Modulation of chromium(VI) toxicity by organic and inorganic sulfur species in yeasts from industrial wastes

Two chromium(VI) resistant yeast strains (Candida sp. and Rhodosporidium sp.) were isolated from industrial wastes. Four different yeasts, three from the Industrial Yeast Collection and one of pharmaceutical origin, were also studied in relation to chromate toxicity and its alleviation by sulfur species. The growth of yeasts from industrial wastes was inhibited by 50% by high concentrations of Cr(VI): Candida sp. by 4 mM Cr(VI) and Rhodosporidium sp. by 10 mM Cr(VI) in Sabouraud Broth medium. The other Cr(VI)-sensitive yeasts were inhibited by 0.1 mM Cr(VI). The general mechanism of chromium resistance in Candida sp. and Rhodosporidium sp. was due to reduced uptake of chromium, but not to biological reduction from Cr(VI) to Cr(III). In Cr(VI)-sensitive yeasts, chromium was accumulated as much as 10-fold, as in Saccharomyces cerevisiae. Cr(VI) toxicity in Candida sp. was modulated from Cr(VI)-resistance to Cr(VI)-hypersensitivity depending on the addition of methionine, cysteine, sulfate and djenkolic acid. If Candida sp. was grown in the presence of S-amino acids, especially methionine, it was more resistant than if the sulfur source was sulfate. When sulfate transport was enhanced by addition of djenkolic acid, Candida sp. became hypersensitive. Rhosporidium sp. was always resistant to Cr(VI) because sulfate transport was inefficient and it assimilated sulfur as S-amino acids. Cr(VI)-sensitive yeasts required larger amounts of S-amino acids, especially methionine, to tolerate Cr(VI) toxicity. Cysteine was toxic for C.famata 6016 above 50 microM.     

Textile-dye polluted waters as a source for selecting chromate-reducing yeasts through Cr(VI)-enriched microcosms

Chromate-resistant microorganisms able to reduce toxic Cr(VI) into insoluble Cr(III) are seen as promising candidates for alleviating Cr(VI)-contamination. In this study, chromate-reducing yeasts could be isolated from a textile-dye effluent and associated biofilm by using microcosm methodology with periodical 1 mM Cr(VI)-pulses. Viable cell count seemed to reveal a progressive tolerance increase to Cr(VI). However, fungal colony numbers decreased after 108 h of cultivation most likely as a consequence of the accumulated toxic effects of metal during enrichment. From 49 different Cr(VI)-tolerant fungal morphotypes isolated under selective conditions, 12 yeasts showed resistance up to 50 mM and 6 filamentous fungi up to 2 mM. These highly tolerant yeasts could be subsequently grouped into 8 OTUs (Operational Taxonomic Units) according to the ITS1-NL4 RFLP (Restriction Fragment Length Polymorphism) analysis. From them, microsatellite amplification, sequencing and Cr(VI)-removal ability allowed to select two representative isolates. A polyphasic approach including morphological, physiological/biochemical characterization and molecular taxonomy analysis allowed to identify these isolates as Cyberlindnera jadinii M9 (previously Pichia jadinii) and Wickerhamomyces anomalus M10 (previously Pichia anomala). Cy. jadinii M9 and W. anomalus M10 were grown in YNB' medium plus 1 mM Cr(VI) at 25 °C and pH 5.0, causing complete chromium removal before reaching 48 h of cultivation. Flame Atomic Absorption Spectroscopy (FAAS) assays suggested that Cr(VI) withdrawal was coupled to Cr(III) appearance. Electron microscopy studies indicated absence of precipitates on the cell wall region or microprecipitates into the cellular cytoplasm. These complementary results revealed that biospeciation to Cr(III) would the main detoxification mechanism in selected chromate-resistant yeasts, which could be thus envisaged as promising tools for future biological treatment of Cr(VI) pollution.     

The effects of Fe(II) and Fe(III) concentration and initial pH on microbial leaching of low-grade sphalerite ore in a column reactor

In this study the effects of initial concentration of Fe(II) and Fe(III) ions as well as initial pH on the bioleaching of a low-grade sphalerite ore in a leaching column over a period of 120 days with and without bacteria were investigated. Four different modifications of medium were used as column feed solutions to investigate the effects of initial concentration of Fe(II) and Fe(III) ions on zinc extraction. The experiments were carried out using a bench-scale, column leaching reactor, which was inoculated with mesophilic iron oxidizing bacteria, Acidithiobacillus ferrooxidans, initially isolated from the Sarcheshmeh chalcopyrite concentrate (Kerman, Iran). The effluent solutions were periodically analyzed for Zn, total Fe, Fe(II) and Fe(III) concentrations as well as pH values. Bacterial population was measured in the solution (free cells). Maximum zinc recovery in the column was achieved about 76% using medium free of initial ferrous ion and 11.4 g/L of ferric ion (medium 2) at pH 1.5. The extent of leaching of sphalerite ore with bacteria was significantly higher than that without bacteria (control) in the presence of ferrous ions. Fe(III) had a strong influence in zinc extraction, and did not adversely affect the growth of the bacteria population.     

