Advanced Materials Research Vols. 71-73

Abstract: This study focused on the soils of sofataric region and acid mine drainage from a copper mine. Based on cultivation, 8 and 6 strains that grow on Fe(II) and sulfur compounds, respectively, were obtained from samples from these two environments. Analysis of 16S rRNA genes of the 14 strains indicated that they were affiliated to Acidithiobacillus, Alicyclobacillus, Sulfobacillus and Leptospirillum. Physiological and phylogenetic studies indicated that three strains (TC-34, TC-71 and ZJ-6) might represent three novel members of Alicyclobacillus. These strains showed 94.8-97.1% 16S rRNA gene identity to other species of Alicyclobacillus. Otherwise, strain TC-34, TC-71 and ZJ-6 showed a range of phenotypic characteristics that differentiated them from previously recognized Alicyclobacillus species, including the growth temperature, assimilation of carbon sources and production of acids from a range of compounds. Chemoautotrophic growth using Fe2+, elemental sulfur and tetrathionate as sole energy source was observed. Especially strain TC-71 was obligately dependent on Fe(II) for growth and quickly oxidized Fe2+. It is concluded that the Fe(II)-oxidizers are metabolically diverse and represent novel Alicyclobacillus species. These are proposed to take part in biogeochemical cycling of iron and sulfur in the solfataric region and could be relevant for biomining.

Abstract: Sulfobacillus thermosulfidooxidans cultures adapted to ferrous or tetrathionate ions were inoculated into media containing both substrates. In batch culture, cells showed a preference for oxidising ferrous ion followed by tetrathionate. Tetrathionate oxidation was slower in ferrous-adapted cells. Biomass formation exhibited an exponential growth phase during ferrous ion oxidation followed by an exponential growth phase during tetrathionate oxidation. Sequential utilisation of ferrous ion followed by tetrathionate ion was observed when equimolar amounts of substrates or electron-equivalent amounts were provided.

Abstract: The effects of temperature, pH and iron concentration on the kinetics of ferrous iron biooxidation by a free suspended culture of Leptospirillum ferriphilum were studied in shake flasks and a circulating bed bioreactor at moderate to high total iron concentration. The kinetic study showed that there are two distinct modes of iron biooxidation: growth associated and non-growth associated, depending on the pH of the medium. There were also distinctive maxima of the effect of temperature and pH on the rate of biooxidation. A kinetic model of the process was proposed, based on an electrochemical-enzymatic model. The proposed model indicates that at moderate to high concentrations (above ~12 g/L), the total iron concentration becomes the single most prominent inhibiting factor.

Abstract: The kinetics of microbial ferrous-iron oxidation have been well studied as it is a critical sub-process in bioleaching of sulphide minerals. Exhaustive studies in continuous culture have been carried out recently, investigating the effects of conditions relevant to heap bioleaching on the microbial ferrous-iron oxidation by Leptospirillum ferriphilum [1-3]. It was postulated that ferric-iron, which is known to be inhibitory, also acts as a stress stimulus, promoting microbial growth at higher total iron concentration. This paper investigates this phenomenon further, by comparing tests run with pure ferrous-iron feeds against those where the feed is partially oxidised to ferric at comparable concentrations. The findings clearly suggest that, contrary to reactor theory, it is indeed ferrous iron concentration in the reactor feed that determines biomass concentration and that ferric iron concentration has little effect on microbial growth. Further mathematical analysis shows that the phenomenon can be explained on the basis of the Pirt equation and the particular reaction conditions employed in the test work.

Abstract: The process of biological oxidation of ferrous iron by the microorganism Leptospirillum ferriphilum was studied in different types of biofilm reactors. A total of 12 bioreactor types with both fixed and mobile support were used. The biofilm supports used had different shapes and were made of: polyethylene, nylon, polystyrene, polyvinylidene difluoride, polyester, ceramics, carbon and PVC. The pH of the microbial medium was maintained around 1.0, the temperature was 40°C while the influent ferrous iron concentration was 20 g/L. Iron oxidation rates of up to 4.5 g/L/h were obtained. The most efficient were the bioreactors with polyethylene and nylon woven fabric support. However, they oxidized ferrous iron at high rate for relatively short time periods - 40 to 60 days. While the bioreactor with a fixed bed of Raschig rings support had lower iron oxidation rate, their long-term stability was much higher. Of all the biofilm support materials tested, it was found that only polyvinylidene difluoride did not allow the formation of biofilm. Since no significant amounts of jarosite were formed at pH of 1.0 and below, the biofilm formed was very weak mechanically. For that reason the moving support, such as inverse fluidized bed, was not very appropriate because of the high shear stress.

