Bivalent copper ions presence triggers removal and homeostatic mechanisms in the metal-resistant microorganism Apiotrichum loubieri M12.

Microorganisms, especially those habiting mining environments, are of great importance for the retention of toxic metals in the environment. This work aimed to isolate a copper removing-microorganism from sediments of an Acid Mine Drainage-affected environment and to study the cellular responses trigger by metal presence. Apiotrichum loubieri M12 was able to tolerate and remove Cu(II) from liquid culture media, reaching a 30-35% removal capacity when it was exposed to 40 µg mL-1 Cu(II) after 48 h. Analysis of the biomass exposed to the metal through SEM-EDS showed copper presence on the cell surface and variations in the proportion of other biomass constituent elements. Proteomics revealed that the presence of Cu(II) induces differential expression of intracellular proteins involved in a wide variety of metabolic processes. Interestingly, a specific response to the metal was detected in cell-free supernatants, in which copper binding proteins were identified. A large number of proteins with metal ion binding sites were detected both at intra and extracellular levels. The microorganism responds not only by adjusting intracellular protein expression, but also by adjusting expression of proteins in the extracellular space.

Characterization of copper stress response in Fusarium tricinctum M6: A metal-resistant microorganism isolated from an acid mine drainage-affected environment.

Acid mine drainage-affected environments are interesting microbial niches for the isolation of metal-resistant microorganisms. In this sense, the aim of the present work is to isolate and characterize metal-resistant microorganisms from sediments of an abandoned gold mine located in San Luis (Argentina). For these purposes, the metal removal capacity and the microelemental composition of the biomass exposed to metals were evaluated. Likewise, proteomic techniques were applied to understand the removal and resistance mechanisms. Fusarium tricinctum M6 was isolated and identified as tolerant to Cu(II), Fe(II) and Cr(VI). When faced with 40 µg mL-1 Cu(II), the growth was affected by 60% and the removal capacity was 30-35%. Copper was found uniformly distributed in the biomass (5.23% w/w) and variations in the proportion of other biomass constituent elements were detected. When exposed to Cu(II), F. tricinctum M6 showed differential expression of intra and extracellular proteins involved in different metabolic processes. A large number of proteins with metal ion binding sites were detected both at intra and extracellular levels. The results obtained in the present work indicated bioadsorption of the metal on the cell surface and an important readjustment of the protein expression to counteract the stress produced by Cu(II).

Impact of anthropogenic activities on an urban river through a comprehensive analysis of water and sediments.

The aim of this study was to assess the impact of urban and industrial areas on an urban river through a comprehensive analysis of water and sediments. Six different sites along the San Luis River, Argentina, were characterized by measuring 12 physical-chemical parameters and nine heavy metals according to standard protocols. Metal pollution in sediment samples was evaluated with several indices. Cluster analysis was applied to standardized experimental data in order to study spatial variability. As, Cu, Cr, Mn, Pb, and Zn were the main contributors to sediment pollution, and the industrial zone studied showed moderate enrichment of Co, Cu, and Zn, probably due to anthropogenic activities. Cluster analysis allowed the grouping of the sites: sediment samples were classified into two clusters according to the metal content; water samples were arranged into three groups according to organic matter content. The results were compared with sediment and water quality guidelines. They indicated progressive deterioration of water and sediment quality compared with the background area, mainly in the sites following the industrial park and domestic discharge areas. Moreover, the results showed that the analysis of both water and sediment should be considered to achieve a watershed contamination profile.

Development and Performance of Dengue Diagnostic Clinical Algorithms in Colombia.

Diagnosing dengue in endemic areas remains problematic because of the low specificity of the symptoms and lack of accurate diagnostic tests. This study aimed to develop and prospectively validate, under routine care, dengue diagnostic clinical algorithms. The study was carried out in two phases. First, diagnostic algorithms were developed using a database of 1,130 dengue and 918 non-dengue patients, expert opinion, and literature review. Algorithms with > 70% sensitivity were prospectively validated in a single-group quasi-experimental trial with an adaptive Bayesian design. In the first phase, the algorithms that were developed with the continuous Bayes formula and included leukocytes and platelet counts, in addition to selected signs and symptoms, showed the highest sensitivities (> 80%). In the second phase, the algorithms were applied on admission to 1,039 consecutive febrile subjects in three endemic areas in Colombia of whom 25 were laboratory-confirmed dengue, 307 non-dengue, 514 probable dengue, and 193 undetermined. Including parameters of the hemogram consistently improved specificity without affecting sensitivity. In the final analysis, considering only confirmed dengue and non-dengue cases, an algorithm with a sensitivity and specificity of 65.4% (95% credibility interval 50-83) and 40.1% (34.7-45.7) was identified. All tested algorithms had likelihood ratios close to 1, and hence, they are not useful to confirm or rule out dengue in endemic areas. The findings support the use of hemograms to aid dengue diagnosis and highlight the challenges of clinical diagnosis of dengue.

