids were no longer detected and the rise in pH corresponded with an increase in the concentration of ammonia. (The detailed NH3/NH+ 4 data can be found in the supplemental material.) During the Cladophora decomposition experiment, the chemical formations/transformations appeared to follow aspects of acidogenesis and solventogenesis pathways. In low oxygen conditions, early bacterial log growth phase takes place, and microbes produce short chain carboxylic acids from carbohydrate and protein sources in a process termed acidogenesis. In the later log phase of anaerobic bacterial growth, a second metabolic process commences, solventogenesis, and certain reduced carbon compounds are created: acetone, butanol and ethanol (ABE fermentation) (Amador-Noguez et al., 2011). In this study, the
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Cladophora decomposition in situ Liquid from decomposing Cladophora mats was collected and transferred to 5 mL vials for SPME analyses, in a similar manner to the in vitro samples. The extraction procedure was performed within 1 h of sample collection and subsequently tested using gas chromatography–mass spectrometry (GC–MS) to determine the predominant organic compounds present.
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Please cite this article as: Peller, J.R., et al., Notable decomposition products of senescing Lake Michigan Cladophora glomerata, J Great Lakes Res (2014), http://dx.doi.org/10.1016/j.jglr.2014.04.012
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short chain carboxylic acids acetic acid, propionic acid, butyric acid, methyl butyric acids and pentanoic acid were readily detected 24 h into the Cladophora decay. Acetic acid was detected in samples throughout the first month, but not in the 61 and 91 day samples. Butyric acid reached a peak at 7 days, which also corresponded to the lowest solution pH measurement measurement. Fig. 3 shows the measured concentrations for acetic acid and butyric acid, over the course of the experiment. These compounds contribute to the putrid odor of the decomposing Cladophora, but are eventually transformed in the anaerobic environment, according to their decrease and eventual absence at t = 61 days. Proteins are a substantial portion of Cladophora algae (AmadorNoguez et al., 2011; Fleurence, 1999). Protein fermentation generates amino acids, which are transformed into ammonia and smaller carbon compounds, including 4-methylphenol, indole and 3-methylindole. These three organic decay products were prominently present at t = 3 h and t = 24 h and throughout the algal decomposition. All are considered environmental pollutants at certain concentrations (Table 1). Fig. 4 shows a cluster analysis of the major detected organic compounds (80) and summarizes their presence and persistence. Compounds categorized in groups 1–6 had relatively long durations (21–91 days), typically in the t = 6 to 46 day range. The persistent organics 4-methylphenol, indole and 3-methylindole belong to group 2, the compounds detected in most of the samples. Compounds in group 7 had relatively short durations (0–14 days) centered around t = 0 to 14 days. Compounds in groups 8–10 had very short durations (0 days, meaning they were never detected more than once), centered around t = 28 to 91 days (R C, 2013). The electronic supplemental material (ESM Figs. S1, S2 and S3) contains the full listing of the predominant, detected compounds, their timeframes and their durations. The compound 4-methylphenol (p-cresol) is a product of protein breakdown and was one of the most predominant and persistent compounds of Cladophora decomposition. Under reduced oxygen conditions, the anaerobic bacterium Desulfobacterium cetonicum can oxidize p-cresol, if sulfate is available as an electron acceptor (Grayburn et al., 2013). Also, pure cultures of nitrate-reducing and sulfate-reducing bacteria can metabolize p-cresol under anaerobic conditions through methyl group oxidation (Londry et al., 1997; Muller et al., 2001). Several anaerobic bacteria also produce p-cresol from tyrosine and its degradation has been shown to be catalyzed by nitrate-, iron-, and sulfatereducing bacteria. In our study, sulfate concentrations in the algae solutions were in the range of 4.2–30.2 mM, indicating this oxidation might have been possible, especially in the later stages of decomposition. However, methyl oxidation was not obvious in our mesocosm experiments, since only very low concentrations of oxidized p-cresol
Acetic and butyric acid (mg/L)
0.7
compounds were detected. P-cresol was measured at 0.48 mg/L at t = 3 days, and from days 4 through 7 p-cresol increased in concentration, which remained fairly consistent at about 1.5 mg/L. Another rise in the concentration was measured through day 28 (Fig. 5a). P-cresol is excreted in human urine daily from protein metabolism, and can alter bacterial cell membrane permeability (Keweloh et al., 1991). Indole and 3-methylindole were the other two metabolites prominently detected throughout the course of the study; the measured concentrations in mg/L are shown in Fig. 5b. The indole heterocycle is part of the structure of certain alkaloids, and the amino acid tryptophan contains the indole moiety. Indole is produced by many bacteria via metabolism of tryptophan (Snell, 1975). Similar to p-cresol, these compounds are considered toxins at certain levels (see LD50 values in Table 1), and are known to be abundant in sewage waters (Hwang et al., 1995). Indole appears to play important roles in microorganism growth inhibition, physiology, chemical resistance, biofilm formation and ecological balance (Lee and Lee, 2010). This relatively simple organic compound is believed to be crucial as an intercellular signaling molecule and may have multiple roles in decomposition chemistry and microbiology, including bacterial pathogenesis. For example, indole increases the pathogenicity of the enterohemorrhagic E. coli O157:H7 (Lee et al., 2007). Its presence and that of the methylated metabolite throughout the experiment may indicate essential roles in microbial functioning within the decomposing algal environment. The 22-carbon, polyunsaturated fatty acid cis-4,7,10,13,16,19docosahexanoic acid (doconexent) was prominently detected in several of the mesocosm samples. Other frequently detected long chain fatty acids were n-hexadecanoic acid, oleic acid and tetradecanoic acid. These compounds were present in both freshly collected samples (i.e., prior to incubation) and throughout the incubation process, suggesting that they are not the preferred carbon source for metabolism (and fermentation) of the microscopic communities. They are represented by groups 2 and 3 on the cluster analysis graph (Fig. 4), which highlights their stability in the anaerobic environment. Algae are considered potentially important resources due to the lipid, or oil, content in the cells, although certain human populations use algae for food, food additives, or compost/fiber due to its other valuable components. A study from 2003 reported the percentage of lipids in Cladophora to be 15.6% dry weight (Orhan et al., 2003). In our study, highly reduced, fatty acid compounds were present in both fresh and decomposing Cladophora samples, indicating that many of these natural compounds can be harvested from both fresh and decomposed algae, as a source of beneficial oils. These highly reduced carbon compounds are also potentially valuable as biofuel components.
