Biochemical and microbiological characterization of a thermotolerant bacterial consortium involved in the corrosion of Aluminum Alloy 7075.

Microorganisms can play a significant role in material corrosion, with bacterial biofilms as major participants in microbially influenced corrosion (MIC). The exact mechanisms by which this takes place are poorly understood, resulting in a scarcity of information regarding MIC detection and prevention. In this work, a consortium of moderately thermophilic bacteria isolated from a biofilm growing over aluminum alloy 7075 was characterized. Its effect over the alloy was evaluated on a 40-day period using Electron Microscopy, demonstrating acceleration of corrosion in comparison to the abiotic control. The bacterial consortium was biochemically and microbiologically characterized as an attempt to elucidate factors contributing to corrosion. Molecular analysis revealed that the consortium consisted mainly of members of the Bacillus genus, with lower abundance of other genera such as Thermoanaerobacterium, Anoxybacillus and Paenibacillus. The EPS polysaccharide presented mainly mannose, galactose, rhamnose and ribose. Our observations suggest that the acidification of the culture media resulting from bacterial metabolism acted as the main contributor to corrosion, hinting at an unspecific mechanism. The consortium was not sulfate-reducing, but it was found to produce hydrogen, which could also be a compounding factor for corrosion.

Characterization of the EPS from a thermophilic corrosive consortium.

Biofilm forming microorganisms are known to contribute to the corrosion of metallic materials, as they can attach to surfaces and influence the electrochemical behavior. Extracellular polymeric substances (EPS) produced by these microorganisms play a major role in adhesion and resistance of the biofilm, thus also contributing to corrosion. A better understanding of the composition of EPS could help mitigate the impact of bacterial mediated corrosion. Here, a preliminary characterization of the EPS from a thermophilic consortium isolated from a corroded airplane engine is presented. Analysis revealed five different monosaccharides, with predominance of glucose and manose, but also a significant amount of rhamnose. Glycosyl linkage analysis was also performed. On the lipid fraction, three types of fatty acids were found. The predominant protein found by peptide finger printing was S-Layer protein, related to bacterial adhesion. Morphological characterization of the biofilm forming consortium was carried using confocal and scanning electron microscopy.

Inhibiting Pathogen Surface Adherence by Multilayer Polyelectrolyte Films Functionalized with Glucofuranose Derivatives.

Inhibiting pathogenic bacterial adherence on surfaces is an ongoing challenge to prevent the development of biofilms. Multilayer polyelectrolyte films are feasible antibacterial materials. Here, we have designed new films made of carbohydrate polyelectrolytes to obtain antibacterial coatings that prevent biofilm formation. The polyelectrolyte films were constructed from poly(maleic anhydride- alt-styrene) functionalized with glucofuranose derivatives and quaternized poly(4-vinylpyridine) N-alkyl. These films prevent Pseudomonas aeruginosa and Salmonella Typhimurium, two important bacterial contaminants in clinical environments, from adhering to surfaces. When the film was composed of more than 10 layers, the bacterial population was greatly reduced, while the bacteria remaining on the film were morphologically damaged, as atomic force microscopy revealed. The antibacterial capacity of the polyelectrolyte films was determined by the combination of thickness, wettability, surface energy, and most importantly, the conformation that polyelectrolytes adopt the function of nature of the carbohydrate group. This polyelectrolyte film constitutes the first green approach to preventing pathogenic bacterial surface adherence and proliferation without killing the bacterial pathogen.

Design aspects of compliant, soft layer bearings for an experimental hip prosthesis.

Currently, an artificial hip joint can be expected to last, on average, in excess of 15 years with failure due, in the majority of cases, to late aseptic loosening of the acetabular component. A realistic alternative to the problem of wear in conventional joints is the introduction of bearing surfaces that exhibit low wear and operate in the full fluid-film lubrication regime. Contact analyses and friction tests were performed on compliant layer joints (metal-on-polyurethane) and the design of a prototype ovine arthroplasty model was investigated. When optimized, these components have been shown to achieve full fluid-film lubrication.