Intracellular Proteomic Analysis of Streptomyces sp. MC1 When Exposed to Cr(VI) by Gel-Based and Gel-Free Methods.

The actinobacterium Streptomyces sp. MC1 has previously shown the capacity to resist and remove Cr(VI) from liquid culture media. The aim of this work is to analyze the differential expression pattern of intracellular proteins when Streptomyces sp. MC1 is exposed to Cr(VI) in order to explain the molecular mechanisms of resistance that this microorganism possesses. For this purpose, 2D-PAGE and shotgun proteomic analyses (2D-nanoUPLC-ESI-MS/MS) were applied. The presence of Cr(VI) induced the expression of proteins involved in molecular biosynthesis and energy generation, chaperones with a key role in the repair of misfolded proteins and stress response, transcription proteins, proteins of importance in the DNA supercoiling, repair and replication, and dehydrogenases involved in oxidation-reduction processes. These dehydrogenases can be associated with the reduction of Cr(VI) to Cr(III). The results of this study show that proteins from the groups mentioned before are important to face the stress caused by the Cr(VI) presence and help the microorganism to counteract the toxicity of the metal. The use of two proteomic approaches resulted in a larger number of peptides identified, which is also transduced in a significant number of protein ID. This decreased the potential complexity of the sample because of the protein dynamic range, as well as increased the recovery of peptides from the gel after digestion.

Biogenic nanoparticles: Synthesis, stability and biocompatibility mediated by proteins of Pseudomonas aeruginosa.

The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae, fungi and bacteria for the production of nanomaterials are viable alternatives due to their low cost, the absence of toxic waste production and their highly energy efficiency. It is also known that biosynthesized silver nanoparticles (AgNPs) show higher biocompatibility compared to the chemically-synthesized ones. In previous results, biosynthesized AgNPs were obtained from the supernatant of Pseudomonas aeruginosa, and they showed a bigger antimicrobial activity against different bacterial species compared to the chemically-synthesized ones. The aim of this work was to analyze the capping of biosynthesized AgNPs using techniques such as transmission electron microscopy (TEM), infrared spectroscopy (IR), and protein identification through mass spectrometry (MS) in order to identify the compounds responsible for their formation, stability and biocompatibility. The TEM images showed that AgNPs were surrounded by an irregular coverage. The IR spectrum showed that this coverage was composed of carbohydrates and/or proteins. Different proteins were identified in the capping associated to biosynthesized AgNPs. Some proteins seem to be important for their formation (Alkyl hydroperoxide reductase and Azurin) and stabilization (Outer membrane protein OprG and Glycine zipper 2 T M domain-containing protein). The proteins identified with the capability to interact with some biomolecules can be responsible for the biocompatibility and may be responsible for the bigger antimicrobial activity than AgNPs have previously shown. These results are pioneers in the identification of proteins in the capping of biosynthesized AgNPs.

The potential application of an autochthonous fungus from the northwest of Argentina for treatment of sugarcane vinasse.

Vinasse is a waste material from distillery industries, which causes major environmental problems around the world. Argentina alone produces about 4 billion liters of vinasse annually; consequently, diverse biological eco-friendly treatments are evaluated for their ability to reduce the detrimental effects. The present study reports on the degradation of a 50% (v/v) local vinasse sample by an autochthonous fungus identified as Aspergillus sp. V1. The Bioprocess was conducted for 15 d at 30 °C after inoculation of spores at an end concentration of 1 × 106 CFU/mL. Effluent neutralization was detected after 6 d of treatment, with maximum COD and BOD removal after 12 d (49% and 59%, respectively). Effects of vinasse before and after treatment were predicted using Caco-2 cells and Triticum aestivum L. (wheat) seeds as toxicological indicators. Only 13% viability was observed for Caco-2 cells exposed to untreated vinasse, but this percentage increased more than 3-fold for cells exposed to the treated effluent. While vinasse without treatment completely inhibited germination of seeds, exposure to treated effluent demonstrated a germination percentage of 60%. The present study highlights the use of a dual-purpose biotechnological process that aimed at reducing the detrimental effects of vinasse, enhancing its quality for agricultural practices.

