Hostname: page-component-78c5997874-m6dg7 Total loading time: 0 Render date: 2024-11-17T21:22:13.183Z Has data issue: false hasContentIssue false

Combined Confocal Laser Scanning Microscopy Techniques for A Rapid Assessment of the Effect and Cell Viability of Scenedesmus sp. DE2009 Under Metal Stress

Published online by Cambridge University Press:  24 June 2019

Laia Millach*
Affiliation:
Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain
Eduard Villagrasa
Affiliation:
Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain
Antonio Solé
Affiliation:
Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain
Isabel Esteve
Affiliation:
Departament de Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain
*
*Author for correspondence: Laia Millach, E-mail: lmillach@gmail.com
Get access

Abstract

Phototrophic microorganisms are the dominant populations in microbial mats, which play an important role in stabilizing sediments, such as happens in the Ebro Delta. These microorganisms are exposed to low metal concentrations over a long period of time. Distinct methods have been used to evaluate their toxic effect on the preservation of these ecosystems. Nevertheless, most of these techniques are difficult to apply in isolated phototrophs because (i) they usually form consortia with heterotrophic bacteria, (ii) are difficult to obtain in axenic cultures, and (iii) do not grow on solid media.

In this study, and for the first time, a combination of fast, non-invasive, and in vivo Confocal Laser Scanning Microscopy (CLSM) techniques were applied in a consortium of Scenedesmus sp. DE2009 to analyze its physiological state and viability under metal stress conditions. Microalga was more resistant to Pb followed by Cr and Cu. However, in multimetal combinations, the presence of Cu negatively affected microalga growth. Additionally, the inhibitory concentration (IC) values were also calculated by CLSM pigment analysis. The result determines a higher degree of toxicity for Cu and Cr in comparison to Pb. The high sensitivity of these CLSM-methods to detect low concentrations allows consideration of Scenedesmus sp. DE2009 as a good bioindicator of metal pollution in natural environments.

