Alternative Methods for Analysis of Cyanobacterial Populations in Drinking Water Supplies: Fluorometric and Toxicological Applications Using Phycocyanin
The management of cyanobacteria and potential exposure to associated biotoxins requires the allocation of scarce resources across a range of freshwater resources within various jurisdictions. Cost effective and reliable methods for sample processing and analysis form the foundation of the protocol yielding reliable data from which to derive important decisions. In this study the utilization of new methods to collect, process and analyze samples enhanced our ability to evaluate cyanobacterial populations. Extraction of phycocyanin using the single freeze thaw method provided more accurate and precise measurements (CV 4.7% and 6.4%), offering a simple and cost-effective means to overcome the influence of morphological variability. In-vacuo concentration of samples prior to ELISA analysis provided a detection limit of 0.001 μg·L？1 MC. Fractionation of samples (<0.2 μm, <2.0 μm, <50 μm, WLW and BFC) influenced our interpretations and improved our ability to establish a causative relationship between phycocyanin and microcystin levels in two aquatic systems with distinctly different cyanobacterial populations. In a Microcystis spp. dominant system Log MC (ng·L？1) = ？0.279 + (1.368 ？ Log PC (μg·L？1) while in an Aphanizomemon spp. dominant system Log MC (ng·L？1) = 0.385 + (0.449 ？ Log PC (μg·L？1). These methods and sampling protocol could be used in other aquatic systems across a broader regional landscape to estimate the levels of microcystins.
References
[1]
Marion, J.W., Lee, J., Wilkins III, J.R., Lemeshow, S., Lee, C., Waletzko, E.J. and Buckley, T.J. (2012) In Vivo Phycocyanin Fluorometry as a Potential Rapid Screening Tool for Predicting Elevated Microcystin Concentrations at Eutrophic Lakes. Environmental Science and Technology, 46, 4523-4531. https://doi.org/10.1021/es203962u
[2]
Macario, I.E., Castro, B., Nunes, M.S., Antunes, S., Pizarro, C., Coelho, C. and Goncalves, F. (2015) New Insights Towards the Establishment of Phycocyanin Concentration Thresholds Considering Species-Specific Variability of Bloom-Forming Cyanobacteria. Hydrobiologia, 757, 155-165. https://doi.org/10.1007/s10750-015-2248-7
[3]
Izydorczyk, C., Tarczynska, M., Jurczak, T., Mrowczynski, J. and Zalewski, M. (2005) Measurement of Phycocyanin Fluorescence as an Online Early Warning System for Cyanobacteria in Reservoir Intake Water. Environmental Toxicology, 20, 425-430. https://doi.org/10.1002/tox.20128
[4]
McQuaid, N., Zamyadi, A., Prevost, M., Bird, D.F. and Dorner, S. (2011) Use If in Vivo Phycocyanin Fluorescence to Monitor Potential Microcystin-Producing Cyanobacterial Biovolume in a Drinking Water Source. Journal of Environmental Monitoring, 13, 455-463. https://doi.org/10.1039/C0EM00163E
[5]
Ahn, C.-Y., Joung, S.-H., Yoon, S.-K. and Oh, H.-M. (2007) Alternative Alert System for Cyanobacterial Bloom, Using Phycocyanin as a Level Determinant. The Journal of Microbiology, 45, 98-104.
