A comparative study of characteristic properties ofThiobacillus ferrooxidans strains

Several cultures ofThiobacillus ferrooxidans were purified by isolation of single colonies and simple physiological and biochemical properties of the purified cultures, such as colony morphology, kinetics of ferrous ion oxidation, growth on elemental sulfur and thiosulfate, oxidation of ferrous ion by growing cells in the presence of organic compounds and metal ions, were examined. These properties may be used to characterize a range ofT. ferrooxidans isolates and for numerical taxonomy. This work was part of a project sponsored by theHindustan Copper Limited, Calcutta. The authors are thankful to Dr. P.N. Kundu and Dr. S. Duttagupta for their help in the initiation of this work.     

A Combined Immunofluorescence-DNA-Fluorescence Staining Technique for Enumeration of Thiobacillus ferrooxidans in a Population of Acidophilic Bacteria

An antiserum raised against whole cells of Thiobacillus ferrooxidans was allowed to react with a variety of acidophilic and nonacidophilic bacteria in an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay. Both experiments demonstrated that the antiserum was specific at the species level. This preparation was used to evaluate the role of T. ferrooxidans in the microbial desulfurization process. Leaching experiments were performed, and the numbers of T. ferrooxidans cells and other bacteria were estimated by using a combined immunofluorescence-DNA-fluorescence staining technique that was adapted for this purpose. Nonsterile coal samples inoculated with T. ferrooxidans yielded high concentrations of soluble iron after 16 days. After this period, however, T. ferrooxidans cells could no longer be detected by the immunofluorescence assay, whereas the DNA-fluorescence staining procedure demonstrated a large number of microorganisms on the coal particles. These results indicate that T. ferrooxidans is removed by competition with different acidophilic microorganisms that were originally present on the coal.     

The potential for diazotrophy in iron-and sulfur-oxidizing acidophilic bacteria

Acetylene reduction was observed with ferrousiron-oxidizingThiobacillus ferrooxidans, as expected from previous studies with this bacterium. Acetylene reduction was also found during the growth ofT. ferrooxidans on tetrathionate. OnlyLeptospirillum ferrooxidans, one of several other phylogenetically diverse, ferrous-iron-and/or sulfur-oxidizing acidophilic microorganisms, also reduced acetylene. A reduction of the oxygen concentration in the culture atmosphere was necessary to alleviate inhibition of nitrogenase activity. DNA sequences homologous tonif structural genes were found in both organisms. Diazotrophic growth ofL. ferrooxidans was inferred from an increase in iron oxidation in ammonium-free medium when the oxygen concentration was limited and from apparent inhibition by acetylene under these conditions.     

Effect of oxyanions of sulfur onThiobacillus ferrooxidans: Ferrous ion oxidation,oxygen uptake,and cytochrome reduction

Ferrous ion oxidation byThiobacillus ferrooxidans was completely inhibited by 10 mM each of thiosulfate, sulfite, metabisulfite, bisulfite, and tetrathionate. The inhibition was enhanced in a low pH medium (pH 1.5 versus pH 2.5). Oxygen uptake measurements with Fe²⁺ as the electron donor confirmed the toxicity of thiosulfate, but also indicated its dependency on the concentration of Fe²⁺. Cytochrome spectra of intact cells ofT. ferrooxidans showed that metabisulfite, and thiosulfate to a lesser extent, directly reduced electron transport components, in contrast to no direct reduction of cytochromes by tetrathionate and sulfite.     

Sebastiano Genovese: In Memoriam

Abstract

Laboratory simulations have helped resolve several problems concerning the role of bacteria in producing acidic drainage from active and abandoned coal mines. It is well established that the bacterium Thiobacillus ferrooxidans oxidizes pyrite in synthetic liquid media and in flooded or agitated experimental simulations of coal mine environments. However, many geologists remain skeptical regarding the role of T. ferrooxidans in producing acidity below a near‐surface belt of soil water. We have demonstrated that T. ferrooxidans is capable of colonizing and acidifying a near‐neutral pH environment of crushed coal or overburden, without prior establishment of a pH‐dependent succession of bacteria. We have suggested that T. ferrooxidans may accomplish this by direct oxidation of pyrite. We have also shown that T. ferrooxidans catalyzes pyrite oxidation in the intermediate belt of the zone of aeration, although only for a limited period of time after rainfall infiltration. T. ferrooxidans was not found to be significant in the simulated zone of groundwater saturation.     

