Improved Methodology for Bioremoval of Black Crusts on Historical Stone Artworks by Use of Sulfate-Reducing Bacteria

An improved methodology to remove black crusts from stone by using Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, a sulfate-reducing bacterium, is presented. The strain removed 98% of the sulfates of the crust in a 45-h treatment. Precipitation of black iron sulfide was avoided using filtration of a medium devoid of iron. Among three cell carriers, Carbogel proved to be superior to both sepiolite and Hydrobiogel-97, as it allowed an easy application of the bacteria, kept the system in a state where microbial activity was maintained, and allowed easy removal of the cells after the treatment.     

Influence of heat processing on the bioaccessibility of zinc and iron from cereals and pulses consumed in India.

Influence of heat processing on the bioaccessibility of zinc and iron from food grains consumed in India was evaluated. Cereals - rice (Oryza sativa), finger millet (Eleusine coracana), sorghum (Sorghum vulgare), wheat (Triticum aestivum), and maize (Zea mays), and pulses - chickpea (Cicer arietinum) - whole and decorticated, green gram (Phaseolus aureus) - whole and decorticated, decorticated black gram (Phaseolus mungo), decorticated red gram (Cajanus cajan), cowpea (Vigna catjang), and French bean (Phaseolus vulgaris) were examined for zinc and iron bioaccessibility by employing an in vitro dialysability procedure. Both pressure-cooking and microwave heating were tested for their influence on mineral bioaccessibility. Zinc bioaccessibility from food grains was considerably reduced upon pressure-cooking, especially in pulses. Among cereals, pressure-cooking decreased zinc bioaccessibility by 63% and 57% in finger millet and rice, respectively. All the pressure-cooked cereals showed similar percent zinc bioaccessibility with the exception of finger millet. Bioaccessibility of zinc from pulses was generally lower as a result of pressure-cooking or microwave heating. The decrease in bioaccessibility of zinc caused by microwave heating ranged from 11.4% in chickpea (whole) to 63% in cowpea. Decrease in zinc bioaccessibility was 48% in pressure-cooked whole chickpea, 45% and 55% in pressure-cooked or microwave-heated whole green gram, 32% and 22% in pressure-cooked or microwave-heated decorticated green gram, and 45% in microwave-heated black gram. Iron bioaccessibility, on the other hand, was significantly enhanced generally from all the food grains studied upon heat treatment. Thus, heat treatment of grains produced contrasting effect on zinc and iron bioaccessibility.     

Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments

This study compares two cleaning methods, one involving an ammonium carbonate-EDTA mixture and the other involving the sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, for the removal of black crust (containing gypsum) on marble of the Milan Cathedral (Italy). In contrast to the chemical cleaning method, the biological procedure resulted in more homogeneous removal of the surface deposits and preserved the patina noble under the black crust. Whereas both of the treatments converted gypsum to calcite, allowing consolidation, the chemical treatment also formed undesirable sodium sulfate.     

The influence of incubation temperature and pH on the antimicrobial properties of hen egg albumen

T ranter H.S. & B oard , R.G. 1984. The influence of incubation temperature and pH on the antimicrobial properties of hen egg albumen. Journal of Applied Bacteriology 56 , 53–6.
Gram positive bacteria, including lysozyme-resistant strains, and yeasts were killed in hen egg albumen with or without iron at 30 of 39.5.dGC. The albumen was more toxic at 39.5.dGC than at 30C for Gram negative bacteria. With the exceptions of Pseudomonas fluorescens, Acinetobacter sp. and Proteus vulgaris , iron caused the growth of Gram negative bacteria or protected them from being killed in hen albumen at 39.5.dGC. At this temperature, however, maximal growth of and glucose utilization by Escherichia coli C20 only occurred in albumen supplemented with growth factors, trace metals, additional nitrogen and sufficient iron to quench ovotransferrin. The bactericidal properties of albumen could be negated by changing its pH from 90 or above to 7.5 or below. At 39.5C, enterochelin allowed growth of E. coli in albumen at pH 7.9, but not at 9.4, whereas iron allowed growth at both pH values.     

Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. vulgaris chelatases complement an E. coli cysG mutant strain showing that, in vivo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiK(C), likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiK(P), is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. vulgaris to produce vitamin B 12 was also demonstrated in this work.     