[6]
Brient, L., Lengronne, M., Bertrand, E., Rolland, D., Sipel, A., Steinmann, D., Baudin, I., Legeas, M., Le Rouzic, B. and Bormans, M. (2008) A Phycocyanin Probe as a Tool for Monitoring Cyanobacteria in Freshwater Bodies. Journal of Environmental Monitoring, 10, 248-255. https://doi.org/10.1039/B714238B
[7]
Kasinak, J.-M.E., Holt, B., Chislock, M. and Wilson, A.E. (2015) Benchtop Fluorometry of Phycocyanin as a Rapid Approach for Estimating Cyanobacterial Biovolume. Journal of Plankton Research, 37, 248-257. https://doi.org/10.1093/plankt/fbu096
[8]
Reynolds, C.S., Oliver, R.L. and Walsby A.E. (1987) Cyanobacterial Dominance: The Role of Buoyancy Regulation in Dynamic Lake Environments. New Zealand Journal of Marine and Freshwater Research, 21, 379-390. https://doi.org/10.1080/00288330.1987.9516234
[9]
Kurmayer, R., Christiansen, G. and Chorus, I. (2003) The Abundance of Microcystin-Producing Genotypes Correlates Positively with Colony Size in Microcystis spp. and Determines Its Microcystin Net Production in Lake Wansee. Applied and Environmental Microbiology, 69, 787-795. https://doi.org/10.1128/AEM.69.2.787-795.2003
[10]
Graham, J.L. and Jones, J.R. (2007) Microcystin Distribution in Physical Size Class Separations of Natural Plankton Communities. Lake and Reservoir Management, 23, 161-168. https://doi.org/10.1080/07438140709353919
[11]
Callieri, C., Cronberg, G. and Stockner, J.G. (2012) Freshwater Picocyanobacteria: Single Cells, Microcolonies and Colonial Forms. In: Whitton, B., Ed., Ecology of Cyanobacteria II, Springer, Dordrecht, 229-269.
[12]
Wang, X., Sun, M., Xie, M., Liu, M., Luo, L., Li, P. and Kong, F. (2013) Differences in Microcystin Production and Genotype Composition among Microcystis Colonies of Different Sizes in Lake Taihu. Water Research, 47, 5659-5669. https://doi.org/10.1016/j.watres.2013.06.040
[13]
Rinta-Kanto, J.M., Konopka, E.A., DeBruyn, J.M., Bourbonniere, R.A., Boyer, G.L. and Wilhelm, S.W. (2009) Lake Erie Microcystis: Relationship between Microcystin Production, Dynamics of Genotypes and Environmental Parameters in a Large Lake. Harmful Algae, 8, 665-673. https://doi.org/10.1016/j.hal.2008.12.004
[14]
Chan, F., Pace, M., Howarth, R.W. and Marino, R.M. (2004) Bloom Formation in Heterocystic Nitrogen-Fixing Cyanobacteria: The Dependence on Colony Size and Zooplankton Grazing. Limnology and Oceanography, 49, 2171-2178. https://doi.org/10.4319/lo.2004.49.6.2171
[15]
Leland, N.J. (2015) A Method and Device for Plankton Separation. US Provisional Patent No. 62/249,633.
[16]
Leland, N.J. (2017) A Method and Device for Plankton Separation. US Patent No. 9,540,632.
[17]
Leland, N.J. (2015) A Method and Device for Plankton Separation. US Provisional Patent No. 62/174,027.
[18]
Leland, N.J. (2016) A Method and Device for Plankton Separation. US Provisional Patent No 15/345,823.
[19]
Zar, J.H. (1996) Biostatistical Analysis. Prentice-Hall, Upper Saddle River, NJ.
[20]
Systat Software (2010) Sigmaplot 12.
[21]
Lawrenz, E., Fedewa, E.J. and Richardson, T.L. (2011) Extraction Protocols for the Quantification of Phycobilins in Aqueous Phytoplankton Extracts. Journal of Applied Phycology, 23, 865-871. https://doi.org/10.1007/s10811-010-9600-0
[22]
Chaffin, J.D., Bridgeman, T.B., Heckathorn, S.A. and Mashra, A. (2011) Assessment of Microcystis Growth Rate Potential and Nutrient Status across a Trophic Gradient in Western Lake Erie. Journal of Great Lakes Research, 37, 92-100. https://doi.org/10.1016/j.jglr.2010.11.016
[23]
Chaffin, J.D., Bridgeman, T.B., Heckathorn, S.A. and Krause, A.E. (2012) Role of Suspended Sediments and Mixing in Reducing Photoinhibition in the Bloom-Forming Cyanobacterium Microcystis. Journal of Water Resource and Protection, 4, 1029-1041. https://doi.org/10.4236/jwarp.2012.412119
[24]
Horvath, A., Kovacs, A.W., Riddick, C. and Presing, M. (2013) Extraction Methods for Phycocyanin Determination in Freshwater Filamentous Cyanobacteria and their Application in a Shallow Lake. European Journal of Phycology, 48, 278-286. https://doi.org/10.1080/09670262.2013.821525
[25]
Sarada, R., Pillai, M.G. and Ravishankar, G.A. (1999) Phycocyanin from Spirulina sp: Influence of Processing of Biomass on Phycocyanin Yield, Analysis of Efficacy of Extraction Methods and Stability Studies on Phycocyanin. Process Biochemistry, 34, 795-801. https://doi.org/10.1016/S0032-9592(98)00153-8
[26]
Sivasankari, S., Naganandhini and Ravindran, D. (2014) Comparison of Different Extraction Methods for Phycocyanin Extraction and Yield from Spirulina platensis. International Journal of Current Microbiology and Applied Sciences, 3, 904-909.
