Rapid PaperMicrobial Oxygenation of Organo-metallic Bissulfides Leading to Formation of Planar Chirality and C2 Symmetry

1,2-Bis(methylthiomethyl)ferrocene (3) was oxidized by Corynebacterium equi IFO 3730 to give monosulfoxide 4 in two diastereomeric forms with (1S,2R,S_S) and (1S,2R,R_S) configurations in a ratio of 4:1, while 1,1′-bis(methylthiomethyl)ferrocene (5) was oxidized by Penicillium frequentans IFO 5692 to (R)-monosulfoxide 6 and then preferentially to (R,R)-bissulfoxide 7. Thus, the bacterial monooxygenase generated specific planar chirality in the metallocenic monosulfoxide, and the fungal enzyme formed C₂ symmetry in the bissulfoxide.     

Microbial asymmetric oxygenation of an organometallic sulfide

Summary Penicillium frequentans IFO 5692 oxidized enantioselectively (methylthiomethyl)ferrocene (1) to (R)-(–)-(methylsulfinylmethyl)ferrocene (2) (98 %e.e.) andCorynebacterium equi IFO 3730 to (S)-(+)-2 (69 %e.e.).     

Biooxidation of Gold-, Silver,and Antimony-Bearing Highly Refractory Polymetallic Sulfide Concentrates,and its Comparison with Abiotic Pretreatment Techniques

Effectiveness of different pure and mixed cultures of three moderately thermophilic, extremely acidophilic bacterial strains (Acidimicrobium ferrooxidans ICP, Sulfobacillus sibiricus N1, Acidithiobacillus caldus KU) were investigated for biooxidation of highly refractory polymetallic gold ore concentrates. Despite of its complex mineralogy and the presence of a mixture of potentially inhibitory metals and metalloids, the concentrate was readily dissolved in defined mixed cultures including both iron and sulfur oxidizers, releasing as much as 80% of soluble Fe and 61% of soluble As. Factors to affect microbial mineral dissolution efficiencies (i.e. microbial As(III) oxidation ability, formation of secondary mineral precipitation (e.g. jarosite, elemental sulfur, scorodite, anglesite), and microbial population dynamics during biooxidation) were studied, based on which roles of individual microbes and their synergistic interactions during biooxidation were discussed. Applying the biooxidation pretreatment using the most efficient mixed cultures containing all three strains significantly improved the recovery of both Au (from 1.1% to 86%) and Ag (from 3.2% to 87%). Finally, this study provides one of the very few available comparisons of the effectiveness of different pretreatment techniques for refractory gold ore concentrates: Compared with other abiotic pretreatment approaches (roasting, pressure oxidation, and alkali dissolution), biooxidation was shown to be one of the most effective options in terms of the recovery of Au and Ag.     

Production of volatile alkyl sulphides by microbial degradation of synthetic alliin and alliin-like compounds, in relation to germination of sclerotia of Sclerotium cepivorum Berk

Onion distillands proved more stimulatory than distillates towards germination of sclerotia of Sclerotium cepivorum. Synthetic allylcysteine and n-propylcysteine and their sulphoxides stimulated germination of sclerotia when added to soil. Solutions of these compounds in the presence of unsterile soil produced gaseous alkyl mercaptans, sulphides and disulphides which were detected by gas-liquid chromatography and which stimulated sclerotial germination when drawn through soil containing sclerotia. No volatile sulphides were detected above autoclaved solutions but the atmosphere over propyl and allylcysteines was slightly stimulatory although the corresponding autoclaved sulphoxides showed no such activity. The gases above all four solutions sterilized by membrane-filtration were not stimulatory. Six common soil bacteria were shown to be capable of degrading the synthetic precursors, causing the evolution of appropriate volatile alkyl sulphides.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of the dominant bacterium of two-stage biooxidation of gold-arsenic concentrate

In the process of biooxidation at 39°C in a continuous mode of the gold-arsenic concentrate from the Olympiadinskoe deposit, which was pretreated by chemical leaching with ferric ions, by a microbial association from the BIO department reactors of the Polyus gold mining company, a bacterial culture designated as strain HT-4 was isolated. The bacterium was a spore-forming rod 0.5–0.6 × 1.4–2.0 μm with a flagellum. The optimal temperature for growth and Fe²⁺ oxidation was 55°C. The strain grew in the pH range from 1.21 to 2.10 with the optimum at pH 1.6. The organism was incapable of lithotrophic and organotrophic growth. It grew mixotrophically by Fe²⁺ oxidation in the presence of 0.02% yeast extract. The DNA G+C base content was 48.6 mol %. Based on comparative phylogenetic analysis of 1472-bp nucleotide sequences of 16S rRNA genes, strain HT-4 was classified as Sulfobacillus thermosulfidooxidans. Analysis by pulse-field gel electrophoresis revealed a unique profile of the NotI fragments of the chromosomal DNA. These results demonstrate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of concentrates in different technological conditions. The strain “S. olympiadicus S-5” dominated in the process of biooxidation of original concentrate not treated with ferric iron, while S. thermosulfidooxidans HT-4 was predominant in biooxidation of the chemically leached concentrate.     