Displacement of iron by zinc at the diiron site of Desulfovibrio vulgaris rubrerythrin: X-ray crystal structure and anomalous scattering analysis

X-ray crystal structures of recombinant Desulfovibrio (D.) vulgaris rubrerythrin (Rbr) have shown a diiron site, whereas the crystal structure of Rbr "as-isolated" from D. vulgaris was reported to contain a mixed Zn,Fe binuclear site. To investigate the possibility that zinc had displaced iron during isolation or crystallization of the "as-isolated" D. vulgaris Rbr, the X-ray crystal structure of recombinant D. vulgaris all-iron Rbr that had been incubated with excess zinc sulfate prior to crystallization, yielding a protein labeled Zn,FeRbr, was solved. Analysis of the anomalous scattering data obtained at two different wavelengths showed that zinc had displaced a significant proportion of iron from both iron centers of the diiron site, and that no iron had been displaced from the [Fe(SCys)(4)] site. UV-visible absorption spectra of the redissolved Zn,FeRbr crystals showed 30-40% retention of oxo-bridged diferric sites, and the redissolved crystals had 37% of the peroxidase specific activity of the starting all-iron Rbr, which, together with the crystallographic results, indicate a predominant mixture of Fe1,Fe2 and Zn1,Zn2 sites. The structure of the Zn(Fe)1,Fe(Zn)2 binuclear site in the Zn,FeRbr crystals was very similar to that of the Zn,Fe binuclear site reported for the "as-isolated" D. vulgaris Rbr, including tetrahedral four-coordination at the Zn(Fe)1 site. The diiron sites in the recombinant Zn,FeRbr crystals were likely at least partially reduced during synchrotron irradiation. Our results suggest that the mixed-metal binuclear site reported for the "as-isolated" D. vulgaris Rbr could be due to displacement of iron from a native diiron site by adventitious zinc during isolation and/or crystallization, and that reduced diiron and dizinc sites can adopt very similar structures in Rbr.     

Demonstration of Elastic Tissue and Iron or Elastic Tissue and Calcium Simultaneously

For the concomitant demonstration of iron and elastic tissue Perls' test solution was used, followed by Verhoeff's stain or Gomori's aldehyde fuchsin. When Perls' and Verhoeff's stain were used in sequence, the iron deposits were greenish blue and the elastic lamellae were black. When Perls' test solution was combined with aldehyde fuchsin the iron deposits were blue and elastic tissue purple. Calcium salts and elastic tissue were demonstrated concomitantly by using von Kossa's method followed by Gomori's aldehyde fuchsin. With such combined staining, the calcium salts appeared brownish black and elastic tissue purple. With these procedures, it was possible to see the exact relationship of calcium and iron deposits to the elastic tissue.     

Overproduction of prismane protein in Desulfovibrio vulgaris (Hildenborough): evidence for a second S = 1/2-spin system in the one-electron reduced state.

The gene encoding the prismane protein from Desulfovibrio vulgaris (Hildenborough) was inserted into broad-host-range vector pSUP104. The recombinant plasmid, pJSP104, was transferred to D. vulgaris by conjugal plasmid transfer. In the transconjugant D. vulgaris cells the prismane protein was 25-fold overproduced. The overproduced prismane protein was characterized by molecular mass, isoelectric point, iron content and spectroscopical properties. Both the iron content and the ultraviolet/visible spectrum are identical to the wild-type protein indicating that iron incorporation in the overproduced protein is complete. EPR spectra of the dithionite-reduced form of the overproduced protein indicated that the Fe-S cluster might occur in a similar structure as found in inorganic model compounds containing a [6Fe-6S] prismane core. The as-isolated overproduced protein showed the presence of a second S = 1/2 spin system that was also detected in the corresponding prismane protein from D. desulfuricans (ATCC 27774), but not in the protein from wild-type D. vulgaris. This additional signal was irreversibly transformed to the 'wild-type' high-spin and low-spin systems upon two reduction/re-oxidation cycles. It is shown that the EPR spectroscopy of the overproduced prismane protein is very similar to that of the D. desulfuricans enzyme and, with the exception of the second S = 1/2 spin system, to that of the prismane protein from wild-type D. vulgaris. Contrary to claims for the D. desulfuricans protein, it is shown here that all data can be fully explained assuming a single [6Fe-6S] cluster, that might be titrated into four different redox states and occurs in up to three different spin systems in the one-electron reduced state.     

Rapid differentiation between Octopus vulgaris Cuvier (1797) and Octopus mimus Gould (1852), using randomly amplified polymorphic DNA

Seventeen specimens of Octopus vulgaris and Octopus mimus were investigated using randomly amplified polymorphic DNA (RAPD). Clearly differentiable RAPD fingerprints allowed a fast and reliable genotypic discrimination of these species. Thus, molecular genetic evidence from total DNA complements recent results of a comparative analysis of mitochondrial sequence data supporting the taxonomic separation of Octopus mimus and Octopus vulgaris .     

The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II

The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.     

Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.     

Behaviour of iron chelates in calcareous soils

Summary Fe-EDTA and Fe-HEEDTA, labelled with Fe⁵⁹, were applied at rates of 10 and 100 ppm iron to 50-g samples of dry soil, which were then stored for periods ranging from one to fifteen days before extracting with water.The analyses of the extracts lead to the following general conclusions.1. The decrease in soluble iron could be attributed to both sorption of chelate anions by the clay and to replacement of iron by calcium in the chelate molecule.2. The amount of each chelate sorbed changed little with time and was proportional to the quantity applied. More HEEDTA was sorbed than EDTA.3. The initial decrease in the concentration of soluble iron was rapid for both chelates, but was the greater for Fe-HEEDTA.4. The precipitation of iron from the soluble Fe-HEEDTA was slower than from Fe-EDTA, so that fifteen days after treatment more soluble iron was obtained from the Fe-HEEDTA treatments.5. Marked increases in the recoveries of Fe-EDTA were made when the treated soils were sealed within small containers, compared with those allowed free access to the air. Comparable treatments using Fe-HEEDTA had a much smaller effect.6. Isotopic exchange of Fe⁵⁹ with natural soil iron was greater in the treatments with 10 ppm chelated iron than the 100 ppm level. Fe-HEEDTA was subject to more exchange than Fe-EDTA at both levels.These results are discussed in relation to the treatment of soils with iron chelates for the control of lime-induced chlorosis, and to the importance of isotopic exchange when using chelates labelled with radioactive iron in soil.     

