Biodesulphurization of coal: rate enhancement by sulphur-grown cells

Pyritic sulphur was removed from coal by growing Thiobacillus ferrooxidans in a 250 ml batch bioreactor. Thiobacillus ferrooxidansgrown on sulphur and which was added 5 days after initial inoculation, enhanced the iron solubilization rate by 35% as compared to control (without addition of sulphur-grown cells). About 93% pyritic sulphur was removed in presence of sulphur-grown cells as compared to 77% in the control.     

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

Dependence of effectiveness of leaching of metallic sulphides on enzymes involved in inorganic sulphur metabolism in Thiobacillus ferrooxidans

Summary The leaching activity of five batches of Thiobacillus ferrooxidans, strain F26-77, cultivated under various conditions, towards elemental sulphur, ferrous ions, pyrite, covellite, chalcopyrite and sphalerite was studied. The activities of sulphite oxidase, thiosulphate oxidase and rhodanese were determined in crude, cell-free bacterial extracts. The effectiveness of leaching was directly correlated with the enzymic activity of the cultures. The results suggest that the activities of the enzymes metabolizing sulphur and its inorganic compounds in Thiobacillus ferrooxidans, or bacterial leaching activity on sulphur and sulphides, rather than the rate of oxidation of ferrous ions, should be taken as the criterion of usefulness for the leaching of sulphide minerals.     

Grazing of acidophilic bacteria by a flagellated protozoan

A biflagellated protozoan was isolated from an acidic drainage stream located inside a disused pyrite mine. The stream contained copious amounts of acid streamer bacterial growths, and the flagellate was observed in situ apparently grazing the streamer bacteria. The protozoan was obligately acidophilic, growing between pH 1.8 and 4.5, but not at pH 1.6 or 5.0, with optimum growth between pH 3 and 4. It was highly sensitive to copper, molybdenum, silver, and uranium, but tolerated ferrous and ferric iron up to 50 and 25 mM, respectively. In the laboratory, the protozoan was found to graze a range of acidophilic bacteria, including the chemolithotrophs Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, and the heterotroph Acidiphilium cryptum. Thiobacillus thiooxidans and Thiobacillus acidophilus were not grazed. Filamentous growth of certain acidophiles afforded some protection against being grazed by the flagellate. In mixed cultures of T. ferrooxidans and L. ferrooxidans, the protozoan isolate displayed preferential grazing of the former. The possibility of using acidophilic protozoa as a means of controlling bacteria responsible for the production of acid mine drainage is discussed.Offprint requests to: Dr. D. B. Johnson.     

Pyrite oxidation in acid sulphate soils: The role of miroorganisms

Summary A study has been made of microbial processes in the oxidation of pyrite in aicd sulphate soil material. Such soils are formed during aeration of marine muds rich in pyrite (FeS₂). Bacteria of the type ofThiobacillus ferrooxidans are mainly responsible for the oxidation of pyrite, causing a pronounced acidification of the soil. However, becauseThiobacillus ferrooxidans functions optimally at pH values bellow 4.0, its activity cannot explain the initial pH drop from approximately neutral to about 4. This was shown to be a non-biological process, in which bacteria play an insignificant part. AlthoughThiobacillus thioparus andThiobacillus thiooxidans were isolated from the acidifying soil, they did not stimulate oxidation of FeS₂, but utilized reduced sulphur compounds, which are formed during the non-biological oxidation of FeS₂.Ethylene-oxide-sterilized and dry-sterilized soil inoculated with pure cultures of mixtures of various thiobacilli or with freshly sampled acid sulphate soil soil did not acidify faster than sterile blanks.Thiobacillus thiooxians. Thiobacillus thioparus. Thiobacillus intermedius andThiobacillus perometabolis increased from about 10⁴ to 10⁵ cells/ml in media with FeS₂ as energy source. However, FeS₂ oxidation in the inoculated media was not faster than in sterile blanks.Attempts to isolate microorganisms other thanThiobacillus ferrooxidans, like metallogenium orLeptospirillum ferrooxidans, which might also be involved in the oxidation of FeS₂ were not successful.Addition of CaCO₃ to the soil prevented acidification but did not stop non-biological oxidation of FeS₂.     