Prokaryotic and eukaryotic community structure affected by the presence of an acid mine drainage from an abandoned gold mine.

The acid mine drainage that originates in the abandoned gold mine in San Luis, Argentina, is released into La Carolina stream. The aim of this study was to determine the influence of this mine drainage on the physicochemical parameters of the area studied and on both prokaryotic and eukaryotic community structure. In addition, specific relationships between microbial taxonomic groups and physicochemical parameters were established. The drainage that flows into La Carolina stream acidifies the stream and increases its sulfate, Zn, Cd and Te concentrations. Microbial analysis showed that prokaryotic community structure is mainly affected by pH values. Actinobacteria and Gammaproteobacteria were abundant in samples characterized by low pH values, while Nitrospirae, Chloroflexi, Deltaproteobacteria, Thaumarchaeota and Euryarchaeota were associated with high concentrations of heavy metals. Otherwise, Alphaproteobacteria was present in samples taken in sunlit areas. Regarding eukaryotic community structure, the sunlight had the greatest impact. Inside the mine, in the absence of light, fungi and protists members were the most abundant microorganisms, while those samples taken in the presence of light displayed algae (green algae and diatoms) as the most abundant ones. After receiving the mine drainage, the stream showed a decrease in the diatom abundance and green algae predominated.

Raw sugarcane bagasse as carbon source for xylanase production by Paenibacillus species: a potential degrader of agricultural wastes.

Paenibacillus species isolated from a variety of natural sources have shown to be important glycoside hydrolases producers. These enzymes play a key role in bio-refining applications, as they are central biocatalysts for the processing of different types of polymers from vegetal biomass. Xylanase production by three native isolates belonging to the genus Paenibacillus was approached by utilizing mineral-based medium and agricultural by-products as a convenient source to produce biocatalysts suitable for their degradation. While varieties of alkali pretreated sugarcane bagasse were useful substrates for the strains from Paenibacillus genus evaluated, raw sugarcane bagasse was the most effective substrate for endoxylanase production by Paenibacillus sp. AR247. This strain was then selected to further improvement of its enzyme production by means of a two-step statistical approach. It was determined that the carbon source, provided as an inexpensive agro-waste, as well as phosphate and magnesium were the culture media components that most influenced the enzyme production, which was improved three times compared to the screening results.

Simultaneous chromate and sulfate removal by Streptomyces sp. MC1. Changes in intracellular protein profile induced by Cr(VI).

The purpose of this study was to investigate the influence of increasing sulfate concentrations on chromium removal, to evaluate the effect of the presence of Cr(VI) on sulfate removal by Streptomyces sp. MC1 and to analyze the differential protein expression profile in the presence of this metal for the identification of proteins repressed or overexpressed. In the presence of Cr(VI) but in the absence of sulfate ions, bacterial growth was negligible, showing the Cr(VI) toxicity for this bacterium. However, the sulfate presence stimulated bacterium growth and Cr(VI) removal, regardless of its concentrations. Streptomyces sp. MC1 showed ability to remove chromium and sulfate simultaneously. Also, the sulfate presence favored the decrease of total chromium concentration from supernatants reaching a decrease of 50% at 48 h. In presence of chromium, seven proteins were down-expressed and showed homology to proteins involved in protein biosynthesis, energy production and free radicals detoxification while two proteins involved in oxidation-reduction processes identified as dihydrolipoamide dehydrogenase and S-adenosyl-l-methionine synthase were overexpressed.

ICPMS analysis of proteins separated by Native-PAGE: Evaluation of metaloprotein profiles in human synovial fluid with acute and chronic arthritis.

