Immunofluorescence of sulphate-reducing bacteria

An indirect fluorescent antibody technique was used as a method of rapidly assessing and identifying sulphate-reducing bacteria. Five specific antisera and one polyvalent serum were raised and tested against 44 strains of the genera Desulfovibrio and Desulfotomaculum along with 4 control organisms. Immunofluorescence was found to be mainly strain specific with the sulphate-reducing bacteria although weak fluorescence was seen both within and between recognised groups. A polyvalent antiserum was successfully used to detect sulphate-reducing bacteria. No interference from 4 control organisms was found.     

Two different hydrogenase enzymes from sulphate-reducing bacteria are responsible for the bioreductive mechanism of platinum into nanoparticles

A mixed consortium of sulphate-reducing bacteria was used to investigate the enzymatic mechanism for the total bioreduction of platinum (IV) into platinum (0) nanoparticles. It was established that two different hydrogenase enzymes were involved. First the platinum (IV) was reduced to platinum (II) by a two-electron bioreduction using an oxygen-sensitive novel cytoplasmic hydrogenase. Second the platinum (II) ion was reduced to platinum (0) nanoparticle by another two-electron bioreduction involving an oxygen-tolerant/protected periplasmic hydrogenase. The enzyme was identified from its reaction with Cu(II), an active inhibitor of periplasmic hydrogenases. No exogenous electron donors were necessary as endogenous production of hydrogen/electrons, via the oxidation of metabolites, was generated in situ by the cytoplasmic hydrogenase. The hydrogen then dispersed through the cell to the periplasm where it became available for use by the periplasmic hydrogenase. The endogenous electrons were used, in the absence of sulphate, for the reduction of platinum (II) by the periplasmic hydrogenase. It was found that the Pt(IV) ion must be fully reduced before reduction of the Pt(II) ion would begin. Transmission electron microscopy and energy dispersive X-ray analysis confirmed the deposit of platinum particles into the periplasmic space.     

Dynamics of bacterial community in up-flow anaerobic packed bed system for acid mine drainage treatment using wine wastes as carbon source

The dynamics of the bacterial populations in an up-flow anaerobic packed bed system (UAPB), applied in acid mine drainage treatment using wine wastes as carbon and nutrients source was elucidated by temperature gradient gel electrophoresis (TGGE) analysis. Moreover, TGGE fingerprints of the bacterial communities developed in a UAPB fed with wine wastes and a UAPB fed with pure ethanol were compared. TGGE fingerprinting and phylogenetic analysis showed that the composition of the community in the UAPB fed with wine wastes remained stable during whole time of operation and its bacterial diversity was higher. The bacterial community of the UAPB fed with wine wastes was composed by bacteria affiliated with Desulfovibrio, Clostridium, Citrobacter and Cronobacter genera and with Bacteroidales order, sp. The dominant community developed in the UAPB fed with ethanol was composed by bacteria affiliated with Desulfovibrio sp. The presence of several bacterial groups in the bioreactor fed with wine wastes suggests a synergistic interaction between the different populations. Syntrophic interaction may be the key factor for the utilization of wine wastes, a complex organic substrate, as carbon and electron source for sulphate reduction.     

In situ spectroelectrochemical investigation of Pt(II/IV) oxidation in aqueous solution using X-ray absorption spectroscopy

The oxidation from to in HCl aq. was studied in situ by combining electrochemistry with XAFS spectroscopy. During the oxidation of , isosbestic points were observed in Pt L_III and L_II XANES spectra as a function of time, indicating that the Pt(II/IV) redox equilibrium is the only reaction in the system. The Pt L_III and L_II X-ray absorption edge energies of the initial Pt^IICl₄²⁻ are 11562.9 and 13271.8 eV, respectively, while those of the electrolyzed species are 11564.6 and 13273.7 eV which are identical with those of a reference sample. The coordination of the electrolyzed species was characterized by structural parameters derived from the EXAFS curve fit, and identified to .     