Purification and characterization of an iron-nickel hydrogenase from Thermococcus celer.

Thermococcus celer cells contain a single hydrogenase located in the cytoplasm, which has been purified to apparent homogeneity using three chromatographic steps: Q-Sepharose, DEAE-Fast Flow, and Sephacryl S-200. In vitro assays demonstrated that this enzyme was able to catalyze the oxidation as well as the evolution of H2. T. celer hydrogenase had an apparent MW of 155,000+/-30,000 by gel filtration. When analyzed by SDS polyacrylamide gel electrophoresis a single band of 41,000+/-2,000 was detected. Hydrogenase activity was also detected in situ in a SDS polyacrylamide gel followed by an activity staining procedure revealing a single band corresponding to a protein of apparent Mr 84,000+/-3,000. Measurements of iron and acid-labile sulfide in different preparations of T. celer hydrogenase gave values ranging from 24 to 30 g-atoms Fe/mole of protein and 24 to 36 g-atoms of acid-labile sulfide per mole of protein. Nickel is present in 1.9-2.3 atoms per mole of protein. Copper, tungsten, and molybdenum were detected in amounts lower than 0.5 g-atoms per mole of protein. T. celer hydrogenase was inactive at ambient temperature, exhibited a dramatic increase in activity above 70 degrees C, and had an optimal activity above 90 degrees C. This enzyme showed no loss of activity after incubation at 80 degrees C for 28 h, but lost 50% of its initial activity after incubation at 96 degrees C for 20 h. Hydrogenase exhibited a half-life of approximately 25 min in air. However, after treating the air-exposed sample with sodium dithionite, more than 95% of the original activity was recovered. Copper sulfate, magnesium chloride and nitrite were also inactivators of this enzyme.

Direct electrochemical characterization of hyperthermophilic Thermococcus celer metalloenzymes involved in hydrogen production from pyruvate.

The reduction potentials of the metalloproteins pyruvate ferredoxin oxidoreductase (POR), ferredoxin, and hydrogenase isolated from hyperthermophilic Thermococcus celer (Topt = 88 degrees C) were determined as a function of temperature from 10 to 85 degrees C. Square-wave voltammetry experiments were carried out on 15 microL samples directly at an unmodified "edge-polished" pyrolytic graphite electrode using MgCl2 as an electrode promoter. POR exhibited two voltammetric waves with peaks at -280 and -403 mV at room temperature, indicating multiple redox centers, and a single wave at -420 mV at 85 degrees C. These waves displayed different temperature-dependent peak positions and peak heights, indicating that these redox centers have different thermodynamic and kinetic properties. Ferredoxin displayed a single linear temperature-dependent voltammetric wave at -280 mV at room temperature and -327 mV at 85 degrees C. Hydrogenase displayed a single biphasic temperature-dependent voltammetric wave at -197 mV at room temperature and -211 mV at 85 degrees C. Thermodynamic parameters associated with electron transfer, namely standard enthalpies and entropies for the redox centers in the various proteins, are reported.

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.

Omeprazole, a specific gastric secretion inhibitor on oxynticopeptic cells, reduces gizzard erosion in broiler chicks fed with toxic fish meals.

The relation between gizzard erosion-black vomit (GE-BV) and gastric secretion is not completely understood. A pharmacological approach to reduce the presence of GE-BV in chicks due to fish meal in diets is also unknown. In this study the use of omeprazole, a H+/K+ ATPase inhibitor, and fish meals of different biotoxicological characteristics, showed that: 1) Omeprazole decreased total gastric acid content, GE scores and severe GE (SGE) cases, in a dose-dependent manner. This reduction was significant at levels higher than 20 mg omeprazole/Kg body weight (BW)/day (p < 0.01). The addition of 50 mg omeprazole/kg BW/day almost completely prevented the incidence of SGE cases and reduced in 50% GE score in chicks (p < 0.01). 2) A significant reduction in specific mortality, near 90%, was also seen with all toxic fish meals when omeprazole (50 mg/Kg BW/day) was added to experimental diets in comparison to control groups. However, no mortality was observed when omeprazole was added to diets containing non-toxic fish meals. 3) In chicks fed with toxic fish meals, addition of different amounts of omeprazole to diets changed the relative weight of proventriculus (p < 0.01) and gizzard (p < 0.05). Maximum effect was obtained with omeprazole concentration higher than 50 mg/Kg BW/day. 4) Omeprazole did not change feed intake in chicks fed with toxic fish meal. However, in some fish meal a reduction on weight gain was observed with the addition of omeprazole.