Yeasts in malting,with special emphasis on <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> (synonym <Emphasis Type="Italic">Pichia anomala</Emphasis>)

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.     

Combined effect of vanadium(V) and chromium(III) on lipid peroxidation in liver and kidney of rats

Since chromium(III) was demonstrated to have antioxidative action, we have decided to study the effect of this element on V-induced LPO in liver and kidney of rats. Outbred 2-month-old, albino male Wistar rats received daily, for a period of 12 weeks: group I (control), deionized water to drink; group II, sodium metavanadate (SMV) solution at a concentration of 0.100mgV/mL; group III, chromium chloride (CC) solution at a concentration of 0.004mgCr/mL and group IV, SMV-CC solution at a concentration of 0.100mgV and 0.004mgCr/mL. The particular experimental groups took up with drinking water about 8.6mgV/kg b.w./24h (group II), 0.4mgCr/kg b.w./24h (group III), 9mgV and 0.36mgCr/kg b.w./24h (group IV). The V- or Cr-treated groups had higher concentrations of these two elements in liver and kidney compared to the controls. The administration of vanadium alone caused a significant decrease in fluid intake and in body weight gain compared to the controls. In liver supernatants obtained from all tested rats a statistically significant increase in MDA concentration was demonstrated in spontaneous LPO in comparison with the control rats. Moreover, in rats intoxicated with vanadium alone a statistically significant increase in liver MDA level was observed in the presence of 100microM NaVO(3). Instead, in supernatants of liver received from rats treated with chromium alone, a statistically significant increase in MDA concentration in comparison with the controls was found in the presence of 400microM NaVO(3). In kidney supernatants obtained from rats treated with chromium alone, a statistically significant increase in lipid peroxidation was shown in the presence of 30microM FeSO(4) and 400microM NaVO(3). These results show that the tested doses of vanadium(V) and chromium(III) ingested by rats with their drinking water caused significant alterations in internal organs, especially in liver. Under the conditions of our experiment, Cr(III) did not demonstrate antioxidant action, it rather had an oxidant effect.     

N‐butylamine functionalized graphene oxide for detection of iron(III) by photoluminescence quenching

An N‐butylamine functionalized graphene oxide nanolayer was synthesized and characterized by ultraviolet (UV)–visible spectrometry, Fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, and transmission electron microscopy. Detection of iron(III) based on photoluminescence spectroscopy was investigated. The N‐butylamine functionalized graphene oxide was shown to specifically interact with iron (III), compared with other cationic trace elements including potassium (I), sodium (I), calcium (II), chromium (III), zinc (II), cobalt (II), copper (II), magnesium (II), manganese (II), and molybdenum (VI). The quenching effect of iron (III) on the luminescence emission of N‐butylamine functionalized graphene oxide layer was used to detect iron (III). The limit of detection (2.8 × 10^?6 M) and limit of quantitation (2.9 × 10^?5 M) were obtained under optimal conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

Bioaccumulation of copper(II), lead(II) and chromium(VI) by growing Aspergillus niger

The effect of copper(II), lead(II) and chromium(VI) ions on the growth and bioaccumulation properties of Aspergillus niger was investigated as a function of initial pH and initial metal ion concentration. The optimum pH values for growth and metal ion accumulation were determined as 5.0, 4.5 and 3.5 for copper(II), lead(II) and chromium(VI) ions, respectively. Although all metal ion concentrations caused an inhibition effect on the growth of A. niger, it was capable of removing of copper(II) and lead(II) with a maximum specific uptake capacity of 15.6 and 34.4 mg g⁻¹ at 100 mg dm⁻³ initial copper(II) and lead(II) concentration, respectively. Growth of A. niger was highly effected by chromium(VI) ions and inhibited by 75 mg dm⁻³ initial chromium(VI) concentration since some inhibition occurred at lower concentrations.     