The removal of pyritic sulphur from coal by Leptospirillum-like bacteria

Summary The microbial oxidation of pyritic sulphur was studied in a 4.5-l airlift fermentor at pH 1.5 and 100 g/l pulp density. By microbial leaching with Leptospirillum-like bacteria 85% of the pyritic sulphur was removed within 40 days; 30% of the removed pyrite was oxidized to elemental sulphur, the rest being transformed to soluble sulphate. Accumulation of elemental sulphur could be avoided by using a mixed culture of Leptospirillum-like bacteria and Thiobacillus ferrooxidans. Apart from oxidation of elemental sulphur neither the pure nor the mixed culture showed a significant difference as to removal of pyrite.     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

Bulk and surface characterization of crystalline and plastic sulphur oxidized by two Thiobacillus species

This work studies the surface interaction between Thiobacillus ferrooxidans and Thiobacillus thiooxidans with crystalline and plastic elemental sulphur. The interaction mechanisms were analysed by fractal geometry which describes textural modifications of the substrate caused by bacterial action. The results demonstrated that the bacteria are able to produce two different effects depending on the substrates. Only surface smoothing (decrease on fractal dimension values) was detected on crystalline sulphur (this effect being stronger with T. ferrooxidans than with T. thiooxidans), but, perforation of the bulk was also observed in plastic sulphur     

The role of higher polythionates in the reduction of chromium(VI) by Acidithiobacillus and Thiobacillus cultures

In this paper, we report the chromium(VI) reduction by filtrates of Acidithiobacillus and Thiobacillus cultures. Chromium(VI) reduction by filtrates of A. ferrooxidans cultures under acidic conditions was higher than that observed for A. thiooxidans. However, at pH close to 7, chromium(VI) reduction by filtrates of T. thioparus cultures was as high as that by filtrates of A. thiooxidans cultures and much higher than that observed for A. ferrooxidans cultures at the same pH. The capability of these cultures to reduce chromium(VI) was associated specifically with the fraction of cultures (cells, sulphur and associated sulphur compounds) retained by filtration through a 0.45mum filter. In the fraction that comes from A. thiooxidans culture, polythionates (S(x)O(6)(2-)) with 3-7 sulphur atoms were detected and identified (by HPLC with MS as detector). The model of vesicles containing polythionates, sulphur and water agrees with our results.     

Leaching of chalcopyrite byThiobacillus thiooxidans and oxidized copper ore byThiobacillus ferrooxidans isolated from local environments

Summary Feasibility for bacterial leaching of two different copper ores from Pakistan was investigated with locally isolated, acidophilic thiobacilli in shake flask culture at 28±2°C. After a lag period of 16 d,Thiobacillus thiooxidans solubilized up to 19% of copper present in chalcopyrite ore in 14 d before entering stationary phase. In oxidized copper ore amended with chalcopyrite ore and supplemented with sulphur to provide an energy source forT. ferrooxidans, the pH dropped as a result of bacterial growth and was accompanied by copper solubilization. The highest copper release, 61% of total, occurred in flasks having oxidized copper ore, sulphur and calcopyrite (111, by weight) and inoculated withT. ferrooxidans. Supplementation used in these tests resulted in nearly six times more solubilization as compared to tests conducted without supplementation.