Aerobic heterotrophic bacteria and petroleum-utilizing bacteria from cow dung and poultry manure

In this study, enumeration and identification of total aerobic heterotrophic bacteria and petroleum-utilizing bacteria as well as the degradative potential of petroleum-utilizing bacterial isolates were carried out. The average counts of total aerobic heterotrophic bacteria in cow dung and poultry manure were 74.25 × 10⁵ c.f.u. g⁻¹ and 138.75 × 10⁵ c.f.u. g⁻¹ respectively. Acinetobacter sp, Bacillus sp, Pseudomonas sp, and Serratia spp. occurred as aerobic heterotrophs in both cow dung and poultry manure. However, Alcaligenes spp. occurred only in cow dung while, Flavobacterium sp, Klebsiella sp, Micrococcus sp, and Nocardia spp. occurred only in poultry manure as aerobic heterotrophs. The average counts of petroleum-utilizing bacteria in cow dung and poultry manure were 9.25 × 10⁵ c.f.u. g⁻¹ and 17.25 × 10⁵ c.f.u. g⁻¹ respectively. Pseudomonas spp. occurred as petroleum utilizer in both cow dung and poultry manure. However, Bacillus spp. occurred only in cow dung while Acinetobacter spp. and Micrococcus spp. occurred only in poultry manure as petroleum utilizers. Relative abundance of petroleum utilizers in total aerobic heterotrophs ranged from 6.38% to 20.00% for cow dung and from 9.38% to 17.29% for poultry manure. Introduction of pure cultures of petroleum-utilizing bacteria from cow dung and poultry manure into sterile oil-polluted soil revealed oil degradation in one week period.     

Photogeneration of titanium(III) from titanium(IV) citrate in aqueous solution

Current interest in the biochemistry of Ti(IV) arises from its widespread use in white pigments and its potential in therapeutic agents. Citrate is known to form strong complexes with Ti(IV). We show here that Ti(III) citrate is generated in a facile manner and in good yield by the action of UV radiation on Ti(IV) citrate in aqueous solution. The Ti(III)-citrate species formed was isolated and characterised by UV-Visible spectroscopy, showing an absorption at 547 nm (epsilon=100 M(-1)cm(-1)), and by electron paramagnetic resonance (EPR) spectroscopy giving a resonance at g=1.949 (linewidth=60G) . An X-ray structure of the parent Ti(IV) complex in the form [TiNa(3)(C(6)H(6)O(7))(2)(C(6)H(5)O(7))(H(2)O)(6.8)].2H(2)O is reported along with a study of the reaction of Ti(IV)-citrate with N,N-ethylenebis(o-hydroxytoluene)glycine (EHTG), which was more rapid than those of other related Ti(IV) complexes.     

Influence of EPS isolated from thermophilic sulphate-reducing bacteria on carbon steel corrosion

Extracellular polymeric substances (EPS) were isolated by centrifugation of thermophilic sulphate-reducing bacteria (SRB) grown in API-RP38 culture medium. The protein and polysaccharide fractions were quantified and the highest concentrations were extracted from a 14-day old culture. The effect of EPS on carbon steel corrosion was investigated by electrochemical techniques. At 30°C, a small amount of EPS in 3% NaCl solution inhibited corrosion, whilst excessive amounts of EPS facilitated corrosion. In addition, the inhibition efficiency of EPS decreased with temperature due to thermal desorption of the EPS. The results suggest that adsorbed EPS layers could be beneficial to anti-corrosion by hindering the reduction of oxygen. However, the accumulation of an EPS film could stimulate the anodic dissolution of the underlying steel by chelation of Fe²⁺ ions.     

Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic ciliates using 16S rRNA binding oligonucleotide probes

The identity of ectosymbiotic bacteria of some marine, free-living anacrobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.Abbreviations DMSO dimethyl sulfoxide - PBS phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH: 7.3 - TEAA triethylamonium acetate - 1 x SSC standard sodium citrate buffer, 150 mM NaCl, 15 mM Na₂ citrate, pH: 7.0 - 1 x Denh Denhardts solution, 0.02% ficoll, 0.02% bovine serum albumine, 0.02% polyvinol-pyralidone     

Molecular analysis of a sulphate-reducing consortium used to treat metal-containing effluents

A sulphate-reducing consortium used in a bioprocess to remove toxic metals from solution as insoluble sulphides, was characterised using molecular (PCR-based) and traditional culturing techniques. After prolonged cultivation under anoxic biofilm-forming conditions, the mixed culture contained a low diversity of sulphate-reducing bacteria, dominated by one strain closely related to Desulfomicrobium norvegicum, identified by three independent PCR-based analyses. The genetic targets used were the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region and the disulfite reductase (dsr) gene, which is conserved amongst all known sulphate-reducing bacteria. This organism was also isolated by conventional anaerobic techniques, confirming its presence in the mixed culture. A surprising diversity of other non-sulphate-reducing facultative and obligate anaerobes were detected, supporting a model of the symbiotic/commensal nature of carbon and energy fluxes in such a mixed culture while suggesting the physiological capacity for a wide range of biotransformations by this stable microbial consortium.     

