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

Microorganisms can mediate in heavy metal sequestration through several cellular strategies and pathways. This offers an efficient way to remediate heavy metal polluted environments. This paper describes the ability of Escherichia coli K-12 to capture chromium(III) (Cr(III)) and the ultrastructural effects of this metal on cells, as well as the cellular metal localization and the possible sequestration strategy uses for it. The study was mainly performed by using several electron microscopy techniques and is based on the chromium trivalent concentration and the related exposure time. Transmission electron microscopy (TEM) assay was performed along with field emission scanning electron microscopy (FESEM) for morphological responses. Furthermore, TEM was coupled with an energy dispersive X-ray (TEM-EDX) and TEM with selected area electron diffraction (TEM-SAED) to conduct analytical assays. The exposed cultures to 10 and 12 mM Cr(III) at 12 h and to 5, 7, 10, 12, 13, and 15 mM of Cr(III) at 24 h indicated the presence of multiple electrodense granules that were significantly enriched in chromium and phosphorus content via EDX analysis. Moreover, these granules were observed to be attached to external membrane and/or surrounding cells in the respective ultrathin sections analyzed under TEM. According to these results, E. coli K-12 possesses the ability to immobilize Cr(III) in external polyphosphate granules through a strategy of accumulation, where cell response to Cr(III) toxicity seems to have a dose-dependent and time-dependent relation, thereby offering significant potential for bioremediation in Cr(III)-contaminated areas.

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

Cytotoxic, chemical, biochemical, compositional, and morphometric responses were analyzed against heavy metal exposure in Ochrobactrum anthropi DE2010, an heterotrophic bacterium isolated from Ebro Delta microbial mats (Tarragona, NE Spain). Several parameters of effect and exposure were evaluated to determine tolerance to a range of cadmium (Cd), lead (Pb(II)), copper (Cu(II)), chromium (Cr(III)), and zinc (Zn) concentrations. Additionally, removal efficiency, polyphosphate production and metal localization patterns were also analyzed. O. anthropi DE2010 showed high resistance to the tested metals, supporting concentrations of up to 20 mM for Zn and 10 mM for the rest of the elements. The bacterium also demonstrated a high removal capacity of metals-up to 90 % and 40 % for Pb(II) and Cr(III), respectively. Moreover, polyphosphate production was strongly correlated with heavy metal concentration, and three clear cell localization patterns of metals were evidenced using compositional and imaging techniques: (i) extracellular in polyphosphate granules for Cu(II); (ii) in periplasmic space forming crystals with phosphorus for Pb(II); and (iii) intracytoplasmic in polyphosphate inclusions for Pb(II), Cr(III), and Zn. The high resistance and metal sequestration capacity of O. anthropi DE2010 both highlight its great potential for bioremediation strategies, especially in Pb and Cr polluted areas.

Genomic and biotechnological insights on stress-linked polyphosphate production induced by chromium(III) in Ochrobactrum anthropi DE2010.

The resistance of microorganisms to heavy metals in polluted environments is mediated by genetically determined mechanisms. One such mechanism includes the intracellular sequestration of heavy metals in polyphosphate (polyP) inclusions. In Cr(III) contaminated mediums, Ochrobactrum anthropi DE2010 is able to bind and sequester Cr(III) in polyP inclusions. In order to further study the relationship between Cr(III) tolerance and polyP production in O. anthropi DE2010, we carried out whole genomic sequencing, analysis of single nucleotide polymorphisms (SNPs), polyP chemical quantification, and determination of the relative abundance and morphometry of polyP inclusions. In the O. anthropi DE2010 genome, six polyP and pyrophosphate (PPi) metabolic genes were found. Furthermore, genomic analysis via SNPs calling revealed that O. anthropi ATCC49188 and DE2010 strains had average variations of 1.51% in their whole genome sequences and 1.35% variation associated with the principal polyP metabolic gene cluster. In addition, the accumulation of polyP in the DE2010 strain and number of polyP inclusions found were directly correlated with the concentration of Cr(III) in contaminated cultures. The results presented in this study may enhance the understanding of polyP production in response to Cr(III) toxicity in the O. anthropi DE2010 strain. This knowledge may facilitate the successful removal of Cr(III) from the natural environment.

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.

Biosorption of lead and copper by heavy-metal tolerant Micrococcus luteus DE2008.

Micrococcus luteus DE2008 has the ability to absorb lead and copper. The effect of these metals on biomass and viability of this microorganism were investigated and removal of the metals from culture media was determined. Lead had no effect on the biomass expressed as mg Carbon/cm(3) of M. Iuteus DE2008, but in the case of copper, the minimum metal concentration that affected the biomass was 0.1 mM Cu(II). According to these results this microorganism shows a greater tolerance for lead. The minimum metal concentration that affected viability (expressed as the percentage of live cells) was 0.5 mM for both metals. M. luteus DE2008 exhibited a specific removal capacity of 408 mg/g for copper and 1965 mg/g for lead. This microorganism has a greater ability to absorb Pb(II) than Cu(II). M. luteus DE2008 could be seen as a microorganism capable of restoring environments polluted by lead and copper.

Viability and biomass of Micrococcus luteus DE2008 at different salinity concentrations determined by specific fluorochromes and CLSM-image analysis.

In previous studies, our group developed a method based on Confocal Laser Scanning Microscopy and Image Analysis (CLSM-IA) to analyze the diversity and biomass of cyanobacteria in microbial mats. However, this method cannot be applied to heterotrophic microorganisms, as these do not have autofluorescence. In this article, we present a method that combines CLSM-IA and Hoechst 33342 and SYTOX Green fluorochromes (FLU-CLSM-IA) to determine the viability and biomass of Micrococcus luteus DE2008, isolated from a saline microbial mat (Ebro Delta, Tarragona, Spain). The method has been applied to assess the effect of salinity on this microorganism. A reduction in viability and biomass (live cells) was observed as the salt concentration increases. The largest effect was at 100‰ NaCl with a cell death of 27.25% and a decrease in total and individual biomass of 39.75 and 0.009 mgC/cm(3), respectively, both with respect to optimal growth (10 ‰ NaCl). On the other hand, another important contribution of this article was that combining the FLU-CLSM-IA results with those achieved by plate counts enabled us to determine, for first time, the viability and the total biomass of the "dormant cells" (66.75% of viability and 40.59 mgC/cm(3) of total biomass at 100‰ NaCl). FLU-CLSM-IA is an efficient, fast, and reliable method for making a total count of cells at pixel level, including the dormant cells, to evaluate the viability and the biomass of a hetetrophic microorganism, M. luteus DE2008.