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Resumen Se ha estudiado la posibilidad de realizar un lixiviado mediante bacterias de dos minerales de cobre del Pakistan. Las bacterias utilizadas son cepas, aísladas localmente, de tiobacilos acidófilos que se hacen crecer en un medio líquido en agitación a 28±2°C. Después de una fase lag de 16 dThiobacillus thiooxidans solubilizó hasta 19% del cobre presente en la calcopirita en 14 d, antes de alcanzar la fase estacionaria. El mineral de cobre oxidado se modificó con calcopirita y se suplementó con azufre afín de sumministrar una fuente de energía paraT. ferrooxidans, a resultas del crecimiento bacteriano el pH disminuyó solubilizándose cobre. La mayor cantidad de cobre sulubilizado se cencontró en frascos con mineral de cobre oxidado, azufre y calcopirita (111 en peso) e inoculados conT. ferrooxidans. La adición de suplementos incrementó 6 veces la solubilización comparada con la obtenida en ensayos sin adición de suplementos.

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Résumé On a testé la faisabilité de la lixiviation bactérienne de deux minerais différents de cuivre du Pakistan au moyen de thiobacilli acidophiles,isolés localement, en culture en flaçons agités à 28 ±2°C. Après une période de latence de 16 jours,Thiobacillus thiooxidans a solubilisé jusqu'à 19% du cuivre présent dans le minerai de chalcopyrite en 14 jours avant d'entrer en phase stationnaire de croissance. Dans un minerai de cuivre amendé par le minerai de chalcopyrite et additionné de soufre de manière à fournir une source d'énergie pourT. ferrooxidans, le pH a baissé comme suite de la croissance bactérienne, ce qui a entraîné la solubilisation du cuivre. La libération la plus élevée de cuivre, 61% au total, a eu lieu dans les flaçons qui avaient à oxyder le minerai de cuivre, le soufre et la chalcopyrite oans les proportions de 111 en poids, et qui avaient été inoculés parT. ferrooxydans. La supplémentation effectuée dans ces tests a eu pour effet de dissoudre six fois plus de cuivre que dans les tests conduits sans supplémentation.

     

The ultrastructure of chemolithoautotrophic Gallionella ferruginea and Thiobacillus ferrooxidans as revealed by chemical fixation and freeze-etching

By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly--hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.     

Short communication: Adverse effect of surface-active reagents on the bioleaching of pyrite and chalcopyrite by Thiobacillus ferrooxidans

Oxidation of Fe(II) iron and bioleaching of pyrite and chalcopyrite by Thiobacillus ferrooxidans was adversely affected by isopropylxanthate, a flotation agent, and by LIX 984, a solvent-extraction agent, each at 1 g/l. The reagents/l were adsorbed on the bacterial surface, decreasing the bacteria's development and preventing biooxidation. Both reagents inhibited the bioleaching of pyrite and LIX 984 also inhibited the bioleaching of chalcopyrite.     

Leaching of iron and toxic heavy metals from anaerobically-digested sewage sludge

Summary Heavy metal-loaded sewage sludge was leached abiotically using FeCl₂ and FeCl₃ which are applied in waste water treatment plants to eliminate phosphate and for coagulation. Due to the hydrolyzing nature of ferric iron, ferric chloride (100 mmll L^–1) was able to solubilize more than 90% of copper and zinc and more than 80% of cadmium, with an optimal pulp density of 3% (w/v), after 10 h of exposition at 25°C. Chromium, lead and nickel were solubilized to an extent of 40–70%. With the exception of copper (redoxolysis), all heavy metals monitored were leached following the principle of acidolysis. Chemical leaching with iron resulted in a secondary contamination of sewage sludge (96 g iron per kg dry weight). The insoluble iron compounds which were precipitated for adsorbed to sludge flocks could be resolubilized with oxalic acid (100 mM, pH<3.3) up to an extent of 90%. Iron was leached by acidolysis and held in solution by complexation with oxalic acid. The pH optimum for the treatment of sewage sludge with 100 mmol L^–1 oxalic acid was pH 3.3. At this pH an excessive solubilization of nutrient elements and compounds (phosphorus, nitrogen, alkali and alkali earth elements) could be avoided concomitantly leaching 75% iron. Furthermore the hydrophobicity of the sewage sludge was significantly reduced as a result of treatment with iron chloride.Thiobacillus ferrooxidans (isolated from arsenopyrite and adapted on sewage sludge) utilized ferrous iron as an energy source in the presence of chloride ions (FeCl₂) as efficiently as ferrous sulphate. No toxic effects of oxalic acid onT. ferrooxidans were observed at the prevailing concentration.     