Differentiation of acidophilic thiobacilli by cell density in renografin gradients

Thiobacillus acidophilus andT. ferrooxidans were separated by centrifugation on the basis of their cell density in Renografin gradients. For both species, growth history was the largest factor influencing cell density. Density was greatest forT. acidophilus andT. ferrooxidans grown with tetrathionate, followed byT. acidophilus grown with glucose.T. ferrooxidans grown with ferrous sulfate was the least dense.T. acidophilus was isolated from iron-grownT. ferrooxidans by separation in a Renografin gradient. Plasmid patterns ofT. acidophilus andT. ferrooxidans were used to confirm the separation of the two species in mixed gradients.     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.     

Enhancement of uranous-ion oxidation byThiobacillus ferrooxidans

U(IV)-ion oxidation was monitored manometrically in cell suspensions ofThiobacillus ferrooxidans. The bacteria accelerated the oxygen uptake associated with U⁴⁺-oxidation, possibly resulting from cyclic regeneration of residual amounts of ferric ion in the cell suspension. The rate of chemical oxidation in dilute sulfuric acid was not affected by addition of autoclaved cells.     

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.     

Microbial Communities in Acid Mine Drainage and Their Interaction with Pyrite Surface

Microbes such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans have been investigated a lot, because of their important role in acid mine drainage (AMD) generation. In this article, the composition of microbial communities in two AMD samples was studied. A culture-independent 16S rDNA-based cloning approach, restriction fragment length polymorphism has been used. The interaction between microbes and natural pyrite specimen surface was researched by scanning electrode microscopy (SEM) and fluorescence in situ hybridization (FISH). The phylogenetic analysis revealed bacteria in these two samples fell into three major groups: Proteobacteria, Nitrospira, and Firmicutes. Archaea was also detected in these two samples. Thermoplasma and Ferroplasma lineages were abundant. From SEM and FISH, a number of A. ferrooxidans, a few cells of Archaea and Acidiphilium were detected adsorbed on the pyrite specimen surface. Leptospirillum sp. (hybridize with the probe LF655) has not been detected to be present on the pyrite specimen surface.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Immobilisation of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulfate oxidation

The immobilisation of the iron-oxidising bacteria Thiobacillus ferrooxidans on nickel alloy fibre as support is described. This matrix showed promise for application in iron oxidation under strongly acidic conditions. The influence on the colonisation process of T. ferrooxidans exerted by the initial pH of the medium and by temperature has also been studied. Results showed that immobilisation of T. ferrooxidans cells was affected by changes of temperature between 30 °C and 40 °C and in pH from 1.4 to 2.0. Received: 25 January 2000 / Received version: 20 April 2000 / Accepted: 1 May 2000     

Proteomic insights into cold adaptation of psychrotrophic and mesophilic <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains

Cold tolerant strains of Acidithiobacillus ferrooxidans play a role in metal leaching and acid mine drainage (AMD) production in northern latitude/boreal mining environments. In this study we used a proteomics and bioinformatics approach to decipher the proteome changes related to sustained growth at low temperatures to increase our understanding of cold adaptation mechanisms in A. ferrooxidans strains. Changes in protein abundance in response to low temperatures (5 and 15°C) were monitored and protein analyses of a psychrotrophic strain (D6) versus a mesophilic strain (F1) showed that both strains increased levels of 11 stress-related and metabolic proteins including survival protein SurA, trigger factor Tig, and AhpC-Tsa antioxidant proteins. However, a unique set of changes in the proteome of psychrotrophic strain D6 were observed. In particular, the importance of protein fate, membrane transport and structure for psychrotrophic growth were evident with increases in numerous chaperone and transport proteins including GroEL, SecB, ABC transporters and a capsule polysaccharide export protein. We also observed that low temperature iron oxidation coincides with a relative increase in the key iron metabolism protein rusticyanin, which was more highly expressed in strain D6 than in strain F1 at colder growth temperatures. We demonstrate that the psychrotrophic strain uses a global stress response and cold-active metabolism which permit growth of A. ferrooxidans in the extreme AMD environment in colder climates.     

Studies on the growth of Thiobacillus ferrooxidans

Summary A method for enumeration of viable numbers of Thiobacillus ferrooxidans using membrane filters on ferrous-iron agar is presented. Factors affecting colony production were the concentration and brand of agar, pH of the medium, and type of membrane filter. The results suggest that inhibition of T. ferrooxidans by agar is a result of the acid hydrolysis of agar, the main product of which is d-galactose. Colony development was suppressed by aged medium, by acid-hydrolysed agar and by 0.1% galactose. Sartorius and Millipore membrane filters were suitable for the experiments, whereas Oxoid MF-50 membranes virtually suppressed the production of colonies. The method was employed to follow growth of T. ferrooxidans in pH 1.3 medium. The viable cell numbers were correlated with ¹⁴CO₂-fixation and ferrous iron oxidation. Generation time was 6 h 22 min with a yield of 2.2×10¹² organisms/g atom Fe²⁺ oxidized. Growth of T. neapolitanus on thiosulphate medium was not affected by agar-type or membrane filters and yield of the organism was 1.5×10¹³ organisms/g molecule Na₂S₂O₃ oxidized.     

