Mauricio Diaz

Biofilm formation, communication and interactions of leaching bacteria during colonization of pyrite and sulfur surfaces

Bioleaching of metal sulfides is an interfacial process where biofilm formation is considered to be important in the initial steps of this process. Among the factors regulating biofilm formation, molecular cell-to-cell communication such as quorum sensing is involved. A functional LuxIR-type I quorum sensing system is present in Acidithiobacillus ferrooxidans. However, cell-to-cell communication among different species of acidophilic mineral-oxidizing bacteria has not been studied in detail. These aspects were the scope of this study with emphasis on the effects exerted by the external addition of mixtures of synthetic N-acyl-homoserine-lactones on pure and binary cultures. Results revealed that some mixtures had inhibitory effects on pyrite leaching. Some of them correlated with changes in biofilm formation patterns on pyrite coupons. We also provide evidence that A. thiooxidans and Acidiferrobacter spp. produce N-acyl-homoserine-lactones. In addition, the observation that A. thiooxidans cells attached more readily to pyrite pre-colonized by living iron-oxidizing acidophiles than to heat-inactivated or biofilm-free pyrite grains suggests that other interactions also occur. Our experiments show that pre-cultivation conditions influence A. ferrooxidans attachment to pre-colonized pyrite surfaces. The understanding of cell-to-cell communication may consequently be used to develop attempts to influence biomining/bioremediation processes.

Effect of Exogenous Galactose on EPS Production during Bioleaching of Pyrite by Acidithiobacillus ferrooxidans

Extracellular polymeric substances (EPS) play an important role in the attachment of bacteria to sulphide minerals, biofilm formation and efficiency of the bioleaching process. Previous studies have suggested a potential connection between galactose and EPS formation. In this context, the influence of exogenous galactose on EPS formation during the bioleaching of pyrite was studied. In order to fully adapt the microorganism to bioleaching conditions it was performed a total of five consecutive sub cultures, one every fifteen days, taking for each one inocula from previous culture in shake flasks with 200 ml of fermentation medium at 30°C, 200 rpm, 40 gL-1 mineral and an initial pH of 1,8. Assays were performed in a medium supplemented with exogenous galactose (0.25% w/v) and without exogenous galactose (control), both with an initial concentration of ferric sulphate in the first three sub cultures of 5 gL-1, decreasing in the last two sub cultures to 2.5 gL-1. Samples of three cultures in both conditions were analyzed using confocal laser scanning microscopy (CLSM) labelling the cells with propidium iodide and EPS carbohydrates with Wheat Germ Agglutinin (WGA). Samples obtained on the last day of the fifth culture showed that the EPS layer on the particle surface was 5.00 μm3/μm2 in the case of the control condition and 6.10 μm3/μm2 when bioleaching was carried out in the presence of exogenous galactose. Also it was observed that in the fifth sub culture the volumetric productivity of total iron in the control experiment was 0.0065 gL-1.h-1 compared with 0.0076 gL-1.h-1 obtained in presence of galactose. The results reveal that the presence of galactose in the bioleaching solution stimulates EPSs formation and apparently also favour the pyrite bioleaching process.

Molecular Regulatory Network Involved in Biofilm Structure Development by Acidithiobacillus thiooxidans Includes Pel Exopolysaccharide Machinery

The Acidithiobacillus genus plays a relevant role in bioleaching. The molecular understanding of biofilm formation has been pointed out to design biological strategies to improve the efficiency of this industrial process and to prevent environmental damages caused by acid mine/rock drainages. In Acidithiobacillus spp., the molecular mechanisms involved in biofilm formation are currently emerging. The second messenger cyclic diguanylate (c-di-GMP) appears as a key player for biofilm formation by Acidithiobacillus sp. Here, results obtained from genomic analysis to characterize c-di-GMP pathway in At. thiooxidans are reported. Intracellular levels of c-di-GMP have been previously measured and data indicated that they are higher in adhered cells than planktonic ones. During the course of characterization of c-di-GMP effectors, a complete pel-like gene cluster has been identified in At. thiooxidans. By using total RNA obtained from planktonic and adhered sulfur-grown cells, transcriptomic analysis revealed that pelA belonging to the pel-like gene cluster is overexpressed in adhered cells. Moreover, genetic experiments were performed to compare wild type and null-mutant strains of At. thiooxidans for assessing the role of Pel exopolysaccharide. All together, the results obtained suggest a specific role for Pel machinery in the attachment to solid energy substrates by At. thiooxidans.