[27]
Salama, A., Ghany, A., Osman, A. and Sitohy, M. (2015) Maximising Phycocyanin Extraction from a Newly Identified Egyptian Cyanobacteria Strain: Anabaena oryzae SOS13. International Food Research Journal, 22, 517-525.
[28]
Dufresne, J. (2015) Pers. comm.
[29]
Chang, D.-W., Hobson, P., Burch, M. and Lin, T.-F. (2012) Measurement of Cyanobacteria Using In-Vivo Fluoroscopy-Effect of Cyanobacterial Species, Pigments and Colonies. Water Research, 46, 5037-5048. https://doi.org/10.1016/j.watres.2012.06.050
[30]
Murby, A.L. (2009) Assessing Spatial Distributions of Cyanobacteria and Microcystins in N.H. Lakes with Implications for Lake Monitoring. Master’s Thesis, University of New Hampshire, Durham, New Hampshire.
[31]
Graham, J.L., Loftin, K.A., Meyer, M.T. and Ziegler, A.C. (2010) Cyanotoxin Mixtures and Taste-and-Odor Compounds in Cyanobacterial Blooms from the Midwestern United States. Environmental Science and Technology, 44, 7361-7368. https://doi.org/10.1021/es1008938
[32]
Rolland, A., Bird, D.F. and Giani, A. (2005) Seasonal Changes in Composition of the Cyanobacterial Community and the Occurrence of Hepatotoxic Blooms in the Eastern Townships, Quebec, Canada. Journal of Plankton Research, 27, 683-694. https://doi.org/10.1093/plankt/fbi042
[33]
Lyck, S. and Christoffersen, K. (2003) Microcystin Quota, Cell Division and Microcystin Net Production of Precultured Microcystis aeruginosa CYA 228 (Chroococcales, Cyanophyceae) under Field Conditions. Phycologia, 42, 667-674. https://doi.org/10.2216/i0031-8884-42-6-667.1
[34]
Izydorczyk, C., Carpentier, C., Mrowczynski, J., Wagenvoort, A., Jurczak, T. and Tarczynska, M. (2009) Establishment of an Alert Level Framework for Cyanobacteria in Drinking Water Resources by Using the Algae Online Analyzer for Monitoring Cyanobacterial Chlorophyll A. Water Research, 43, 989-996. https://doi.org/10.1016/j.watres.2008.11.048
[35]
Sommer, U., Gliwicz, Z.M., Lampert, W. and Duncan, A. (1986) The PEG-Model of Seasonal Succession of Planktonic Events in Fresh Water Arch. Hydrobiologia, 106, 433-471.
[36]
Francy, D.S., Brady, A.M.G., Ecker, C.D., Graham, J.L., Stelzer, E.A., Struffolino, P., Dwyer, D.F. and Loftin, K.A. (2016) Estimating Microcystin Levels at Recreational Sites in Western Lake Erie and Ohio. Harmful Algae, 58, 23-34. https://doi.org/10.1016/j.hal.2016.07.003
[37]
Bastien, C., Cardin, R., Veilleux, E., Deblois, C., Warren, A. and Laurion, L. (2011) Performance Evaluation of Phycocyanin Probes for the Monitoring of Cyanobacteria. Journal of Environmetal Monitoring, 13, 110-118. https://doi.org/10.1039/C0EM00366B
[38]
Zamyadi, A., Choo, F., Newcombe, G., Stuetz, R. and Henderson, R.K. (2016) A Review of Monitoring Techniques for Real-Time Management of Cyanobacteria: Recent Advances and Future Direction. Trends in Analytical Chemistry, 85, 83-96. https://doi.org/10.1016/j.trac.2016.06.023
[39]
US Environmental Protection Agency, Office of Water (2015) Recommendations for Public Water Systems to Manage Cyanotoxins in Drinking Water. EPA 815-R-15-010.
