Moderate thermophiles including “Ferroplasma cupricumulans” sp. nov. dominate an industrial-scale chalcocite heap bioleaching operation

This study describes the microbiology of the MICCL Monywa chalcocite heap bioleaching operation. Microorganisms were detected in the heap using culture-based techniques and culture-independent PCR-DGGE analysis of the 16S rRNA gene. The metabolic requirements, temperature and pH optima were determined for selected strains isolated from the heap material. Six strains (BH1-BH6) were enriched and isolated from heap solids and leachate samples. Phylogenetic analysis of the 16S rRNA genes showed that strains BH3 and BH4 were closely related to the acidophilic bacterium Acidithiobacillus caldus and strains BH5 and BH6 were closely related to Leptospirillum ferriphilum. The optimum growth temperature for one of the L. ferriphilum strains was 41.3 °C and the optimum pH range was 1.1 to 1.5. A new species of Archaea was isolated from the heap samples. Cells of the proposed species, "Ferroplasma cupricumulans" (formerly "Ferroplasma cyprexacervatum"), were non-motile pleomorphic cocci, capable of chemomixotrophic growth with ferrous sulphate and yeast extract. Cells grew anaerobically on potassium tetrathionate and yeast extract as electron donors with ferric iron as the electron acceptor. Growth occurred from 22 °C to 63 °C, with an optimum temperature of 53.6 °C. The optimal pH for growth was 1.0-1.2. Phylogenetic analysis based on the 16S rRNA gene showed the most closely related described species to strain BH2 was Ferroplasma acidiphilum YT (95% sequence similarity). Molecular analysis of mine samples (16S rRNA PCR-DGGE) detected the above-mentioned cultured strains as well as species of Sulfobacillus and unknown representatives of the Proteobacteria. The microbial community of the Myanmar bioleach heaps contained similar levels of diversity to a dump bioleach operation in Chile, but contained greater amounts of biomass than another heap bioleach operation in Australia. The operating parameters of the Myanmar heaps have selected for the growth of moderately thermophilic microorganisms that are more commonly found in extremely low pH acidic mine drainage sites than in heap bioleach systems. The novel species of Ferroplasma is considered to be involved in the aerobic and anaerobic cycling of iron within the heap bioleaching environment

Microbiology of an industrial-scale chalcocite heap bioleaching operation

A study was conducted to describe the microbiology of the MICCL Monywa chalcocite heap bioleaching operation. Microorganisms were detected in the heap using culture-based techniques and culture-independent PCR-DGGE analysis of the 16S rRNA gene. The metabolic requirements, temperature and pH optima were determined for selected strains isolated from the heap material. Six strains (BH1-BH6) were enriched and isolated from heap solids and leachate samples. Phylogenetic analysis of the 16S rRNA genes showed that strains BH3 and BH4 were closely related to the acidophilic bacterium Acidilhiobacillus caldus and strains BH5 and BH6 were closely related to Leptospirillum ferriphilum. The optimum growth temperature, for L. ferriphilum was 41.3°C and the optimum pH range was 1.1 to 1.5. A new species of Archaea was isolated from the heap samples. Cells of the proposed species, Ferroplasma cyprexacervatum ('cyprus' L.n. meaning copper; 'exacervo' L.v. meaning 'to heap up'), are non-motile pleomorphic cocci, capable of chemomixotrophic growth with ferrous sulfate and yeast extract. Cells grow anaerobically on ferric iron in the presence of potassium tetrathionate and yeast extract as electron donors. Growth occurs from 14°C to 63°C, with an optimum temperature of 55.2°C. The optimal pH for growth was 1.0-1.2. The species was phylogenetically most closely related to Ferroplasma acidiphilum with a 16S rRNA gene sequence similarity of 95 per cent. Molecular analysis of mine samples (16S rRNA PCR-DGGE) detected the above-mentioned cultured strains of microorganisms as well as species of Sulfobacillus and other unknown Proteobacteria. The microbial community of the Myanmar bioleach heaps contained similar levels of diversity to a dump bioleach operation in Chile, but contained greater amounts of biomass than another heap bioleach operation in Australia. The operating parameters of the Myanmar heaps (low pH and moderate temperature) have, selected for the growth of moderately thermophilic microorganisms that are more, commonly found in extremely low pH acidic mine drainage sites than in heap systems. The novel species of Ferroplasma is thought to be involved in the aerobic and anaerobic cycling of iron within the. heap bioleaching environment

Geographical distribution and diversity of moderately thermophilic members of the thermoplasmatales