The role of trace elements bound to proteins in the etiology and pathogenesis of rheumatoid arthritis (RA) remains unclear. In this sense, the identification and detection of metalloproteins has a strong and growing interest. Metalloprotein studies are currently carried out by polyacrylamide gel electrophoresis (PAGE) associated to inductively coupled plasma mass spectrometry (ICPMS), and despite that complete information can be obtained for metals such as Fe, Cu and Zn, difficulties due to poor sensitivity for other trace elements such as Sn, As, etc, are currently faced. In the present work, a simple and fast method for the determination of trace metals bound to synovial fluid (SF) proteins was optimized. Proteins from SF (long and short-term RA) were separated in ten fractions by native PAGE, then dissolved in nitric acid and peroxide hydrogen, and analyzed by ICPMS. Fifteen metals were determined in each separated protein fraction (band). Adequate calibration of proteins molecular weight allowed stablishing which protein type were bound to different metals.

Production of bioemulsifiers by Amycolatopsis tucumanensis DSM 45259 and their potential application in remediation technologies for soils contaminated with hexavalent chromium.

In recent years, increasing interest has been shown in the use of bioemulsifiers as washing agents that can enhance desorption of soil-bound metals. However, high production costs derived from the use of expensive substrates for formulation of the fermentation media represent the main challenge for full, large-scale implementation of bioemulsifiers. This work reports on a first study of bioemulsifier production by the actinobacterium Amycolatopsis tucumanensis DSM 45259 using different carbon and nitrogen sources. Preliminary results on the potential use of these compounds as washing agents for soils contaminated with Cu(II) and Cr(VI) are also presented. The best specific production was detected using glycerol and urea as carbon and nitrogen substrates, respectively. However, with all of the substrates used during the batch assay, the bioemulsifiers showed high levels of stability at extreme conditions of pH, temperature, and salt concentration. Under the current assay conditions, the bioemulsifiers were not effective in removing Cu(II) from soil. However, they were able to mediate Cr(VI) recovery, with the removal percentage doubled compared to that seen when using deionized water. These findings appear promising for the development of remediation technologies for hexavalent chromium compounds based upon direct use of these microbial emulsifiers.

Production and partial characterization of bioemulsifier from a chromium-resistant actinobacteria.

Surface-active compounds such as synthetic emulsifiers have been used for several decades, both for the degradation of hydrocarbons and increasing desorption of soil-bound metals. However, due to their high toxicity, low degradability, and production costs unaffordable for use in larger ecosystems, synthetic emulsifiers have been gradually replaced by those derived from natural sources such as plants or microbes. In previous studies, the bacterium Streptomyces sp. MC1 has shown the ability to reduce and/or accumulate Cr(VI), a highly promising advance in the development of methods for environmental clean-up of sites contaminated with chromium. Here, new studies on the production of emulsifier from this strain are presented. The cultivation factors that have a significant influence on emulsifier biosynthesis, as well as the interactions among them, were studied by factorial design. Based upon optimization studies, maximum bioemulsifier production was detected in the culture medium having an initial pH of 8 with phosphate 2.0 g L(-1) and Ca(+2) 1.0 g L(-1) added, with an emulsification index about 3.5 times greater compared to the basal value. Interestingly, in the presence of 5.0 g L(-1) Cr(VI), Streptomyces sp. MC1 retained about 65% of its emulsifier production ability. Partially purified emulsifier presented high thermo-stability and partial water solubility. These findings could have promising future prospects for the remediation of organic- and metal-contaminated sites.

Cu(II) removal by Rhodotorula mucilaginosa RCL-11 in sequential batch cultures.

The present study explored the ability of the yeast Rhodotorula mucilaginosa RCL-11 to adapt to increasing Cu(II) concentrations, measuring oxidative stress through superoxide dismutase and catalase activity in two parallel sequential batch assays. One assay was performed in Erlenmeyer flasks without aeration and a second in a fermentor in which the dissolved oxygen was maintained at 30% saturation. Both assays were carried out by increasing Cu(II) concentrations in five sequential steps: 0; 0.1; 0.2; 0.5 and 1 mM. Each assay was incubated at 30 degrees C, 250 rpm and pH 5.5. While growth parameters of R. mucilaginosa RCL-11 decreased 90-95% with increasing Cu(II) concentration in the culture medium, the oxidative stress level increased from 30 to 55% in both assays. Cells grown under controlled oxygen conditions showed 30% more copper bioaccumulation and 10% glucose consumption when compared with cells grown without aeration. SOD activity was higher under controlled than without aeration, whereas CAT activity was similar under both test conditions. Cu(II) bioaccumulation by R. mucilaginosa RCL-11 and a possible increase in this capacity by adaptation of the strain under controlled aeration represent a potential valuable tool for treatment of effluents or water bioremediation with high copper contents.