[40]
Park, H.-D., Iwami, C., Watanabe, M.F., Harada, K.-I., Okino, T. and Hayashi, H. (1998) Temporal Variabilities of the Concentrations of Intra and Extracellular Microcystin and Toxic Microcystis Species in a Hypereutrophic Lake, Lake Suwa, Japan (1991-1994). Environmental Toxicology and Water Quality, 13, 61-72. https://doi.org/10.1002/(SICI)1098-2256(1998)13:1<61::AID-TOX4>3.0.CO;2-5
[41]
Oh, H.M., Lee, S.J., Kim, J.H., Kim, H.S. and Yoon, B.D. (2001) Seasonal Variation and Indirect Monitoring of Microcystin Concentration in Daechung Reservoir, Korea. Applied Environmental Microbiology, 67, 1484-1489. https://doi.org/10.1128/AEM.67.4.1484-1489.2001
[42]
Sakai, H., Hao, A., Iseri, Y., Wang, S., Kuba, T., Zhang, Z. and Katayama, H. (2013) Occurrence and Distribution of Microcystins in Lake Taihu, China. The Scientific World Journal, 2013, Article ID: 838176. https://doi.org/10.1155/2013/838176
[43]
Su, X., Steinman, A.D., Xue, Q., Zhao, Y. and Xie, L. (2018) Evaluating the Contamination of Microcystins in Lake Taihu, China: The Application of Equivalent MC-LR Concentration. Ecological Indicators, 89, 445-454. https://doi.org/10.1016/j.ecolind.2017.11.042
[44]
Stockner, J.G. and Porter, KG. (1988) Microbial Food Webs in Fresh-Water Planktonic Ecosystems. In: Carpenter, S.R., Ed., Complex Interactions in Lake Communities, Springer-Verlag, New York, 69-83. https://doi.org/10.1007/978-1-4612-3838-6_5
[45]
Yoshida, T., Gurung, T.B., Kagami, M. and Urabe, J. (2001) Contrasting Effects of Cladoceran (Daphnia galeata) and Calanoid Copepod (Eudiaptomus japonicas) on Algal and Microbial Plankton in a Japanese Lake, Lake Biwa. Oecologia, 129, 602-610. https://doi.org/10.1007/s004420100766
[46]
Han, B.P., Lin, X., Lei, L.M. and Gu, J.G. (2012) Survival of D. galeata in Sub-Tropical Reservoirs: Harmful Effects of Toxic Cyanobacteria in Food Source. Ecotoxicology, 21, 1692-1705. https://doi.org/10.1007/s10646-012-0940-1
[47]
Nakamura, T., Adachi, Y. and Suzuki, M. (1993) Floatation and Sedimentation of a Single Microcystis Floc Collected from a Surface Bloom. Water Research, 27, 979-983. https://doi.org/10.1016/0043-1354(93)90061-L
[48]
Rowe, M.D., Anderson, E.J., Wynne, T.T., Stumpf, R.P., Fanslow, D.L., Kijanka, K., Vanderploeg, H.A., Strickler, J.R. and Davis, T.W. (2016) Vertical Distribution of Buoyant Microcystis Blooms in a Lagrangian Particle Tracking Model for Short-Term Forecasts in Lake Erie. Journal of Geophysical Research and Oceans, 121, 5296-5314. https://doi.org/10.1002/2016JC011720
[49]
Capron, S.A. (1995) Occurrence of Microcystins Produced by Microcystis aeruginosa (Blue-Green Algae) and Accumulation in Zooplankton. Master’s Thesis, University of New Hampshire, Durham, New Hampshire.
[50]
Reynolds, C.S., Jaworski, G.H.M., Cmiech, H.A. and Leedale, G.F. (1981) On the Annual Cycle of the Blue-Green Alga, Microcystis aeruginosa Kutz. Emend. Elenkin. Philosophical Transactions of the Royal Society B, 293, 419-477. https://doi.org/10.1098/rstb.1981.0081