Effect of iron (III) and its hydrolysis products (jarosites) onThiobacillus ferrooxidans growth and on bacterial leaching

Summary There is a significant inhibition of the growth ofThiobacillus ferrooxidans in the presence of iron (III), but this does not affect bacterial leaching. Moreover, the insoluble hydrolytic products (jarosites) have no influence, except from a mechanical point of view when they are generated in situ.     

Enhancement of Copper Dissolution from a Sulfide Ore by Using Thiobacillus thiooxidans

Copper dissolution from a sulfide ore (with covellite as the main copper phase) was investigated in cultures of Thiobacillus ferrooxidans or Thiobacillus thiooxidans and in abiotic controls. In unsupplemented media, T. ferrooxidans was more efficient than T. thiooxidans. In the presence of ferric iron, the dissolution of covellite was not significantly different in cultures inoculated with T. ferrooxidans or T. thiooxidans. However, the most extraction was found in T. thiooxidans cultures supplemented with ferrous sulfate. The first results were explained by the mechanism proposed by Schippers and Sand (Appl Envir Microbiol 65:319-321, 1999), which involves polysulfides and sulfur as intermediates. This mechanism was extended to explain the behavior of T. thiooxidans culture supplemented with ferrous iron.     

Effect of mineral nutrients and organic substances on the development of Thiobacillus ferrooxidans

The effect of inorganic nutrients on iron oxidation by Thiobacillus ferrooxidans was studied using a concentration of 2.0 g Fe²⁺/liter. Besides ammonium-nitrogen and phosphorus, the essential nutrients were sulphate and magnesium, with the lowest unlimiting concentration being 2.0 g SO/liter and 2.0 mg Mg²⁺/liter for a suspension of about 10⁸ cells/ml. Omitting nitrate, calcium, potassium, and chloride had no influence on the iron oxidation. Nitrate and chloride were inhibitory in high concentrations although the bacteria were able to adapt to their presence. Several organic compounds tested in 0.5% (w/v) concentrations inhibited iron oxidation. Complete inhibition up to 14 days was caused by fructose, lactose, meat extract, yeast, extract, peptone, and tryptone. T. ferrooxidans-strains were able to adapt to the presence of organic material even though they were not necessarily able to use the organic compounds for energy. Some general conclusions are made concerning the role of nutrients in leaching processes.     

Utilization of 35S-Thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans

Differentially labelled ³⁵S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5–4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated K _m value of 0.5 mM for ³⁵S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50°C. In the absence of thiosulphate, the cells bound ³⁵S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-³⁵S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-³⁵S]thiosulphate or ³⁵S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP_sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.Abbreviation APS adenosine-5-sulphatophosphate     

Quantitative measurement of sulphur formation by steady-state and transient-state continuous cultures of autotrophic Thiobacillus species

 Sulphur formation by the obligately chemolithoautotrophic Thiobacillus o and Thiobacillus neapolitanus was studied in aerobic, substrate-limited continuous cultures. The performance of transient-state and steady-state cultures was compared using different methods for measuring sulphur production. Below a dilution rate (D) of 0.3 h^-1 (at 50% air saturation), sulphate-producing steady states were obtained, and cultures grown with sulphide or thiosulphate (at D=0.06 h^-1) showed similar characteristics (e.g. cell yields, oxidation capacities and CO₂-fixation capacities). Elemental sulphur was a major product above D=0.3 h^-1, but steady states were difficult to achieve, because of adherence of sulphur to the fermentor surfaces and the accumulation of sulphide. These problems could be circumvented using transient-state experiments of 1 h. It was then found that elemental sulphur was formed under oxygen limitation or at high substrate load. The rates of sulphur formation obtained by sulphur analysis agreed with the values calculated from stoichiometric balances. Sulphide and thiosulphate proved to be equivalent substrates for both Thiobacillus species during elemental sulphur formation under the conditions tested. It is concluded that transient-state cultures of thiobacilli, pregrown as sulphate-producing steady-state cultures, provide experimental conditions for the quantitative assessment of sulphur formation from (labile) sulphide and from thiosulphate. Received: 15 May 1995 / Received revision: 4 August 1995 / Accepted: 22 August 1995     