Galactose as Inducer of the Production of Extracellular Polymeric Substances by Acidithiobacillus ferrooxidans

The presence of extracellular polymeric substances (EPS) is important in the building of biofilms on mineral surfaces, increasing the bioleaching activity, as well as protecting the cells from adverse environmental conditions. The objective of this work was to study the effect of galactose in EPS production by Acidithiobacillus ferrooxidans. The experiences were performed in shake flask of 250 mL at 30 °C, 200 rpm and at an initial pH of 1.8. In order to establish the natural tolerance of the strain, its growth behaviour was evaluated at high ferric iron concentrations by adding consecutively the equivalent of 9 g/L of ferrous iron each time it was depleted in the broth. Cell growth stopped once ferric iron concentration increased up to 38 g/L. In order to determine the optimal conditions for EPS production, experiments were run in a chemostat of 0.5 L, operated at a constant dilution rate of 0.03 h-1. Different steady states were obtained varying feeding concentrations of galactose (0.15%; 0.25% and 0.35%) and carbon dioxide (180 ppm and 360 ppm). , Cells grown in the chemostat at optimum operation conditions were used as inoculum to determine oxidative capacity of the microorganisms overproducing EPS. The EPS was quantified using confocal laser scanning microscopy (CLSM), labelling the cells with propidium iodide and EPS carbohydrates with wheat germ agglutinin (WGA). The higher volume production of EPS was observed in cells grown using 360 ppm of CO2 and 0.35% of galactose. Also it was observed a size increment of cells, compared to cells grown in culture medium having 9 g/L of ferrous iron where presence of EPS was no detected. The results revealed that EPS overproducing A. ferrooxidans showed a tolerance to ferric iron concentration almost 9.5 g/L higher than the natural tolerance of cells grown in absence of galactose. Presence of galactose in culture medium stimulated the EPS production.

Functional Analysis of c-di-GMP Pathway in Biomining Bacteria Acidithiobacillus thiooxidans

Bioleaching is the solubilization of metals catalyzed by acidophilic microorganisms. Among these, the Gram negative γ-proteobacteria from the genus Acidithiobacillus play a relevant role. Biofilm formation has an impact on bioleaching performance. In Acidithiobacillus sp. the molecular mechanisms involved in biofilm formation are still unknown. However, in many Gram-negative and some Gram-positive bacteria, the intracellular levels of the second messenger cyclic diguanilic acid (c-di-GMP) control the regulation of biofilm formation. These levels depend on the balance between the enzymatic activities of diguanylate cyclases (synthesis) and phosphodiesterases (degradation). Previously, functional c-di-GMP pathways have been characterized in At. ferrooxidans and At. caldus. Here, our main goal was to characterize the presence of a functional c-di-GMP pathway in At. thiooxidans. By bioinformatic analysis, several putative-ORFs encoding DGCs, PDEs and effector proteins have been identified in the genome sequence of At. thiooxidans ATCC 19377. RT-PCR experiments revealed that most of these ORFs are transcribed. In addition, the analysis of nucleotide-enriched fraction extracted from At. thiooxidans cells showed that this bacterium is able to produce c-di-GMP. These results strongly indicate that At. thiooxidans has a functional c-di-GMP pathway.

EPS Production during Adaptation of Acidithiobacillus ferrooxidans to High Ferric Ion Concentration

