Antonio Solé

In Situ Determination of the Effects of Lead and Copper on Cyanobacterial Populations in Microcosms

Background Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth. Methodology/Principal Findings Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine in situ the effect of Pb and Cu stress in cyanobacterial populations. Conclusions/Significance The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and Microcoleus sp. (the dominant filamentous cyanobacterium in microbial mats) was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants.

Confocal laser scanning microscopy as a tool to determine cyanobacteria biomass in microbial mats

We have developed a method based on confocal laser scanning microscopy for detection and quantification of cyanobacteria from the Ebro Delta microbial mats. Cyanobacteria play a major role as primary producers in microbial mats; it is difficult, however, to apply classical methods to estimate their biomass because they establish strong interactions with detritic particles. The protocol described here allows the localization of individual cells or filaments with micrometre precision without the need to either manipulate or stain the samples. This method is suitable for studying biomass ‘in situ’ in microbial mats.

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

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.

A COMPARATIVE STUDY OF CYANOBACTERIAL DIVERSITY IN POLLUTED AND UNPOLLUTED MICROBIAL MATS BY MEANS CLSM

Abstract This paper is a summary of the results obtained from a study of the diversity of cyanobacteria in oil polluted and unpolluted natural environments. Our work group identified the different genera of cyanobacteria by means of CLSM, as this technique is specifically appropriate for studying the diversity of these microorganisms in stratified bentonic environments (microbial mats). The cyanobacteria identified in pristine ecosystems show that the most abundant cyanobacteria correspond to the genera: Microcoleus chthonoplastes, Oscillatona sp., Lyngbya sp., Leptolyngbya sp., and Limnothrix sp. Phormidium sp.and Pseudanabaena sp. were also identified, although they were less abundant. On the other hand, in the ecosystems heavily polluted with oil (Etang de Bêrre) neither Microcoleus chthonoplastes nor Oscillatoria sp. were detected, although the other genera mentioned were. With respect to coccoid cyanobacteria, the most abundant groups in the Ebro delta were Gloeocapsa sp. and Synechocystis sp. and also the genus Chroococcus sp. with Synechocystis sp. dominating the mats from Saline de Giraud. The microbial mats from Orkney Islands showed a different composition from those above (Pleurocapsa-group and Microcystis sp., Aphanothece sp. and Merismopedia sp.)

A novel method to analyse in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser

Phototrophic microorganisms are very abundant in extreme environments, where are subjected to frequent and strong changes in environmental parameters. Nevertheless, little is known about the physiological effects of these changing environmental conditions on viability of these microorganisms, which are difficult to grow in solid media and have the tendency to form aggregates. For that reason, it is essential to develop methodologies that provide data in short time consuming, in vivo and with minimal manipulating the samples, in response to distinct stress conditions.