A variable-temperature direct electrochemical study of metalloproteins from hyperthermophilic microorganisms involved in hydrogen production from pyruvate.

The hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaeon Pyrococcus furiosus grow optimally at 80 and 100 degrees C, respectively, by the fermentation of carbohydrates to organic acids, CO2, and H2. Pyruvate is a major source of reductant for H2 production during fermentation, and pyruvate ferredoxin oxidoreductase (POR), a 4Fe-type ferredoxin, and hydrogenase have been previously purified from both species. P. furiosus utilizes a copper-iron-containing POR and a nickel-iron-containing hydrogenase, whereas the POR of T. maritima lacks copper and its hydrogenase lacks nickel. For all four enzymes and for the two ferredoxins, we have determined their reduction potentials (E degrees') and, where possible, thermodynamic parameters associated with electron transfer (delta S degrees and delta H degrees), using differential pulse voltammetry at temperatures ranging from 25 to 95 degrees C. At ambient temperature, the E degrees' values for all six proteins were comparable and spanned less than 50 mV, but their temperature dependence varied dramatically, even between analogous proteins, such that in the physiological-relevant temperature range the E degrees' values became widely separated. In most cases, transition points were observed in E degrees'/temperature profiles, and these generally corresponded with significant increases in catalytic activity, but occurred at lower temperatures in T. maritima than in P. furiosus. The two ferredoxins (and also P. furiosus rubredoxin) had much more negative entropy terms than were calculated for POR and hydrogenase, and these values were also more negative than those previously reported for mesophilic redox proteins. The reduction potentials measured at high temperatures and likely efficiencies of electron transfer between the various proteins were consistent with in vitro activity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

The hyperthermophilic glycolytic enzyme enolase in the archaeon, Pyrococcus furiosus: comparison with mesophilic enolases.

High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C). In this organism, the enzyme probably functions in a sugar fermentation pathway. The enzyme was purified to homogeneity. It had a temperature optimum of > 90 degrees C and a pH optimum of 8.1. The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min. In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C. Both the P. furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P. furiosus enolase was equally active in the presence of Mn2+. Both enzymes were competitively inhibited by citrate. P. furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000. A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P. furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals. Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids.

Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster.

Properties of a thermostable 4Fe-ferredoxin from the hyperthermophilic bacterium Thermotoga maritima.

A ferredoxin has been purified from one of the most ancient and most thermophilic bacteria known, Thermotoga maritima, which grows up to 90 degrees C. The reduced protein (M(r) approx. 6300) contains a single S = 1/2 [4Fe-4S]1+ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95 degrees C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria.

Pyruvate ferredoxin oxidoreductases of the hyperthermophilic archaeon, Pyrococcus furiosus, and the hyperthermophilic bacterium, Thermotoga maritima, have different catalytic mechanisms.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from two hyperthermophiles, the archaeon Pyrococcus furiosus (Pf, Topt = 100 degrees C) and the bacterium Thermotoga maritima (Tm, Topt = 80 degrees C). Each catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 near the optimal growth temperature of the organism and are virtually inactive at 25 degrees C. Both PORs contain a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] ferredoxin-type clusters. We have now shown, using EPR spectroscopy and metal analyses, that PfPOR also contains an unusual copper center that is not present in Tm POR. In addition, distinct catalytic intermediates were generated in both enzymes by the addition, separately and in combination, of the substrates pyruvate and CoASH, and these were examined by EPR spectroscopy. The addition of pyruvate to oxidized Pf POR produced an isotropic signal centered at g = 2.01, which was measurably broader in the presence of pyruvate-2(13)C. This signal, which was assigned to a (hydroxyethyl)thiamine pyrophosphate radical intermediate, was not observed in Tm POR under the same experimental conditions. Incubation of the oxidized enzymes with CoASH resulted in the partial reduction of the copper site in Pf POR and the partial reduction of a novel iron-sulfur center in Tm POR, which was not seen in the dithionite-reduced enzyme. The addition of both pyruvate and CoASH to the PORs in their oxidized states resulted in the reduction of the same iron-sulfur centers that are reduced by sodium dithionite.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima.