Modification of Cycloserine Cefoxitin Fructose Agar to Suppress Growth of Yeasts from Stool Specimens

Cycloserine cefoxitin fructose agar is a widely used selective isolation medium for Clostridium difficile from stool specimens. Yeasts often colonize in the intestine of C. difficile disease patients and, if colonized heavily, pure culture of C. difficile can be delayed. The aim of this study was to modify cycloserine cefoxitin fructose agar to suppress the growth of yeasts. Antimicrobial activities of three commonly available antifungal agents were tested against recent clinical isolates of Candida species. Amphotericin B was most active in inhibiting all isolates by ≤0.5 mg/L concentration. Cycloserine cefoxitin fructose agar was modified by adding 2 mg/L of amphotericin B. Serial ten-fold dilution of stool specimens from 126 suspected C. difficile -associated diarrhea patients were cultured both on cycloserine cefoxitin fructose agar plates and modified agar plates. Yeasts grew from 60 specimens on cycloserine cefoxitin fructose agar, but none grew on the modified medium. Growth of C. difficile was detected from 37 and 39 of 126 specimens on cycloserine cefoxitin fructose agar and modified medium, respectively. The number of C. difficile colonies was similar on both media. In conclusion, 2 mg/L of amphotericin B supplementation to cycloserine cefoxitin fructose agar can facilitate the isolation of C. difficile from stool specimens which are densely colonized with yeasts.     

Candida biotypes in patients with oral leukoplakia and lichen planus

Prevalence of yeasts in 35 leukoplakia and 34 oral lichen planus patients was compared with that observed in persons without oral diseases. Serotype and morphotype were determined on Candida albicans isolates. Yeasts were isolated from the oral cavity specimens of 43.7% of the patients. C. albicans (serotype A) was the predominant species (76% in leukoplakia, 88.2% in lichen planus and 60.8% in healthy persons). Sixteen morphotypes were encountered on malt extract agar, being 732, 733, 734, 753 and 754 the most frequently found. Morphotypes SP1N and SP1Y were the most common on Sabouraud-trypheniltetrazolium agar (68.4% of the isolates from leukoplakia and 73.3% from lichen planus, but only 46.6% of the isolates from healthy oral mucosa showed SP1N morphotype). Presence of oral lesions was associated with a marked reduction in the yeast species and C. albicans biotypes, suggesting that C. albicans and particularly some of its biotypes, show a high potential of adaptation to the changes associated with the development of oral leukoplakia and lichen planus.     

Pyridine-2,6-bis(thiocarboxylic acid) Produced by <Emphasis Type="BoldItalic">Pseudomonas stutzeri</Emphasis> KC Reduces Chromium(VI) and Precipitates Mercury,Cadmium, Lead and Arsenic

Interactions of the Pseudomonas stutzeri KC siderophore pyridine-2,6-bis(thiocarboxylic acid) (pdtc) with chromium(VI), mercury(II), cadmium(II), lead(II), and arsenic(III) are described. Pdtc was found to reduce Cr(VI) to Cr(III) in both bacterial cultures and in abiotic reactions with chemically synthesized pdtc. Cr(III) subsequently formed complexes with pdtc and pdtc hydrolysis products, and their presence was confirmed using electrospray ionization-mass spectrometry (ESI-MS). Cr(III):pdtc complexes were found to slowly release Cr(III) as chromium sulfide and possibly Cr(III) oxides. Pdtc also formed poorly soluble complexes with Hg, Cd, Pb, and As(III). Hydrolysis of those complexes led to the formation of their respective metal sulfides as confirmed by energy dispersive X-ray spectroscopy (EDS) elemental analysis. The pdtc-producing strain P. stutzeri KC showed higher tolerance to most of these metals as compared to a pdtc-negative mutant. A novel role of pdtc is postulated as its involvement in providing an extracellular pool of thiols that are used for redox processes in detoxification of the bacterial extracellular environment. These redox processes can be mediated by transition metal:pdtc complexes.     

Patterns of tolerance to heavy metals among methane-utilizing bacteria

The susceptibility of obligate methane-utilizing bacteria, including 14 reference strains and 175 environmental isolates, to five readily available heavy metal pollutants was determined. The chloride salts of Hg(II), Cd(II), Cu(II), Cr(III) and Zn(II) were tested in a system free from organic matter. Methane-utilizers appeared to have relatively discrete metal tolerance patterns with resistances to all metals varying with isolation site. Methanotrophs proved to be quite sensitive to mercury and cadmium but relatively resistant to copper, chromium and zinc     

Complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin. A study of their antimicrobial activity

Cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin (HL(1) and HL(2), respectively) were synthesized and characterized, being the crystal structures of HL(1), HL(2) and [Ni(L(1))(2)].2CHCl(3) elucidated by X-ray diffraction techniques. The in vitro antimicrobial activity of all these compounds was tested against several bacteria and fungi. HL(1)and its complexes exhibited a strong inhibition of the growth of Haemophilus influenzae (MIC 0.15-1.50microg/mL) and good antibacterial properties towards Bacillus subtilis (MIC 3-25microg/mL). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound inhibiting the growth of each strain. The antibacterial effectiveness was confirmed against a number of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Yeasts and moulds showed a low susceptibility, except the dermatophyte mould Epidermophyton floccosum that is inhibited at concentrations ranging from 6 to 50microg/mL. In general, the antimicrobial activity of the thiophene derivatives was greater than that of the isonicotinic analogues.     

