Sequestration and in vivo effect of lead on DE2009 microalga, using high-resolution microscopic techniques

https://doi.org/10.1016/j.jhazmat.2010.06.085Get rights and content

Abstract

Algae are primary producers in a wide variety of natural ecosystems, and these microorganisms have been used in bioremediation studies. Nevertheless, very little is known about the in vivo effect of heavy metals on individual living cells.

In this paper, we have applied a method based on confocal laser scanning microscopy and lambda scan function (CLSM-λscan) to determine the effect of lead (Pb), at different concentrations, on the DE2009 microalga. At the same time, we have optimized a method based on CLSM and image-analysis software (CLSM-IA) to determine in vivo biomass of this microorganism. The results obtained by lambda scan function indicated that the pigment peak decreases while the concentration of metal increases at pH 7. On the other hand at pH 4 there is no good correlation between the concentration of metal and the intensity of the emission of fluorescence of the pigment. Also, in some cases a displacement of the Chl a peak towards 680 nm is produced. Total and individual biomass determined by CLSM-IA shows statistically significant differences between unpolluted and 10 mM polluted cultures.

Complementary studies using electron microscopy techniques coupled to energy dispersive X-ray microanalysis (EDX) demonstrate that the microalga can sequestrate Pb extra- and intracellularly.

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),

References (35)

  • I. Esteve et al.

    Microbial mats: structure, development and environmental significance

  • T. Nakagawa et al.

    Phylogenetic characterization of microbial mats and streamers from a Japanese alkaline hot spring with a thermal gradient

    J. Gen. Appl. Microbiol.

    (2002)
  • A. Wieland et al.

    Microbial mats on the Orkney Islands revisited: microenvironment and microbial community composition

    Microb. Ecol.

    (2003)
  • E. Diestra et al.

    Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

    Scanning

    (2005)
  • O. Sánchez et al.

    Molecular characterization of an oil-degrading cyanobacterial consortium

    Microb. Ecol.

    (2005)
  • M. Burnat et al.

    In situ determination of the effects of lead and copper on cyanobacterial populations in microcosms

    PLoS One

    (2009)
  • R. De Philippis et al.

    Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanobacteria capsulata and Nostoc PCC7936

    J. Appl. Phycol.

    (2003)
  • Cited by (20)

    • Ultrastructural evidences for chromium(III) immobilization by Escherichia coli K-12 depending on metal concentration and exposure time

      2021, Chemosphere
      Citation Excerpt :

      It has been widely reported that ultramicroscopy techniques (TEM and TEM-EDX) can be employed to obtain information on ultrastructural changes from cellular responses to metal exposure and metal localization (Gerber et al., 2016; Kiran et al., 2017; Niu et al., 2018). Extensively, those scientific studies that utilized TEM-EDX analyses focused on (i) localizing toxic heavy metals in the different microbial cell structures and (ii) examining the sequestration strategy (Lengke et al., 2006; Maldonado et al., 2010a, 2010b; Maldonado et al., 2011; Villagrasa et al., 2020a, 2020b). Taking this into consideration, our research group, during the last decade, has isolated several bacteria from the Ebro Delta microbial mats (Tarragona, Spain).

    • Cellular strategies against metal exposure and metal localization patterns linked to phosphorus pathways in Ochrobactrum anthropi DE2010

      2021, Journal of Hazardous Materials
      Citation Excerpt :

      DE2011) and heterotrophic (Paracoccus sp. DE2007, Micrococcus luteus DE2008, Ochrobactrum anthropi DE2010) microorganisms from Ebro Delta mats have been tested in axenic laboratory cultures to analyze their ability capturing metals such as Cr(III), Pb(II), and Cu(II) (e.g. Burnat et al. 2009; Burgos et al., 2013; Maldonado et al., 2010a, b; Puyen et al., 2012; Millach et al., 2015; Villagrasa et al., 2019, 2020a). Interestingly, all these isolated microorganisms have the capacity to sequester metals externally (biosorption) in extracellular polymeric substances (EPS), with this ability becoming especially high in Micrococcus luteus DE2008 for Cu(II) and Pb(II) (Puyen et al., 2012).

    • Bioremoval of heavy metals from metal mine tailings water using microalgae biomass

      2019, Algal Research
      Citation Excerpt :

      These results are consistent with the lower removal efficiency observed during the assay done with the Cu solution. These results are consistent with those of Maldonado et al. [37], who reported that the intensity of pigments' fluorescence emission decreases when the heavy metal concentration increases, as the cellular response of DE2009 microalgae. On the other hand, a larger cell area was evident for the MTW treatment: 33 μm2 on average, compared with the control, which showed an area of only 7.5 μm2.

    • Precipitation of iron-hydroxy-phosphate of added ferric iron from domestic wastewater by an alternating aerobic-anoxic process

      2014, Chemical Engineering Journal
      Citation Excerpt :

      At the low SRP loading rate corresponding to 4.15 mgP h−1, the operation of AAA treatment process could be working at approximately 74% of its optimum efficiency. Further work should thus be carried out to determine if “the polyphosphate overplus” phenomenon [42,43] exists in a wide variety of operating conditions. The removal of TP can vary widely in a range of 55–80%; however, -the operation of AAA treatment process can achieve approximately 80% of its optimum efficiency when the influent concentration of TP in the range of 10.0–19.5 mgP L−1 (see Fig. 6b) even in different influent Fe/P molar ratio.

    View all citing articles on Scopus
    View full text