Chromium (VI)‐induced oxidative stress,apoptotic cell death and modulation of p53 tumor suppressor gene

Chromium (VI) is a widely used industrial chemical, extensively used in paints, metal finishes, steel including stainless steel manufacturing, alloy cast irons, chrome, and wood treatment. On the contrary, chromium (III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been demonstrated to exhibit a significant number of health benefits in rodents and humans. However, the cause for the hexavalent chromium to induce cytotoxicity is not entirely understood. A series of in vitro and in vivo studies have demonstrated that chromium (VI) induces an oxidative stress through enhanced production of reactive oxygen species (ROS) leading to genomic DNA damage and oxidative deterioration of lipids and proteins. A cascade of cellular events occur following chromium (VI)induced oxidative stress including enhanced production of superoxide anion and hydroxyl radicals, increased lipid peroxidation and genomic DNA fragmentation, modulation of intracellular oxidized states, activation of protein kinase C, apoptotic cell death and altered gene expression. In this paper, we have demonstrated concentration and timedependent effects of sodium dichromate (chromium (VI) or Cr (VI)) on enhanced production of superoxide anion and hydroxyl radicals, changes in intracellular oxidized states as determined by laser scanning confocal microscopy, DNA fragmentation and apoptotic cell death (by flow cytometry) in human peripheral blood mononuclear cells. These results were compared with the concentration-dependent effects of chromium (VI) on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Chromium (VI)induced enhanced production of ROS, as well as oxidative tissue and DNA damage were observed in these cells. More pronounced effect was observed on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Furthermore, we have assessed the effect of a single oral LD₅₀ dose of chromium (VI) on female C57BL/6Ntac and p53deficient C57BL/6TSG p53 mice on enhanced production of superoxide anion, lipid peroxidation and DNA fragmentation in the hepatic and brain tissues. Chromium (VI)induced more pronounced oxidative damage in p53 deficient mice. This in vivo study highlighted that apoptotic regulatory protein p53 may play a major role in chromium (VI)induced oxidative stress and toxicity. Taken together, oxidative stress and oxidative tissue damage, and a cascade of cellular events including modulation of apoptotic regulatory gene p53 are involved in chromium (VI)induced toxicity and carcinogenesis.     

Bacterial removal of chromium (VI) and (III) in a continuous system

The capacity of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans to reduce different concentrations of hexavalent chromium in shake flask cultures has been investigated. A. ferrooxidans reduces 100% of chromium (VI) at concentrations of 1, 2.5 and 5 ppm, but in the presence of 10 ppm only 42.9% of chromium (VI) was reduced after 11 days of incubation. A. thiooxidans showed a lower capacity to reduce this ion and total reduction of chromium (VI) was only obtained for concentrations of 1 and 2.5 ppm, whereas 64.7% and 30.5% was reached for 5 and 10 ppm, respectively, after 11 days. A continuous flow mode system was subsequently investigated, in which A. thiooxidans was immobilized on elemental sulphur and the acidic medium obtained was employed to solubilize chromium (III) and to reduce chromium (VI) present in a real electroplating waste [30% of chromium (III) and 0.1% of chromium (VI)]. The system enabled the reduction of 92.7% of hexavalent chromium and represents a promising way to treat this type of waste in the industry.