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.

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.

Comparison of two analgesic protocols for post-tonsillectomy pain control in outpatient adults.

INTRODUCTION AND OBJECTIVES: Tonsillectomy causes a moderate to severe postoperative pain, and its treatment is an unsolved problem. The objective of this study was to compare the effectiveness of 2 analgesic protocols and their related complications. METHODS: Two groups of adult patients submitted to ambulatory tonsillectomy were studied. In group 1, 52 patients received a combination of tramadol and NSAIDs postoperatively; in group 2, 60 patients were treated with prednisone and NSAIDs. Two surgical techniques were used: cold dissection or dissection with electrocautery. Pain was recorded on days 4, 7 and 15, using a numerical scale from 0 to 10. RESULTS: Both groups showed similar pain at postoperative day 4. At day 7, pain was higher in group 2 (P=.049), while at day 15 both groups showed only some discomfort. Sickness and vomiting was more frequent in group 1, and haemorrhage and hospitalisation in group 2. Cold dissection patients showed lower levels of pain at days 4 and 7, independently of analgesic protocol, and had lower haemorrhage and emergency visit rates. CONCLUSIONS: The efficacy of both protocols was similar in terms of control of pain, with the exception of day 7; however, the protocol with prednisone showed fewer secondary effects. Patients operated using cold dissection had less pain and fewer complications.

Protocol for post-tonsillectomy pain control in outpatient adults.

INTRODUCTION: Even though notable advances in anaesthetic and surgical techniques have appeared in recent years, morbidity, and especially pain, associated with tonsillectomy is still an important clinical problem. OBJECTIVES: Assess the influence of a specific protocol for the control of postoperative pain and compare the frequency of complications in patients with and without it. METHODS: This was a descriptive, observational and prospective study on adult tonsillectomy patients in outpatient surgery. There were 2 groups: group 1, with 65 patients to whom a variable analgesic treatment was given; and group 2, with 50 patients with analgesic protocol and preoperative nursing interview. For the evaluation of pain, a numerical scale from 0 to 10 was used. The surgical techniques used were cold dissection or electric dissection. RESULTS: On the 4(th) day, group 1 (without protocol) presented a mean pain of 4.8 points on a numerical scale from 0 to 10, while group 2 (with protocol) presented mean of 3 (P=.0002). From group 1, 22 patients (36%) had to go to the emergency service, while 8 (16%) in group 2 did so (P=.019). On the 4(th) day, patients operated with cold dissection presented 3.7 points, as opposed to those operated with electric dissection, who presented 4.4 points. CONCLUSIONS: A specific protocol applied to adult tonsillectomy patients in outpatient surgery is useful to obtain less pain and fewer complications.

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.

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.

Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria.

In this paper, we determine for the first time the in vivo effect of heavy metals in a phototrophic bacterium. We used Confocal Laser Scanning Microscopy coupled to a spectrofluorometric detector as a rapid technique to measure pigment response to heavy-metal exposure. To this end, we selected lead and copper (toxic and essential metals) and Microcoleus sp. as the phototrophic bacterium because it would be feasible to see this cyanobacterium as a good biomarker, since it covers large extensions of coastal sediments. The results obtained demonstrate that, while cells are still viable, pigment peak decreases whereas metal concentration increases (from 0.1 to 1 mM Pb). Pigments are totally degraded when cultures were polluted with lead and copper at the maximum doses used (25 mM Pb(NO(3))(2) and 10 mM CuSO(4)). The aim of this study was also to identify the place of metal accumulation in Microcoleus cells. Element analysis of this cyanobacterium in the above mentioned conditions determined by Energy Dispersive X-ray microanalysis (EDX) coupled to Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), shows that Pb (but not Cu) accumulates externally and internally in cells.

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.

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

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.

Changes in the composition of polar and apolar crude oil fractions under the action of Microcoleus consortia.

Cultures of Microcoleus consortia polluted with two different types of crude oil, one with high content in aliphatic hydrocarbons (Casablanca) and the other rich in sulphur and aromatic compounds (Maya), were grown for 50 days and studied for changes in oil composition. No toxic effects from these oils were observed on Microcoleus consortia growth. In fact, the interface layer between the oils and the water culture medium proved to be the ideal site for consortia development, leading to a wrapping effect of the oil layers by these organisms. Despite this affinity of cyanobacteria for the oil substrate, the changes in oil composition were small. Microcoleus consortia did not induce transformation in the aliphatic-rich oil, and the modifications in the sulphur and aromatic-rich oil were small. The latter essentially involved degradation of aliphatic heterocyclic organo-sulphur compounds such as alkylthiolanes and alkylthianes. Other groups of compounds, such as the alkylated monocyclic and polycyclic aromatic hydrocarbons, carbazoles, benzothiophenes and dibenzothiophenes, also underwent some degree of transformation, involving only the more volatile and less alkylated homologues.