Oxygen-containing sulfur-rich compounds from the bark of the tropical garlic tree Scorodophloeus zenkeri Harms

Two sulphoxides (2,3,5-trithiahexane 5-oxide and 2,4,5,7-tetrathiaoctane 2-oxide), three novel sulphones [S-(methylthiomethyl)methanesulfonothioate, methylthio(methylthio-methyl)sulfone, 2,3,5,7-tetrathiaoctane3,3-dioxide] and four known sulphones [methylsulphonylmethylthiomethane, methylmethanethiosulfonate, bis-methyl-sulphonylmethane, and bis-(methylthiomethyl)sulfone] were isolated from the bark extracts of Scorodophloeus zenkeri Harms. The structures were determined by spectral methods, essentially MS and NMR experiments.     

Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6

Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni²⁺ and, to some extent, Zn²⁺ and Co²⁺, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities. Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Methane-derived carbonate build-ups and associated microbial communities at cold seeps on the lower Crimean shelf (Black Sea)

In the euxinic waters of the NW’ Black Sea shelf, tower-like carbonate build-ups up to several metres in height grow at sites of cold methane seepage. These structures are part of an unique microbial ecosystem that shows a considerable biodiversity and a remarkable degree of organization. The accretion of the build-ups is promoted by the growth of centimetre-sized, methane-filled spheres constructed by calcifying microbial mats. Progressive mineralization of these spheres involves the early precipitation of strongly luminescent high-Mg-calcite rich in iron sulphides, and closely interfingered aragonite phases that finally create the stable (mega-) thrombolithic fabric of the towers. Within the microbial mats, microorganisms occur in distinctive spatial arrangements. Major players among the microbial consortia are the archaea groups ANME-1 and ANME-2, Crenarchaeota, and sulphate-reducing bacteria (SRB) of the Desulfosarcina/Desulfobacterium group. The intracellular precipitation of iron sulphides (greigite) by some of these bacteria, growing in close association with ANME-2, suggests iron cycling as an additional biogeochemical pathway involved in the anaerobic oxidation of methane (AOM).     

Species-strain dependence of stereoselectivity in microbial oxidation of thioethers

The stereoselectivity in the aerobic, microbial oxidation of thioethers and sulphoxides is shown to be dependent on species and strain. A strain of Aspergillus niger was used to obtain an optically active dialkyl sulphoxide.     

Polymorphism of <Emphasis Type="Italic">Sulfobacillus thermosulfidooxidans</Emphasis> strains dominating in processes of high-temperature oxidation of gold-arsenic concentrate

The composition was studied of the microbial association involved in tank biooxidation of the concentrate of a refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore from the Olympiadinskoe deposit at 50°C. The two Sulfobacillus thermosulfidooxidans strains predominant in the association were phylogenetically different from the strains used as inocula. The isolates were found to differ significantly both from each other and from the strains that dominated in the processes of biooxidation of a similar concentrate by traditional tank technology at 39°C or at 39°C with treatment of the concentrate with ferric iron prior to biooxidation. These results indicate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of the concentrates under different technological modes.     

Two-stage process of bacterial-chemical oxidation of refractory pyrite-arsenopyrite gold-bearing concentrate

A technology for tank biooxidation of refractory gold-bearing concentrate under variable temperature conditions has been improved: the temperature of the first of two stages was changed from 30°C to 34–36°C. Gold in this concentrate is mainly associated with sulfide minerals: arsenopyrite and pyrite, which underlies a low gold recovery (16.68%) as a result of cyanidation. To resolve the problem, an association of mesophilic acidophilic chemolithotrophic microorganisms and moderately thermophilic bacteria of the Sulfobacillus genus were used for the concentrate oxidation. The composition of the used microbial association was studied; it was shown that it depends upon temperature: at 42°C, the population of the mesophilic thiobacteria decreased, whereas that of thermophilic sulfobacilli enhanced as compared to 36°C. The accepted scheme of the process ensures a high extent of gold recovery (94.6%) within a short space of time for biooxidation (96 h).     

Radical-scavenging properties of ferrocenyl chalcones

The radical-scavenging capacities of ferrocenyl group and phenolic hydroxyl group in ferrocenyl chalcone were identified in this work. 1,1′-Diacetylferrocene was applied to condense with benzaldehyde, vanillin, and protocatechualdehyde to produce ferrocenyl chalcones, which were employed to interact with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS⁺), 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively. It was found that ferrocenyl chalcones as well as diacetylferrocene can trap these radicals effectively, and thus, concluded that both iron atom in ferrocene and phenolic hydroxyl group played the radical-scavenging role, and the radical-scavenging capacity of iron atom in ferrocene was even higher than that of phenolic hydroxyl group.     