Type
Biological Applications
Copyright
Copyright © Microscopy Society of America 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Al-Rubeai, M, Welzenbach, K, Lloyd, DR & Emery, AN (1997). A rapid method for evaluation of cell number and viability by flow cytometry. Cytotechnology 24, 161.Google Scholar
Belapurkar, P, Goyal, P & Kar, A (2016). In vitro evaluation of bioremediation capacity of a commercial probiotic, Bacillus coagulans, for chromium (VI) and lead (II) toxicity. J Pharm Bioallied Sci 8(4), 272276.Google Scholar
Burgos, A, Maldonado, J, de los Ríos, A, Solé, A & Esteve, I (2013). Effect of copper and lead on two consortia of phototrophic microorganisms and their capacity to sequester metals. Aquat Toxicol 140(141), 324336.Google Scholar
Burnat, M, Diestra, E, Esteve, I & Solé, A (2010). Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria. Bull Environ Contam Toxicol 84, 5560.Google Scholar
Chakraborty, P, Raghunadh Babu, PV, Acharyya, T & Bandyopadhyay, D (2010). Stress and toxicity of biologically important transition metals (Co, Ni, Cu and Zn) on phytoplankton in a tropical freshwater system: An investigation with pigment analysis by HPLC. Chemosphere 80(5), 548553.Google Scholar
D'ors, A, Pereira, M, Bartolomé, MC, López-Rodas, V, Costas, E & Sánchez-Fortún, S (2010). Toxic effects and specific chromium acquired resistance in selected strains of Dyctiosphaerium chlorelloides. Chemosphere 81, 282287.Google Scholar
Esteve, I, Ceballos, D, Martínez-Alonso, M, Gaju, N & Guerrero, R (1994). Development of versicolored microbial mats: Succession of microbial communities. In Microbial Mats: Structure, Development and Environmental Significance, Stal, LJ & Caumette, P (Eds.), pp. 415420. NATO ASI Series G: Ecological Sciences, Berlin: Springer-Verlag.Google Scholar
Fleeger, JW, Carman, KR & Nisbet, RM (2003). Indirect effects of contaminants in aquatic ecosystems. Sci Total Environ 317, 207233.Google Scholar
Franklin, NM, Stauber, JL, Lim, RP & Petocz, P (2002). Toxicity of metal mixtures to a tropical freshwater alga (Chlorella sp.): The effect of interactions between copper, cadmium, and zinc on metal cell binding and uptake. Environ Toxicol Chem 21(11), 24122422.Google Scholar
Gissi, F, Adams, MS, King, CK & Jolley, DF (2015). Robust bioassay to assess the toxicity of metals to the antarctic marine microalga Phaeocyctis Antarctica. Environ Toxicol Chem 34(7), 15781587.Google Scholar
Guerrero, R, Piqueras, M & Berlanga, M (2002). Microbial mats and the search for minimal ecosystems. Int Microbiol 5, 177188.Google Scholar
Kamala-Kannan, S, Dass Batvari, BP, Lee, KJ, Kanna, N, Krishnamoorthy, R, Shanti, K & Jayaprakash, M (2008). Assessment of heavy metals (Cd, Cr and Pb) in water, sediment and seaweed (Ulva lactuca) in the Pulicat Lake, South East India. Chemosphere 71, 12331240.Google Scholar
Levy, JL, Stauber, JL & Jolley, DF (2007). Sensitivity of marine microalgae to copper: The effect of biotic factors on copper adsorption and toxicity. Sci Total Environ 38, 141154.Google Scholar
Lindemann, S, Moran, J, Dohnalkova, A, Kim, Y, Kennedy, D, Stolyar, S, Majors, PD, Wiley, H, Konopka, AE & Fredrickson, JK (2012). Microbial diversity and biogeochemical function of the phototrophic microbial mats of Epsomitic Hot Lake, WA. Microsc Microanal 18(S2), 1011.Google Scholar
Maldonado, J, de los Rios, A, Esteve, I, Ascaso, C, Puyen, ZM, Brambilla, C & Solé, A (2010 a). Sequestration and in vivo effect of lead on DE2009 microalga, using high-resolution microscopic techniques. J Hazard Mater 183, 4450.Google Scholar
Maldonado, J, Diestra, E, Huang, L, Domènech, AM, Villagrasa, E, Puyen, ZM, Duran, R, Esteve, I & Solé, A (2010 b). Isolation and identification of a bacterium with high tolerance to lead and copper from marine microbial mat in Spain. Ann Microbiol 60, 113120.Google Scholar
Millach, L, Obiol, A, Solé, A & Esteve, I (2017). A novel method to analyze in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser. J Microsc 268, 5365.Google Scholar
Millach, L, Solé, A & Esteve, I (2015). Role of Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 as bioindicators and immobilizers of chromium in a contaminated natural environment. BioMed Res Int 2015, 11, Article ID 519769.Google Scholar
Millán de Kuhn, R, Streb, C, Breiter, R, Richter, P, Neeβe, T & Häder, DP (2006). Screening for unicellular algae as possible bioassay organisms for monitoring marine water samples. Water Res 40, 26952703.Google Scholar
Perkins, RG, Oxborough, K, Hanlon, ARM, Underwood, GJC & Baker, NR (2002). Can chlorophyll fluorescence be used to estimate the rate of photosynthetic electron transport within microphytobenthic biofilms? Mar Ecol Prog Ser 228, 4756.Google Scholar
Pfennig, N & Trüpper, HG (1992). The family Chromatiaceae. In The Prokaryotes, Balows, A, Trüpper, HG, Dworkin, M, Harder, W & Schleifer, KH (Eds.), pp. 32003221. Berlin: Springer-Verlag.Google Scholar
Puyen, ZM, Villagrasa, E, Maldonado, J, Diestra, E, Esteve, I & Sole, A (2012). Biosorption of lead and copper by heavy-metal tolerant Micrococcus luteus DE2008. Bioresour Technol 126, 233237.Google Scholar
Rahman, MA, Hogan, B, Duncan, E, Doyle, C, Krassoi, R, Rahman, MM, Naidu, R, Lim, RP, Maher, W & Hassler, C (2014). Toxicity of arsenic species to three freshwater organisms and biotransformation of inorganic arsenic by freshwater phytoplankton (Chlorella sp. CE-35). Ecotoxicol Environ Saf 106, 126135.Google Scholar
Rajamani, S, Torres, M, Falcao, V, Ewalt Gray, J, Coury, DA, Colepicolo, P & Sayre, R (2014). Noninvasive evaluation of heavy metal uptake and storage in microalgae using a fluorescence resonance energy transfer-based heavy metal biosensor. Plant Physiol 164(2), 10591067.Google Scholar
Roane, TM, Rensing, C, Pepper, IL & Maier, RM (2009). Microorganisms and Metal Pollutants. In Environmental Microbiology, Maier, RM, Pepper, IL & Gerba, CP (Eds.), pp. 421441. Elsevier Inc.Google Scholar
Rugnini, L, Costa, G, Congestri, R & Bruno, L (2017). Testing of two different strains of green microalgae for Cu and Ni removal from aqueous media. Sci Total Environ 601–602, 959967.Google Scholar
Schreiber, U (1998) Chlorophyll fluorescence: New instruments for special applications. In Photosynthesis: Mechanisms and Effects, vol. V, Garag, G (Ed.), pp. 42534258. Dordrecht: Kluwer Academic Publishers.Google Scholar
Seder-Colomina, M, Burgos, A, Maldonado, J, Solé, A & Esteve, I (2013). The effect of copper on different phototrophic microorganisms determined in vivo and at cellular level by confocal laser microscopy. Ecotoxicology 22, 199205.Google Scholar
Shanab, S, Essa, A & Shalaby, E (2012). Bioremoval capacity of three heavy metals by some microalgae species (Egyptian isolates). Plant Sing Behav 7(3), 18.Google Scholar
Thompson, JA (1997). Cellular fluorescence capacity as an endpoint in algal toxicity testing. Chemosphere 35, 20272037.Google Scholar
Trenfield, MA, van Dam, JW, Harford, AJ, Parry, D, Streten, C, Gibb, K & van Dam, RA (2015). Aluminium, gallium and molybdenum toxicity to the tropical marine microalga Isochrysis galbana. Environ Toxicol Chem 34(8), 18331840.Google Scholar
Veldhuis, MJW, Kraay, GW & Timmermans, KR (2001). Cell death in phytoplankton: Correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. Eur J Phycol 36, 167177.Google Scholar
Willemann, RL (2002). Development of an application of the ECOTOX System in the Estuarine Zone of the Baía da Babitonga, SC, Brazil, Diplom, Friedrich-Alexander Universität, Erlangen-Nürnberg, 1–72.Google Scholar