Sequestration and in vivo effect of lead on DE2009 microalga, using high-resolution microscopic techniques
Introduction
Microalgae and cyanobacteria are the most important primary producers in stratified laminated ecosystems, such as microbial mats, which cover large extensions of marine coastal environments [1], [2], [3], [4], [5].
In the last few years, we have isolated a consortium of microorganisms, from Ebro delta microbial mats, dominated by a single cyanobacterium, Microcoleus sp., and different heterotrophic bacteria [6], [7]. Recently we have isolated a new phototrophic microorganism, a microalga (DE2009) from the same habitat. Given that Microcoleus sp. was able to tolerate lead and copper [8] in this study we propose an analysis of whether DE2009 microalga is able to sequestrate heavy metals.
Phototrophic microorganisms have been frequently used in biosorption research [9], [10], [11], [12]. Metals are one group of contaminants frequently involved in marine environmental pollution. It is known that some metals at low concentrations, participate in different metabolic routes (essentials), but at high concentrations they are toxic for many living organisms; while others metals always have a toxic effect [13]. Different methods have been proposed to study the toxic effect of heavy metals on microalgae, but most authors conclude that the metal concentration that affects growth in microalgae is variable and depends of many different factors, including the ability to accumulate heavy metals [14], [15]. Algal surfaces have been found that containing different chemical function groups that differ in affinity and specificity towards these metals [16], [17], [18].
Although the capacity of some microalgae to capture heavy metals has been described, little is known about the effect of these metals in individual living cells, which is needed to predict the impact of heavy metals on natural ecosystems. In this study we selected Pb as a toxic metal and because the microbial mats studied are located in a lead-polluted area of the Ebro delta [19].
Confocal laser scanning microscopy (CLSM) based on natural pigment fluorescence emitted by phototrophic microorganisms is proving to be an excellent methodology for different types of studies related to these microorganisms. This optical microscopy technique avoids the need for either manipulating or staining the samples and allows accurate and non-destructive optical sectioning that generates high-resolution images, where out-of-focus is eliminated. Due to its high resolution, it is easy to differentiate morphotypes of phototrophic microorganisms living in mixed populations, because they emit natural fluorescence.
The CLSM coupled to a spectrofluorometric detector (λscan function), provides simultaneous three-dimensional information on photosynthetic microorganisms and their fluorescence spectra profiles in stratified ecosystems, such as microbial mats and biofilms. The most significant application is the discrimination of cells with specific fluorescence spectra profiles within a colony, and the correlation of morphology and individual cell states [20].
In this paper, we have applied CLSM-λscan, to determine the in vivo effect of Pb (at different concentrations) on DE2009 microalga and CLSM-IA to determine their total and individual biomass.
Complementary studies using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray microanalysis (EDX) coupled to SEM and TEM were also performed to test the capacity of DE2009 microalga for extra- and intracellular uptake of Pb.
Section snippets
Culture conditions
Cultures of DE2009 microalga were grown at 27 °C and 15 μE m−2 s−1 in liquid mineral Pfennig medium at two pHs (7 and 4) and at different concentrations (0, 0.1, 0.5, 0.75, 1, 5 and 10 mM) of lead (Pb(NO3)2) for 9 days.
Confocal laser scanning microscopy
The confocal experiments were performed using a confocal laser scanning microscope (Leica TCS SP5; Leica Heidelberg, Germany).
Characterisation of the DE2009 microalga
DE2009 microalga was isolated from the Ebro delta microbial mats. Cells are spherical, with a diameter of 7–9 μm. Ultrathin sections of cells show the thylakoids grouped into bands (inside the chloroplast); the nucleus and the pyrenoid. High electron-dense inclusions (HE) inside the cytoplasm, were identified as polyphosphate granules (PPG). In pristine cultures (without Pb) no exopolysaccharides (EPS) were detected surrounding the cell wall (Fig. 1).
According to 18S rRNA gene sequence
Conclusions
In conclusion, we consider that the CLSM-λscan could be a rapid technique for studying in vivo the cellular responses to heavy metal pollution. At pH 7 there is and inverse correlation between the intensity of pigment's fluorescence emission and the concentration of essayed metal. At pH 4 there is no good correlation between the concentration of metal and the pigment's intensity of the fluorescence emission.
Moreover, this method combined with the values obtained by means of CLSM-IA enables
Acknowledgments
This research was supported by the following grants: DGICYT (CGL2008-01891/BOS and CTM2009-1238 CO4-O3) and FONCICYT (000000000095887). We express our thanks to the staff of the Servei de Microscòpia at the Universitat Autònoma de Barcelona for technical assistance with the confocal and electron microscopies and to Mª José Malo from Centro de Ciencias Medioambientales for her help in molecular biology work. We also thank Marc Alamany and Francesc Fornells from Ecología Portuaria S. L. (Spain),
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