Growth and Maintenance of Thiobacillus ferrooxidans Cells

A rapid and sensitive spectrophotometric procedure was developed for monitoring the growth of Thiobacillus ferrooxidans in liquid culture. Values determined for the optical densities at 500 nm of washed T. ferrooxidans cell suspensions were directly proportional to both total cell number and total cell protein concentration and provided an accurate measurement of culture growth rate. The utility of this procedure was demonstrated by conducting physiological studies on the influence of CO₂ and FeSO₄ availability on the growth of T. ferrooxidans. In addition, we describe a procedure for the long-term maintenance of cells T. ferrooxidans that ensures culture purity and genetic stability.     

Microbial leaching of arsenic from low‐sulfide gold mine material

Drainages from high‐sulfide tailings near abandoned lode deposits in Alaska, U.S.A., and Yukon, Canada, were found to be acidic, to contain large numbers of Thiobacillus ferrooxidans, and to have high concentrations of dissolved arsenic. Drainages from active placer gold mines are not acidic, but T. ferrooxidans and concentrations of dissolved arsenic exceeding 10 μg/L are found in some streams affected by placer mine drainage. Placer mine material containing low amounts of sulfides (326 (μg/g) and moderately high amounts of arsenic (700 μg/g) was leached with growing cultures of T. ferrooxidans, T. ferrooxidans‐spent filtrate, and acid ferric sulfate. The results showed that while more arsenic was released from this material by growing cultures of T. ferrooxidans than by abiotic controls, acid ferric sulfate released much more arsenic than did either growing cultures of T. ferrooxidans or spent culture filtrate containing oxidized iron. Cation exchange chromatography showed that oxidized iron from T. ferrooxidans culture filtrate is chemically less reactive than the iron in aqueous solutions of ferric sulfate salt. These results indicate that arsenic release from both high‐ and low‐sulfide mine wastes is enhanced biologically, but that rates and amounts of arsenic release are primarily controlled by iron species.     

Seasonal Variations in Microbial Populations and Environmental Conditions in an Extreme Acid Mine Drainage Environment

Microbial populations, their distributions, and their aquatic environments were studied over a year (1997) at an acid mine drainage (AMD) site at Iron Mountain, Calif. Populations were quantified by fluorescence in situ hybridizations with group-specific probes. Probes were used for the domains Eucarya, Bacteria, and Archaea and the two species most widely studied and implicated for their role in AMD production, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans. Results show that microbial populations, in relative proportions and absolute numbers, vary spatially and seasonally and correlate with geochemical and physical conditions (pH, temperature, conductivity, and rainfall). Bacterial populations were in the highest proportion (>95%) in January. Conversely, archaeal populations were in the highest proportion in July and September (~50%) and were virtually absent in the winter. Bacterial and archaeal populations correlated with conductivity and rainfall. High concentrations of dissolved solids, as reflected by high conductivity values (up to 125 mS/cm), occurred in the summer and correlated with high archaeal populations and proportionally lower bacterial populations. Eukaryotes were not detected in January, when total microbial cell numbers were lowest (<10⁵ cells/ml), but eukaryotes increased at low-pH sites (~0.5) during the remainder of the year. This correlated with decreasing water temperatures (50 to 30°C; January to November) and increasing numbers of prokaryotes (10⁸ to 10⁹ cells/ml). T. ferrooxidans was in highest abundance (>30%) at moderate pHs and temperatures (~2.5 and 20°C) in sites that were peripheral to primary acid-generating sites and lowest (0 to 5%) at low-pH sites (pH ~0.5) that were in contact with the ore body. L. ferrooxidans was more widely distributed with respect to geochemical conditions (pH = 0 to 3; 20 to 50°C) but was more abundant at higher temperatures and lower pHs (~40°C; pH ~0.5) than T. ferrooxidans.     

Metabolic reconstruction of sulfur assimilation in the extremophile <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> based on genome analysis

Background  

Acidithiobacillus ferrooxidansis a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences ofA. ferrooxidansare available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism.     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.