Localization and stability of hydrogenases from aerobic hydrogen bacteria

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction, while the second enzyme was found to be particulate and unable to react with NAD.All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gentle methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis.Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 moles H₂ oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme from A. eutrophus H 16 which was shown to be more stable under aerobic conditions.     

Unusual reactivity of cytotoxic cis-dihydrazide Pt(II) complexes in aqueous solution

Complexes of the general structure cis-[PtX(2)(hydrazide)(2)] and cis-[PtX(2)NH(3)(hydrazide)], where X=Cl(-), Br(-) and I(-), and hydrazide=cyclohexylcarboxylic acid hydrazide (chcah), cyclopentylcarboxylic acid hydrazide (cpcah), 3-aminocyclohexanspiro-5-hydantoin (achsh) and 3-aminocyclopentanspiro-5-hydantoin (acpsh), were investigated with respect to aqueous stability, DNA platination rates and cytotoxic activity on a panel of seven human cancer cell lines as well as a cisplatin-resistant cell line. Stabilities in aqueous solution, determined by RP-HPLC and UV-Vis methods, were highly dependent on the type of halide ligand, with stability decreasing in the order I(-)>Cl(-)>Br(-). Added chloride (100 mM) only stabilized the dichloro-Pt(II) complexes containing the hydrazide as part of a hydantoin ring (i.e., achsh). Platination of calf thymus DNA determined by AAS was most rapid with dichloro-Pt(II) complexes containing achsh ligand. The mixed-amine dichloro-Pt(II) complexes with either chcah or cpcah ligands also platinated DNA >80%, but at a slower rate, while dihydrazide dichloro-Pt(II) complexes with either chcah or cpcah ligands resulted in <25% DNA platination at 24 h. cis-[PtX(2)(hydrazide)(2)], where hydrazide=chcah or cpcah, were the most potent compounds (chcah>cpcah), but activity was independent of the halide ligand (I(-)=Cl(-)=Br(-)). These complexes showed no cross-resistance with cisplatin, but they also showed little differentiation in potency over the seven cell lines. Complexes with the hydantoin ligands achsh and acpsh were inactive in all cell lines. Thus, neither stability in aqueous media nor covalent binding to DNA are correlated with biological activity, suggesting that cis-dihydrazide Pt(II) complexes act by a unique mechanism of action.     

Enhancing pozzolana colonization by As(III)-oxidizing bacteria for bioremediation purposes

The colonization of pozzolana by an As(III)-oxidizing bacterial consortium was monitored from the first hours of bacterial adhesion to 6 weeks of development under fed-batch conditions, using adapted ultrasonic dislodging and crystal-violet staining procedures to determine the biofilm adhering to the complex surfaces. The effect of temperature, arsenic concentration, and presence or absence of yeast extract (YE) on the amount of biofilm biomass and on the As(III)-oxidation were assessed to test the biofilm’s resilience and optimize the colonization. Fed-batch cultures allow twice as much pozzolana colonization as that obtained under batch conditions. In addition, As(III) oxidation and the quantities of biomass under fed-batch culture conditions were the same at 14°C and 25°C. Whereas YE improves (+150%) bacterial adhesion during the first 2 h, its impact in the longer term appears to be less significant—biofilm formation in presence of YE after 5 weeks was no greater than biofilm formation in the absence of YE. Finally, YE involves a drastic (−70%) decrease of As(III) oxidation. Preliminary tests for drinking-water bioremediation revealed the ability of Chéni Arsenic Oxidizing 1 biofilms to remain and retain As(III) oxidation activity at low As(III) concentrations (50 μg l⁻¹).     

Treatment of acid mine drainage by sulphate-reducing bacteria using permeable reactive barriers: A review from laboratory to full-scale experiments

Acid mine drainage in-situbioremediation has in the last decades drawnthe attention in the field of environmentalbiotechnology. The most recent treatmenttechnique are the permeable reactive barriersusing sulphate-reducing bacteria. This viewdescribes the basis of many of the currentapproaches to use sulphate-reducing bacteria inacid mine drainage treatment, from laboratoryto full-scale realisations, and the limitationsencountered when applied to full scaleapplications.     

Transformation of metals and metal ions by hydrogenases from phototrophic bacteria

The ability of hydrogenases isolated from Thiocapsa roseopersicina and Lamprobacter modestohalophilus to reduce metal ions and oxidize metals has been studied. Hydrogenases from both phototrophic bacteria oxidized metallic Fe, Cd, Zn and Ni into their ionic forms with simultaneous evolution of molecular hydrogen. The metal oxidation rate decreased in the series Zn>Fe>Cd>Ni and depended on the pH. The presence of methyl viologen in the reaction system accelerated this process. T. roseopersicina and L. modestohalophilus cells and their hydrogenases reduced Ni(II), Pt(IV), Pd(II) or Ru(III) to their metallic forms under H₂ atmosphere. These results suggest that metals or metal ions can serve as electron donors or acceptors for hydrogenases from phototrophic bacteria.     