Oxidation of ferrous iron by moderately thermophilic species of the genus Ferroplasma is of considerable potential value in commercial bioleaching operations. A collection of strains was enriched and isolated from a number of natural or industrial acidic sites at 45 and 55°C. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all strains were members of the Order Thermoplasmatales. Stains BH8 and BH12 showed 98% sequence similarity with Ferroplasma acidiphilum strain YT and DR1 respectively. Strains BH7 and BH10 were closely related to the moderately thermophilic species Ferroplasma cupricumulans. Strain BH9, a moderately thermophilic organotroph, was unrelated to previously described species and probably represents a novel genus. Strains BH8 and BH12 showed some unique physiological differences to the type species F. acidiphilum. Unlike F. acidiphilum, both strains were moderately thermophilic with a temperature range for strains BH8 and BH12 of 24 to 61°C and 27 to 49°C respectively. Strain BH12 grew organotrophically on Darland's Medium containing glucose and yeast extract and chemomixotrophically on Darland's Medium supplemented with ferrous sulphate. Moderately thermophilic species of Ferroplasma and Ferroplasma-like genera appear widely distributed geographically and possess considerable physiological and phylogenetic diversity that may benefit industrial bioleaching processes

Ferroplasma cupricumulans sp. nov., a novel moderately thermophilic, acidophilic archaeon isolated from an industrial-scale chalcocite bioleach heap

A new species of Archaea was isolated from an industrial mineral sulphide bioleach heap. Strain BH2, a non-motile pleomorphic coccus, was capable of chemomixotrophic growth on ferrous sulphate and yeast extract. Growth was not supported in the absence of yeast extract. Phylogenetic analysis based on the 16S rRNA gene showed that strain BH2 was most closely related to the species Ferroplasma acidiphilum; however, it showed only 95% sequence similarity with this species. Strain BH2 had a temperature optimum of 53.6°C and a temperature range for growth between 22 and 63°C. Thus, it is the first moderately thermophilic member of the genus Ferroplasma. The optimum pH for the growth of the strain occurred between pH 1.0 and 1.2 and the lowest pH at which growth was observed was 0.4. Based on 16S rRNA gene sequence analysis and other physiological characteristics, strain BH2 constitutes a new species within the genus Ferroplasma. The name Ferroplasma cupricumulans is proposed for the new species and strain BH2 (DSM 16651) is proposed as the type strain

Degradation of 2-nitrodiphenylamine, a component of Otto Fuel II, by Clostridium spp.

Otto Fuel II, a propellant in torpedoes, is composed of 76% 1,2 propanediol dinitrate (PGDN), 22.5% di-n-butyl sebacate, and 1.5% 2-nitrodiphenylamine (NDPA), and is largely recalcitrant to aerobic microbial degradation. Anaerobic microbial degradation of Otto Fuel II was tested by inoculating anaerobic enrichment media, containing either 2% (vol:vol) complete Otto Fuel II or 2% of a 0.02% solution of Otto Fuel II in methanol, with soil and water from sites contaminated with munitions or with landfill leachate. Anaerobic bacterial growth was completely inhibited by 2% Otto Fuel II. Two mixed bacterial enrichments developed in anaerobic media containing 2% (v/v) of a 0.02% solution of Otto Fuel II in methanol. After incubation, PGDN could not be detected in either enrichment, but was also not detectable in sterile controls, suggesting abiotic degradation of low concentrations of PGDN in reduced anaerobic medium. NDPA did not degrade in either enrichment. Similarly, complete Otto Fuel was recalcitrant to degradation by highly reducing methanogenic biomass collected from an upflow anaerobic sludge blanket bioreactor (UASB). A comparison of the degradative ability of autoclaved and viable biomass showed that low concentrations of PGDN autodegraded, however unlike the autoclaved anaerobic biomass, the viable anaerobic biomass degraded the NDPA component of Otto Fuel II. Two strains of anaerobic clostridia, strains SP3 and SPF, that caused the disappearance of NDPA at its limit of solubility in culture media, were isolated from the UASB bioreactor biomass. SP3 and SPF were shown, by comparison of 16S rDNA sequences, to be most closely related to Clostridium butyricum and Clostridium cochlearium respectively. Although NDPA was lost from cultures of both strains, metabolic endproducts were not identified. Neither strain could degrade NDPA unless supplied with an alternative energy source. In the culture system used, NDPA stimulated the growth of SP3 but it had no appreciable effect on the growth of SPF. Both SP3 and SPF degraded low concentrations of trinitrotoluene (TNT), without the production of detectable concentrations of aromatic amines. A possible method for the remediation of small spills of Otto Fuel II is suggested

Effects of temperature on the rates of iron and sulfur oxidation by selected bioleaching Bacteria and Archaea: Application of the Ratkowsky equation

Bioleaching rates are affected by temperature and temperature is a major selective pressure for the organisms that will inhabit a bioleaching operation. The relationship of microbial activity to temperature is best described by the Ratkowsky equation. Ratkowsky and Arrhenius equations were applied to temperature versus sulfur or ferrous iron oxidation data for selected bioleaching microorganisms. Extrapolated cardinal temperatures for iron and sulfur oxidation, and activation energies were derived from the fitted Ratkowsky and Arrhenius equations. The data provided definition of the temperature operating windows, to a precision not previously available, for the majority of species that are frequently encountered as the biological catalysts in tank and heap bioleaching environments