The ability of Acidithiobacillus ferrooxidans to get its energy from the oxidation of ferrous iron and the inhibitory effect of high ferric iron concentrations on its growth behaviour has been extensively studied. Furthermore it is known that A. ferrooxidans exudes organic substances called extracellular polymeric substances (EPS), which could play a role in its protection against adverse environmental conditions. In this context, the aim of this work was to study the production of EPS during adaptation of A. ferrooxidans to high ferric ion concentrations. The experiments were performed in shake flasks of 250 mL at 30 °C, 200 rpm and at an initial pH of 1.8. In order to establish the natural tolerance of the strain, its growth behaviour was evaluated at high ferric iron concentrations by adding consecutively the equivalent of 9 g/L of ferrous iron each time it was depleted in the broth. Cell growth stopped once ferric iron concentration increased up to 38 g/L. The adaptation consisted in eight sub-cultures run in parallel at initial concentrations of ferrous iron of 18, 27 and 36 g/L. The EPS was quantified as micro volumes using confocal laser scanning microscopy (CLSM), labelling the cells with propidium iodide and EPS carbohydrates with wheat germ agglutinin (WGA). During the adaptation procedure it was observed an increase in the ferric ion volumetric productivity of subcultures run with 27 and 36 g/L, as a result of cell adaptation. The amount of EPS exuded by cells was higher along with those experimental conditions having higher ferric iron concentrations. It was not detected EPS on cells grown on 9 g/L of ferrous iron. This study found that the adapted strain showed higher production of EPS at high ferric ion concentrations and higher ferric ion tolerance than non-adapted ones.

Biofilm Formation by the Acidophile Bacterium Acidithiobacillus thiooxidans Involves c-di-GMP Pathway and Pel exopolysaccharide

Acidophile bacteria belonging to the Acidithiobacillus genus are pivotal players for the bioleaching of metallic values such as copper. Cell adherence to ores and biofilm formation, mediated by the production of extracellular polymeric substances, strongly favors bioleaching activity. In recent years, the second messenger cyclic diguanylate (c-di-GMP) has emerged as a central regulator for biofilm formation in bacteria. C-di-GMP pathways have been reported in different Acidithiobacillus species; however, c-di-GMP effectors and signal transduction networks are still largely uncharacterized in these extremophile species. Here we investigated Pel exopolysaccharide and its role in biofilm formation by sulfur-oxidizing species Acidithiobacillus thiooxidans. We identified 39 open reading frames (ORFs) encoding proteins involved in c-di-GMP metabolism and signal transduction, including the c-di-GMP effector protein PelD, a structural component of the biosynthesis apparatus for Pel exopolysaccharide production. We found that intracellular c-di-GMP concentrations and transcription levels of pel genes were higher in At. thiooxidans biofilm cells compared to planktonic ones. By developing an At. thiooxidans ΔpelD null-mutant strain we revealed that Pel exopolysaccharide is involved in biofilm structure and development. Further studies are still necessary to understand how Pel biosynthesis is regulated in Acidithiobacillus species, nevertheless these results represent the first characterization of a c-di-GMP effector protein involved in biofilm formation by acidophile species.

Impacto da hipotensão e hipoperfusão global sobre o delírio pós‐operatório: um estudo piloto com idosos submetidos à cirurgia aberta do cólon

BACKGROUND Post-operative delirium is a serious complication in patients undergoing major abdominal surgery. It remains unclear whether peri-operative hemodynamic and perfusion variables affect the risk for postoperative delirium. The objective of this pilot study was to evaluate the association between perfusion and hemodynamics peri-operative with the appearance of post-operative delirium. METHODS Prospective cohort study of adults 60 years or older undergoing elective open colon surgery. Multimodal hemodynamic and perfusion variables were monitored, including central venous oxygenation (ScvO2), lactate levels, and non-invasive cerebral oxygenation (rSO2), according to a standard anesthesia protocol. Fishers exact test or Students t-test were used to compare patients who developed post-operative delirium with those who did not (p<0.05). RESULTS We studied 28 patients, age 73±7 years, 60.7% female. Two patients developed post-operative delirium (7.1%). These two patients had fewer years of education than those without delirium (p=0.031). None of the peri-operative blood pressure variables were associated with incidence of post-operative delirium. In terms of perfusion parameters, postoperative ScvO2 was lower in the delirium than the non-delirium group, without reaching statistical significance (65±10% vs. 74±5%; p=0.08), but the delta-ScvO2 (the difference between means post-operative and intra-operative) was associated with post-operative delirium (p=0.043). Post-operative lactate and rSO2 variables were not associated with delirium. CONCLUSIONS Our pilot study suggests an association between delta ScvO2 and post-operative delirium, and a tendency to lower post-operative ScvO2 in patients who developed delirium. Further studies are necessary to elucidate this association.