Demonstration of Elastic Tissue and Iron or Elastic Tissue and Calcium Simultaneously

For the concomitant demonstration of iron and elastic tissue Perls' test solution was used, followed by Verhoeff's stain or Gomori's aldehyde fuchsin. When Perls' and Verhoeff's stain were used in sequence, the iron deposits were greenish blue and the elastic lamellae were black. When Perls' test solution was combined with aldehyde fuchsin the iron deposits were blue and elastic tissue purple. Calcium salts and elastic tissue were demonstrated concomitantly by using von Kossa's method followed by Gomori's aldehyde fuchsin. With such combined staining, the calcium salts appeared brownish black and elastic tissue purple. With these procedures, it was possible to see the exact relationship of calcium and iron deposits to the elastic tissue.     

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.     

Precipitation of iron in windowpane oyster shells by marine shell‐boring Cyanobacteria

Shells of windowpane oyster [Placuna placenta (L)] in the intertidal zone of the Zuari estuary, Goa, were often found to be black in color. Microscopical observation of partially decalcified shells showed the presence of cyanobacterial filaments encrusted with black precipitate. Microchemical test (Prussian blue reaction) and wavelength dispersive x‐ray analysis confirmed this precipitate to be of iron. Mineralogical studies of this black precipitate, using x‐ray diffraction and scanning electron microscopy, suggested the presence of iron as iron oxides. The cyanobacteria from such black shells were cultured in enriched seawater medium. In this medium also they precipitated iron as confirmed by Prussian blue reaction. They were identified as Plectonema terebrans Born et Flah and Phormidium sp.     

Iron Deficiency Induced by Chrysobactin in Saintpaulia Leaves Inoculated with Erwinia chrysanthemi

In this communication, we examine the fate of iron during soft rot pathogenesis caused by Erwinia chrysanthemi on its host, Saintpaulia ionantha. The spread of soft rot caused by this enterobacterium was previously shown to depend on a functional genetic locus encoding a high-affinity iron assimilation system involving the catechol-type siderophore chrysobactin. Leaf intercellular fluid from healthy plants was analyzed with regard to the iron content and its availability for bacterial growth. It was compared to the fluid from diseased plants for the presence of strong iron ligands, using a new approach based on the iron-binding property of an ion-exchange resin. Further characterization allowed the identification of chrysobactin in diseased tissues, thus providing the first evidence for the external release of a microbial siderophore during pathogenesis. Competition for nutritional iron was also studied through a plant-bacterial cell system: iron incorporated into plant ferritin appeared to be considerably reduced in bacteria-treated suspension soybean cells. The same effect was visualized during treatment of soybean cells with axenic leaf intercellular fluid from E. chrysanthemi-inoculated saintpaulia leaves or with chrysobactin.     

Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L(-1) FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed.     

Epigallocatechin gallate (EGCG) (TEAVIGO) does not impair nonhaem-iron absorption in man.

A number of studies have shown that tea catechins can inhibit intestinal iron absorption, mostly iron in the nonhaem form. This randomized, double-blind, placebo-controlled, 3-periods cross-over study examined the degree of inhibition of nonhaem iron absorption by pure crystalline epigallocatechin gallate (EGCG). The study was designed to show the maximum inhibitory action of EGCG by selecting 30 healthy women with low iron stores. Treatments were 150 mg, 300 mg EGCG and placebo each for 8 consecutive study days with a wash-out period of 14 days between treatments. Iron incorporation was assessed by supplying 57Fe orally and 58Fe intravenously. Differences in fractional nonhaem iron absorption between the treatments were evaluated by using two-sided ANOVA. Results showed a relative nonhaem iron absorption reduction of 14% with 150mg EGCG and 27% for 300mg EGCG treatment compared to placebo. Differences were statistically significant (p < or = 0.05) between the placebo and the 300mg EGCG treatments and between the 150 and 300 mg EGCG treatments. The inverse relation between EGCG dose and fractional nonhaem iron absorption was linear (p = 0.0002). In this study the magnitude of the inhibitory action of EGCG on nonhaem iron absorption was found to be much lower than that reported in the literature for black tea and similar compounds. The doses of EGCG in supplements, which will be lower than those used in this study, are not expected to have any health relevant effects on iron absorption in subjects with normal iron stores.