Responses of Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11 to copper stress.

The effect of high Cu(II) concentrations on superoxide dismutase (SOD) and catalase (CAT) activity in Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11, previously isolated from a copper filter at a mine plant in Argentina, was studied. Addition of 0.1, 0.2 and 0.5 mM Cu(II) to the culture medium increased total SOD and CAT activity in both strains. Native polyacrylamide gel electrophoresis revealed two bands with SOD activity for C. fukuyamaensis RCL-3 and only one for R. mucilaginosa RCL-11; the three bands corresponded to MnSOD.Intracellular accumulation of copper and morphological changes was observed using electron microscopy. Dark bodies examined with transmission electron microscopy (TEM) after 48 h of incubation probably corresponded to copper deposits. The number of dark bodies in R. mucilaginosa RCL-11 grew with increasing incubation time, whereas in C. fukuyamaensis RCL-3 the amount decreased. Scanning electron micrographs (SEM) of C. fukuyamaensis RCL-3 did not reveal any differences compared with the control, but R. mucilaginosa RCL-11 cells were bigger than control ones. TEM confirmed absence of compartmentalization mechanisms in Cu(II) detoxification since electron-dense bodies were mainly found in the cytoplasm.

Chromate removal by yeasts isolated from sediments of a tanning factory and a mine site in Argentina.

Twenty-one yeast-like microorganisms were isolated from tannery effluents and from a nickel-copper mine in Argentina. They were tested for their Cu(II), Ni(II), Cd(II) and Cr(VI) tolerance in qualitative assays on solid medium. Three isolates were selected for their multiple tolerance to the different heavy metals and highest tolerance to Cr(VI). According to morphological and physiological analysis and 26S rDNA D1/D2 domain sequences the isolates were characterized as: Lecythophora sp. NGV-1, Candida sp. NGV-9 and Aureobasidium pullulans VR-8. Resistance of the three strains to high Cr(VI) concentrations and their ability to remove Cr(VI) were assessed using YNB-glucose medium supplemented with 0.5 and 1 mM Cr(VI). Chromate removal activity was estimated by measuring remaining Cr(VI) concentration in the supernatant using the colorimetric 1,5-diphenylcarbazide method and total chromium was determined by flame atomic absorption spectroscopy. The results indicate that the initial Cr(VI) concentration negatively influenced growth and the specific growth rate but stimulated the metabolic activity of the three strains; resistance to Cr(VI) by these strains was mainly due to reduction of Cr(VI) rather than chromium bioaccumulation. This study showed the potential ability of these strains as tools for bioremediation of Cr(VI) from contaminated sites.

Copper tolerant yeasts isolated from polluted area of Argentina.

Eleven yeasts were isolated from wastewater sediment samples collected from a copper filter mine plant, located in the province of Tucumán, Argentina, and tested for their heavy metal tolerance. Two isolates were selected based on their multiple tolerance to different heavy metals and their copper biosorption capacity was studied. Analysis of the 26S rDNA D1/D2 domain sequences indicates that isolate RCL-3 showed similarity with Candida sp. and RCL-11 with Rhodotorula mucilaginosa . Growth performance and copper toxicity of both yeasts were evaluated using YNB-glucose medium supplemented with 0.1, 0.2, 0.5 and 1 mM of Cu2+ solutions. Candida sp. RCL-3 was able to grow up to 7 mg ml(-1) biomass in the presence of either 0.1 or 0.2 mM Cu2+, and at 0.5 mM Cu2+ growth reached 5.5 mg ml(-1). R. mucilaginosa RCL-11 reached 8 mg ml(-1) in the presence of 0.1 mM Cu2+, and values of 6.5 and 5.5 mg ml(-1) biomass were obtained at 0.2 and 0.5 mM Cu2+, respectively. Copper accumulation profiles were different: the metal was librated from the intact cells by Candida sp. whereas R. mucilaginosa did not show release from the cells indicating intracellular storage. Specific biosorption of copper by both isolated yeasts showed increase with the initial copper supplied with the medium (up to 11.5 and 8.0 mg Cu g(-1) biomass for Candida sp. and R. mucilaginosa , respectively). However, specific biosorption decreased with time.