Abstract: L. ferrooxidans plays a significant role in bioleaching process of ores. Being a chemoautotrophic bacterium, its sources of carbon and energy are independent. In this study were measured separately in a chemostat, the effect of growth limited by each source (CO2 and Fe(II)), and under conditions of inhibition (Fe(III)) on metabolic parameters of the cell. The runs were carried out in a bioreactor with 1.25 liter of KJ culture medium at 33.5 °C, pH 1.8, agitation rate of 300 rpm and aeration rate of 2 VVM. Using only air as CO2 source, it was established that the cells suffer simultaneous limitation of carbon and energy. It was determined that these limitations are released separately when enriching the air with 4% CO2 in one case and when doubling concentration of Fe(II) in the feed stream in the other. Under double limitation maximum yield (YºX/S) and maintenance coefficient (m) were 6.0•10-3 gcel/gFe(II) and 2.48 gFe(III)/gcel•h respectively. Growth limited only by carbon source and only by energy source gave YºX/S 11.5•10-3 and 21.9•10-3 gcel/gFe(II), whereas m was 1.23 and 0.11 gFe(II)/gcel•h respectively. On the other hand, the lower specific Fe(III) production rate (qP) was obtained when cell growth was limited by the energy source and the higher value was observed during growth in presence of 15 g/L exogenous Fe(III). The qP values at D = 0.04 h-1 were 1.93 and 14.04 gFe(III)/gcel•h respectively. In general, the worse the culture conditions the highest the specific rate of Fe(III) production. The bacterium varied its metabolic parameters quite broadly depending on the growth limiting nutrient and presence of Fe(III).

Abstract: Novel iron- and sulfur-oxidizing, moderate thermophiles were isolated from an acidic geothermal site and from a previously studied, pyrite-enrichment mixed culture (which also contained the related actinobacterium Acidimicrobium ferrooxidans). The novel species (proposed genus “Acidithiomicrobium”) grew autotrophically with ferrous iron at an optimum temperature of about 50°C, efficiently degraded pyrite at 55°C and also grew well autotrophically on sulfur. The extensive dissolution of pyrite during autotrophic growth contrasted with a requirement for yeast extract for significant growth of the related Acidimicrobium ferrooxidans.

Abstract: We applied a kit for bioluminescent ATP measurement based on the luciferase reaction to Acidithiobacillus ferrooxidans cultures. Determination limit for monitoring cell growth in the culture was 1.1 × 106 cells per ml. Determination errors varied between 1.5 and 10 % (RSD). ATP formation correlated with cell growth and iron oxidation up to limitation by iron(II). A maximum ATP content was approximately 1 amol per cell under the growth yield on iron of 1 × 1012 cells per mol Fe(II).

Abstract: Extremophiles such as the acidophilic Sulfolobus metallicus (Archaea) and Acidithiobacillus ferrooxidans (Bacteria) can resist Cu (CuSO4) concentrations of 200 mM and 800 mM respectively. These microorganisms are important in biomining processes to extract copper and other metals. A. ferrooxidans grown at low Cu concentrations (5 mM) expressed genes coding for ATPases most likely involved in pumping the metal from the cytoplasm to the periplasm of the bacterium. At 100 mM Cu the previous systems were repressed and there was a great induction in the expression of efflux systems known to use the proton motive force energy to export the metal outside the cell. These Cu-resistance determinants from A. ferrooxidans were found to be functional since when expressed in Escherichia coli they conferred higher Cu tolerance to it. Novel Cu-resistance determinants for A. ferrooxidans were found and characterized. S. metallicus possessed at least 2 CopM metallochaperones and 2 CopA ATPases whose expressions were induced by Cu (5 to 50 mM). Furthermore, we previously reported that both microorganisms accumulate high levels of inorganic polyphosphate (PolyP) and that intracellular Cu concentration stimulates polyP hydrolysis. The resulting Pi would then be transported out of the cell as a metal-Pi complex to detoxify the cells. In addition, our results suggest that at high Cu concentrations polyP could also provide energy for the metal efflux. All the data suggest that both biomining microorganisms use different systems to respond to Cu depending on the extracellular concentrations of the metal and suggest that the presence of different additional systems to respond to Cu may explain the extremely high metal resistance of these extremophiles.

Abstract: In this study an acidic saline drain in the Western Australian wheat belt was sampled and enriched for salt tolerant chemolithotrophic microorganisms in acidic media containing up to 100 gL-1 NaCl. A mixed consortium was obtained which grows at pH 1.8 and oxidises iron (II) in the presence of up to 30 gL-1 NaCl. In comparative tests (growth rates and iron (II) oxidation rates) it was found that NaCl concentrations >3.5 gL-1 generally cause reduced growth and iron (II) oxidation rates in known biomining organisms. The results help to set a benchmark for NaCl tolerance in known biomining microorganisms and will lead to the generation of a consortium of microorganisms that can oxidise iron (II) effectively in saline process water.