The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90 degrees C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113,000 and comprises four dissimilar subunits with M(r) values of approximately 43,000, 34,000, 23,000, and 13,000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent Km values at 80 degrees C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM, respectively, and the corresponding apparent Vm values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent Km and Vm values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 microM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90 degrees C, and the time for a 50% loss of activity (t50%) at 80 degrees C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25 degrees C of 70 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus grows optimally at 100 degrees C by carbohydrate fermentation. It is thought to contain a novel tungsten-dependent, NAD(P)-independent glycolytic pathway in which one of the oxidation steps is catalyzed by a tungsten-containing aldehyde ferredoxin oxidoreductase. The enzyme that catalyzes the terminal oxidation step, pyruvate ferredoxin oxidoreductase (POR), has now been purified. POR has a molecular mass of 100 kDa and is comprised of three subunits (45, 31 and 24 kDa). It lacks tungsten but contains thiamine pyrophosphate (TPP) and two ferredoxin-type [4Fe-4S] clusters per molecule which, by EPR spectroscopy, can be differentiated by their relaxation properties. The enzyme requires CoASH but not TPP for pyruvate oxidation activity and will not use 2-oxoglutarate, phenyl pyruvate or indole pyruvate as substrates. POR is virtually inactive at 25 degrees C and shows a temperature optimum for pyruvate oxidation above 90 degrees C. The apparent Km values for pyruvate, CoASH and P. furiosus ferredoxin at 80 degrees C are 460, 100 and 70 microM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent Ki = 7 microM). The half-life of activity (t50%) in air at 25 degrees C was 15 min and the t50% value at 80 degrees C (under anaerobic conditions) was 23 min. Based on molecular comparisons with PORs from mesophilic organisms, it is proposed that P. furiosus POR may represent an ancestral form of a pyruvate-oxidizing enzyme.

Soft layered prostheses for arthritic hip joints: a study of materials degradation.

A great deal of interest is being shown in complaint bearing surfaces for artificial joints. These produce very low friction because of the fluid-film lubrication that they exhibit, and therefore should produce lower wear than current prosthetic materials as the two surfaces of the joint are completely separated by a film of synovial fluid. However, one problem with soft elastic materials in vivo is that the elastomers may degrade with time. Specimens of four polyurethanes were kept in Ringer's solution at 37 degrees C for about 4 months to investigate changes in mechanical properties and the mode of lubrication with time of immersion. The materials tested were a polyether-urethane (E57), a polyester-urethane (E58), an aromatic polyether-urethane (P1) and an aliphatic polyether-urethane (A1). Samples of E57 showed a decrease in hardness and elastic modulus of 21% and 28% respectively. This was much better than E58 which showed decreases of 32% and 42% respectively. Better results were achieved with A1 (hardness change 2%) and P1 (decrease in hardness of 14% and modulus 17%). When tested in a hip-function simulator, A1 showed virtually no change in its very low coefficient of friction (0.004), P1 showed an increase of 60%, while E57 and and E58 both showed a doubling in friction over the course of the study. The modes of in vitro degradation were considered and surface effects determined to be most damaging. Currently the best candidate for a compliant material in hip replacement is the aliphatic polyether-urethane which maintains fluid-film lubrication.