Synthesis of steroid-ferrocene conjugates of steroidal 17-carboxamides via a palladium-catalyzed aminocarbonylation--copper-catalyzed azide-alkyne cycloaddition reaction sequence

Steroids with the 17-iodo-16-ene functionality were converted to ferrocene labeled steroidal 17-carboxamides via a two step reaction sequence. The first step involved the palladium-catalyzed aminocarbonylation of the alkenyl iodides with prop-2-yn-1-amine as the nucleophile in the presence of the Pd(OAc)₂/PPh₃ catalyst system. In the second step, the product N-(prop-2-ynyl)-carboxamides underwent a facile azide–alkyne cycloaddition with ferrocenyl azides in the presence of CuSO₄/sodium ascorbate to produce the steroid–ferrocene conjugates. The new compounds were obtained in good yield and were characterized by ¹H and ¹³C NMR, IR, MS and elemental analysis.     

Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination

 For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA ^- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu⁺ transformants (μg pRPLeu10 DNA)^-1 (1×10⁶ viable protoplasts)^-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus. Received: 2 October 1995/Received last revision: 5 December 1995/Accepted: 11 December 1995     

Biodegradation of sulfamethoxazole by individual and mixed bacteria

Antibiotic compounds, like sulfamethoxazole (SMX), have become a concern in the aquatic environment due to the potential development of antibacterial resistances. Due to excretion and disposal, SMX has been frequently detected in wastewaters and surface waters. SMX removal in conventional wastewater treatment plants (WWTPs) ranges from 0% to 90%, and there are opposing results regarding its biodegradability at lab scale. The objective of this research was to determine the ability of pure cultures of individual and mixed consortia of bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii) known to exist in WWTP activated sludge to remove SMX. Results showed that R. equi alone had the greatest ability to remove SMX leading to 29% removal (with glucose) and the formation of a metabolite. Degradation pathways and metabolite structures have been proposed based on the potential enzymes produced by R. equi. When R. equi was mixed with other microorganisms, a positive synergistic effect was not observed and the maximum SMX removal achieved was 5%. This indicates that pure culture results cannot be extrapolated to mixed culture conditions, and the methodology developed here to study the biodegradability of compounds under controlled mixed culture conditions offers an alternative to conventional studies using pure bacterial cultures or inocula from activated sludge sources consisting of unknown and variable microbial populations.     

Biogeochemical controls on microbial diversity in seafloor sulphidic sediments

The ultimate fate of hydrothermal sulphides on the seafloor depends on the nature and rate of abiotic and microbially catalysed reactions where sulphide minerals are exposed to oxic seawater. This study combines organic and inorganic geochemical with microbiological measurements across a suboxic transition zone of highly altered sulphidic sediments from the Trans‐Atlantic Geotransverse hydrothermal field to characterize the reaction products and microbial communities present. There is distinct biogeochemical zonation apparent within the sediment sequence from oxic surface layers through a suboxic transition zone into the sulphide material. The microbial communities in the sediment differ significantly between the biogeochemical horizons sampled, with the identified microbes inferred to be associated with Fe and S redox cycling. In particular, Marinobacter species, organisms associated with circumneutral Fe oxidation, are dominant in a sulphide lens present in the lower core. The dominance of Marinobacter‐related sequences within the relict sulphide lens implies that these organisms play an important role in the alteration of sulphides at the seafloor once active venting has ceased.     

Analysis of an SEIRS epidemic model with two delays

 A disease transmission model of SEIRS type with exponential demographic structure is formulated. All newborns are assumed susceptible, there is a natural death rate constant, and an excess death rate constant for infective individuals. Latent and immune periods are assumed to be constants, and the force of infection is assumed to be of the standard form, namely proportional to I(t)/N(t) where N(t) is the total (variable) population size and I(t) is the size of the infective population. The model consists of a set of integro-differential equations. Stability of the disease free proportion equilibrium, and existence, uniqueness, and stability of an endemic proportion equilibrium, are investigated. The stability results are stated in terms of a key threshold parameter. More detailed analyses are given for two cases, the SEIS model (with no immune period), and the SIRS model (with no latent period). Several threshold parameters quantify the two ways that the disease can be controlled, by forcing the number or the proportion of infectives to zero. Received 8 May 1995; received in revised form 7 November 1995     

The effect of ferric ion on the rate of ferrous oxidation by Thiobacillus ferrooxidans

 In this study, the effect of ferric ion and cell concentrations on the oxidation of ferrous ion by T. ferrooxidans was investigated. Ferric ions competitively inhibited ferrous ion oxidation by the bacteria. The inhibitory effect of ferric ion was, however, reduced by increasing cell concentration. The apparent ferric ion inhibition constant did not change with increasing cell concentration. The ferrous ion oxidation kinetics in the absence and presence of ferric ion changes from the standard Michaelis-Menten type at low cell concentrations to pseudo-first-order kinetics at high cell concentration. Received: 8 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996