Bioremediation of chromium by the yeast Pichia guilliermondii: toxicity and accumulation of Cr (III) and Cr (VI) and the influence of riboflavin on Cr tolerance

A comparative study has been made on the sensitivity of the yeast Pichia guilliermondii to Cr (III) and Cr (VI) as well as on the Cr uptake potential at growth-inhibitory concentrations of chromium. The strains used in the study were either isolated from natural sources or obtained from a laboratory strain collection. The results show that most of the natural strains were more tolerant to chromium and were able to grow in the presence of 5 mM Cr (III) or 0.5 mM Cr (VI), that is at concentrations which substantially inhibited the growth of laboratory strains. The cellular Cr content after treatment was similar for both strain types and ranged from 1.2-4.0 mg/g d.w. and 0.4-0.9 mg/g d.w., for Cr (III) and Cr (VI) forms, respectively, however, in one case of a natural strain it reached the value of 10 mg Cr (III)/g dry mass. Natural-source strains were grouped into four groups based on the yeasts' differential response to Cr (III) and Cr (VI). Hexavalent Cr-resistant mutants of a P. giuilliermondii laboratory strain, which revealed markedly changed capabilities of chromium accumulation, were obtained by means of UV-induced mutagenesis. Cr (VI) treatment triggered oversynthesis of riboflavin and the addition of exogenous riboflavin increased P. guilliermondii resistance to both Cr (III) and Cr (VI). Electrophoretic protein profiles revealed the induction and/or suppression of several proteins in response to toxic Cr (VI) levels.     

Computer simulation of Zn(II) speciation and effect of Gd(III) on Zn(II) speciation in human blood plasma

The speciation and distribution of Zn(II) and the effect of Gd(III) on Zn(II) speciation in human blood plasma were studied by computer simulation. The results show that, in normal blood plasma, the most predominant species of Zn(II) are [Zn(HSA)] (58.2%), [Zn(IgG)](20.1%), [Zn(Tf)] (10.4%), ternary complexes of [Zn(Cit)(Cys)] (6.6%) and of [Zn(Cys)(His)H] (1.6%), and the binary complex of [Zn(Cys)₂H] (1.2%). When zinc is deficient, the distribution of Zn(II) species is similar to that in normal blood plasma. Then, the distribution changes with increasing zinc(II) total concentration. Overloading Zn(II) is initially mainly bound to human serum albumin (HSA). As the available amount of HSA is exceeded, phosphate metal and carbonate metal species are established. Gd(III) entering human blood plasma predominantly competes for phosphate and carbonate to form precipitate species. However, Zn(II) complexes with phosphate and carbonate are negligible in normal blood plasma, so Gd(III) only have a little effect on zinc(II) species in human blood plasma at a concentration above 1.0×10⁻⁴ M.     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure

The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia. The morphology of the yeast cell surface was observed by the scanning electron microscopy. Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds. Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.     

Analysis of non‐Saccharomyces yeast populations isolated from grape musts from Sicily (Italy)

Aims: The aim of this study was to identify the non‐Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Methods and Results: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5·8S‐internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. Conclusions: We isolated non‐Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. Significance and Impact of the Study: This investigation is a first step in understanding the distribution of non‐Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non‐Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.     

Interaction of chromium(III) complexes with model lipid bilayers: implications on cellular uptake

To understand molecular cytotoxicity of chromium(III) and how it affects the stability of biological membranes, studies on the interaction of chromium(III) complexes aquapentaminechromium complex (complex I) and trans- [Cr(5-methoxysalcyclohex) (H(2)O) (2)] ClO(4) (complex II) with model biomembranes have been carried out. Langmuir films of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidic acid (DPPA), dioctadecyldimethylammoniumbromide (DOMA) at air/water interface interacting with the chromium(III) complexes have been characterized using the surface pressure-molecular area (π-A) isotherms. Initial surface pressures changes for the two complexes show that the chromium(III) complexes inserted in the Langmuir films and complex I interacted strongly compared to complex II. Supported bilayers (SB) of the lipids on solid substrates formed by hydrating their Langmuir-Blodgett films (LB films) have been characterized using linear dichroic spectra, low angle X-ray diffraction and steady state fluorescence anisotropy. Depending on the geometry of the ligands and concentration, the complexes either insert in the alkyl or in the head group region of the SB and sometimes in both regions. The Supported lipid bilayers are well-layered and at low concentration, the metal complexes are incorporated near the head group region. Order and increase in lamellar spacing show stronger interaction of complex I with the lipids compared with complex II. This study provides some insights into the mechanism of chromium(III) toxicity and uptake of chromium(III) by the cells.     

Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil

Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and (1)H NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were subsequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes. The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.