Multi-approach analysis to assess the chromium(III) immobilization by Ochrobactrum anthropi DE2010.

Ochrobactrum anthropi DE2010 is a microorganism isolated from Ebro Delta microbial mats and able to resist high doses of chromium(III) due to its capacity to tolerate, absorb and accumulate this metal. The effect of this pollutant on O. anthropi DE2010 has been studied assessing changes in viability and biomass, sorption yields and removal efficiencies. Furthermore, and for the first time, its capacity for immobilizing Cr(III) from culture media was tested by a combination of High Angle Annular Dark Field (HAADF) Scanning Transmission Electron Microscopy (STEM) imaging coupled to Energy Dispersive X-ray spectroscopy (EDX). The results showed that O. anthropi DE2010 was grown optimally at 0-2 mM Cr(III). On the other hand, from 2 to 10 mM Cr(III) microbial plate counts, growth rates, cell viability, and biomass decreased while extracellular polymeric substances (EPS) production increases. Furthermore, this bacterium had a great ability to remove Cr(III) at 10 mM (q = 950.00 mg g-1) immobilizing it mostly in bright polyphosphate inclusions and secondarily on the cellular surface at the EPS level. Based on these results, O. anthropi DE2010 could be considered as a potential agent for bioremediation in Cr(III) contaminated environments.

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

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.

Morphological responses to nitrogen stress deficiency of a new heterotrophic isolated strain of Ebro Delta microbial mats.

Microorganisms living in hypersaline microbial mats frequently form consortia under stressful and changing environmental conditions. In this paper, the heterotrophic strain DE2010 from a microalgae consortium (Scenedesmus sp. DE2009) from Ebro Delta microbial mats has been phenotypically and genotypically characterized and identified. In addition, changes in the morphology and biomass of this bacterium in response to nitrogen deficiency stress have been evaluated by correlative light and electron microscopy (CLEM) combining differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These isolated bacteria are chemoorganoheterotrophic, gram-negative, and strictly aerobic bacteria that use a variety of amino acids, organic acids, and carbohydrates as carbon and energy sources, and they grow optimally at 27 °C in a pH range of 5 to 9 and tolerate salinity from 0 to 70‰ NaCl. The DNA-sequencing analysis of the 16S rRNA and nudC and fixH genes and the metabolic characterization highlight that strain DE2010 corresponds to the species Ochrobactrum anthropi. Cells are rod shaped, 1-3 µm in length, and 0.5 µm wide, but under deprived nitrogen conditions, cells are less abundant and become more round, reducing their length and area and, consequently, their biomass. An increase in the number of pleomorphic cells is observed in cultures grown without nitrogen using different optical and electron microscopy techniques. In addition, the amplification of the fixH gene confirms that Ochrobactrum anthropi DE2010 has the capacity to fix nitrogen, overcoming N2-limiting conditions through a nifH-independent mechanism that is still unidentified.

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. In this paper, we present a novel method using Confocal Laser Scanning Microscopy and a Dual Laser (CLSM-DL) for determining the cell viability of phototrophic microorganisms without the need of either staining or additional use of image treating software. In order to differentiate viable and nonviable Scenedesmus sp. DE2009 cells, a sequential scan in two different channels was carried out from each same xyz optical section. On the one hand, photosynthetic pigments fluorescence signal (living cells) was recorded at the red channel (625- to 785-nm fluorescence emission) exciting the samples with a 561-nm laser diode, and an acousto-optic tunable filter (AOTF) of 20%. On the other hand, nonphotosynthetic autofluorescence signal (dead cells) was recorded at the green channel (500- to 585-nm fluorescence emission) using a 405-nm UV laser, an AOTF of 15%. Both types of fluorescence signatures were captured with a hybrid detector. The validation of the CLSM-DL method was performed with SYTOX green fluorochrome and electron microscopic techniques, and it was also applied for studying the response of distinct light intensities, salinity doses and exposure times on a consortium of Scenedesmus sp. DE2009.

Characterization of transfer function, resolution and depth of field of a soft X-ray microscope applied to tomography enhancement by Wiener deconvolution.

Full field soft X-ray microscopy is becoming a powerful imaging technique to analyze whole cells preserved under cryo conditions. Images obtained in these X-ray microscopes can be combined by tomographic reconstruction to quantitatively estimate the three-dimensional (3D) distribution of absorption coefficients inside the cell. The impulse response of an imaging system is one of the factors that limits the quality of the X-ray microscope reconstructions. The main goal of this work is to experimentally measure the 3D impulse response and to assess the optical resolution and depth of field of the Mistral microscope at ALBA synchrotron (Barcelona, Spain). To this end we measure the microscope apparent transfer function (ATF) and we use it to design a deblurring Wiener filter, obtaining an increase in the image quality when applied to experimental datasets collected at ALBA.

Role of Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 as Bioindicators and Immobilizers of Chromium in a Contaminated Natural Environment.

The aim of this work was to study the potential of the two phototrophic microorganisms, both isolated from Ebro Delta microbial mats, to be used as bioindicators and immobilizers of chromium. The results obtained indicated that (i) the Minimum Metal Concentration (MMC) significantly affecting Chlorophyll a intensity in Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 was 0.25 µM and 0.75 µM, respectively, these values being lower than those established by current legislation, and (ii) Scenedesmus sp. DE2009 was able to immobilize chromium externally in extracellular polymeric substances (EPS) and intracellularly in polyphosphate (PP) inclusions. Additionally, this microorganism maintained high viability, including at 500 µM. Based on these results, we postulate that Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 are good chromium-indicators of cytotoxicity and, further, that Scenedesmus sp. DE2009 plays an important role in immobilizing this metal in a contaminated natural environment.