Role of iron-reducing bacteria in corrosion and protection of carbon steel

The role of iron-reducing bacteria (IRB) in biocorrosion is under discussion. According to some reports, IRB are able to induce protection of carbon steel while others suggest an important enhancement of corrosion through the reduction and removal of passive films of ferric compounds on the metal surface. In this work, we review recent knowledge concerning microbial respiration, the ecology of IRB containing biofilms and the corrosive or protective effect of such biofilms on metal surfaces.     

Hydrogenase of purple bacteria: properties and regulation of synthesis

Physico-chemical properties of homogeneous preparations of soluble and membrane-bound hydrogenases from the purple sulfur bacterium Thiocapsa roseopersicina BBS and membrane-bound hydrogenase of Rhodopseudomonas capsulata, strain B10 have been studied. Compared to the enzymes from other sources, the hydrogenase of Thiocapsa roseopersicina is more stable to O₂ and products of its reduction (O ₂ ^- , H₂O₂), temperature and a number of other factors of the medium. A natural electron donor for T. roseopersicina hydrogenase is a low-potential cytochrome C₃, while the natural electron acceptors for hydrogenases of R. capsulata, T. roseopersicina, Ectothiorhodospira shaposhnikovii and Anabaena cylindrica are cytochromes of groups c and b.In different phototrophs, synthesis of hydrogenase can be inhibited by the presence of high concentrations of O₂. In some microorganisms (e.g. Rhodopseudomonas capsulata, strain B10) the repressing effect on hydrogenase formation is also exhibited by organic compounds. H₂ may not necessarily be present for hydrogenase synthesis by purple bacteria, but its presence may considerable increase the level of the enzyme.Abbreviations SDS sodium dodecylsulfate - Hipip high-potential iron — sulfur protein - R Rhodopseudomonas - T Thiocapsa - Rh Rhodospirillum - C Chromatium This paper is dedicated to Professor Dr. H.G. Schlegel in honour of his sixtieth birthday and in recognition of his great contribution in the field of physiology and biochemistry of microorganisms capable of using H₂. Professor H.G. Schlegel had a profound and most fuitful influence on the progress in the research of the laboratory headed by the author     

Photodynamic inactivation of multiresistant bacteria (KPC) using zinc(II)phthalocyanines

The worldwide increase in antibiotic resistance has led to search of alternatives anti-microbial therapies such as photodynamic inactivation. The aim of this paper was to evaluate the photodynamic activity in vitro of a neutral and two cationic Zn phthalocyanines. Their photokilling activity was tested on Escherichia coli ATCC 25922 and Klebsiella pneumoniae Carbapenemase (KPC)-producing. After treating bacteria with phthalocyanines, the cultures were irradiated with white light. As a result, the bacteria were inactivated in presence of cationic phthalocyanines. The photoinactivation was dependent of the irradiation time and phthalocyanine concentration. The most effective photosensitizer on KPC-producing was Zinc(II)tetramethyltetrapyridino[2,3-b:2′,3′-g:2″,3″-l:2?,3?-q]porphyrazinium methylsulfate (ZnTM2,3PyPz). After irradiation using the water soluble ZnTM2,3PyPz (3 μM) the viability of KPC (30 min of irradiation) and E. coli (10 min of irradiation) decreased ≈99.995%.     

Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution

Aims: The primary objective of this study was to compare the removal of cadmium, lead, aflatoxin B₁ and microcystin‐LR from aqueous solution by Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii shermanii JS and Bifidobacterium breve Bbi99/E8, separately and in combination. Methods and Results: The removal of toxins and heavy metals was assessed in batch experiments. The removal of all compounds was observed to be strain specific. The removal of lead by a combination of all the strains used was observed to be lower than could be predicted from the removal by single strains (P < 0·05). A similar trend was also observed with the other compounds studied. Conclusions: The results show that the toxin‐removal capacity of a combination of strains of lactic acid bacteria is not the sum of their individual capacities. Therefore, pure single strains should be used when the goal is to remove single compounds. The use of combinations of strains may be beneficial when several compounds are removed together. This needs to be studied in future experiments. Significance and Impact of the Study: Lactic acid bacteria have been identified as potent tools for the decontamination of heavy metals, cyanotoxins and mycotoxins. The results of this study should be considered when selecting combinations of bacteria for the simultaneous removal of several toxic compounds.