acetic acid butyric acid
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Please cite this article as: Peller, J.R., et al., Notable decomposition products of senescing Lake Michigan Cladophora glomerata, J Great Lakes Res (2014), http://dx.doi.org/10.1016/j.jglr.2014.04.012
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Table 1 Structures and descriptions of the most frequently detected and persistent organic compounds in the Cladophora decay experiments. Description and LD50 values
Other toxicity
4-Methylphenol (p-cresol)
Metabolite of tyrosine and phenylalanine; EPA Group C — possible carcinogen LD50 (mammal/oral) = 500 mg/kg
EPA Classification C: possible human carcinogen (IRIS) (EPA, 2013)
Indole
Metabolite of tryptophan; Used as a marker for the presence of bacteria (Li and Young, 2013) Insecticide LD50 (rat/oral) = 1 g/kg Metabolite of tryptophan; Fermentation in the intestinal tract; Present in cigarette smoke (Weisburger et al., 1978) LD50 (rat/oral) = 3.4 g/kg
Toxic to several fish species at 10 mg/L (PAN)
Compound
Structure
3-Methylindole (skatole)
mesocosm studies were also found in field samples; these results strengthen our findings, and also demonstrate that our experimental conditions approached midsummer ambient conditions. The massive accumulations of Cladophora along areas of the Great Lakes' shorelines, as a result of uncontrollable algal overgrowth, must be thoroughly understood for safe management of the decaying material. This study examined the main chemical constituents present during the various stages of Cladophora decomposition in order to better evaluate the potential health risks. From a different perspective, the plant matter or the algal decomposition products may be viewed as potential resources. Many organic compounds were identified, some of which are potential components for biofuels, compost or industrial feedstock. Other detected compounds are potential toxins and must be considered in the context of public and environmental health or when decomposed Cladophora is used as compost. While much research has focused on Cladophora ecology, productivity, limiting factors and occurrence, it may be that many important environmental impacts and products occur during the decompositional microbial-mediated phases of its life cycle. Surely, that is true of pathogens and chemical byproducts and thus, this study emphasized the need to understand the senescent and
In situ samples Liquid was sampled directly from Cladophora mats sampled in the field at Jeorse Park beach. While the exact age of the stranded Cladophora was not known, the state of decay suggested that the mats were in the early stages (less than 10 s) of decomposition. Three algal mat environments, which varied in distance from the lake water from approximately half of a meter to 3 m, were sampled. The carbon compounds detected in the field samples included short chain carbon acids, several alcohols and other compounds indicative of carbohydrate break-down products, all of which were also identified in the mesocosm experiment. This collection of compounds further substantiated the likely age of the Cladophora mats to be a few to several days. While studies on decaying Cladophora mats have focused on the pathogenic organisms harbored in the anaerobic environment, our mesocosm experiment studied Cladophora decomposition products. The experiment was designed to mimic the interior environment of stranded Cladophora piles that lack sun and air exposure in order to determine consequential chemical constituents and associated products of anaerobic transformations. Many of the identified products from our
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Mean occurrence (days) Fig. 4. Mean occurrence (the mean day of detection during the experiment) versus duration for 80 compounds detected during decaying algae. Points were slightly displaced to distinguish between overlapping symbols (i.e., a small amount of random noise was added to the coordinates of each point). Each compound is numbered and colored according to its membership in one of 10 groups formed by cluster analysis (R C, 2013). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Peller, J.R., et al., Notable decomposition products of senescing Lake Michigan Cladophora glomerata, J Great Lakes Res (2014), http://dx.doi.org/10.1016/j.jglr.2014.04.012
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Fig. 5. (a) Concentration (mg/L) of p-cresol in the decaying algal water environment, determined by semi-quantitative SPME analyses determined through 61 days. (b) Concentrations of indole and 3-methylindole (mg/L) determined by SPME analyses determined from t = 1 to t = 90 days.
decompositional phase more, as well as the implications of such processes in lake and water quality dynamics. Acknowledgments This work was supported by a grant from the U.S. Environmental Protection Agency (EPAR5-GL2010-1). This article is contribution number 1845 of the USGS Great Lake Science Center. We thank Jean Adams (USGS) for help with the statistical data analyses. We also thank the Center for Sustainable Energy at Notre Dame for the use of the gas chromatography–mass spectrometry facilities. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jglr.2014.04.012. References Amador-Noguez, D., Brasg, I.A., Feng, X.J., Roquet, N., Rabinowitz, J.D., 2011. Metabolome remodeling during the acidogenic–solventogenic transition in Clostridium acetobutylicum. Appl. Environ. Microbiol. 77, 7984–7997.
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