Continuous microbial leaching of a pyritic concentrate by Leptospirillum-like bacteria

Summary Continuous leaching of a pyritic flotation concentrate by mixed cultures of acidophilic bacteria was studied in a laboratory scale airlift reactor. Enrichment cultures adapted to the flotation concentrate contained Thiobacillus ferrooxidans and Thiobacillus thiooxidans. During the late stationary growth phase of these thiobacilli growth of Leptospirillum-like bacteria was observed, too. In discontinuous cultivation no significant influence of Leptospirillum-like bacteria on leaching rates could be detected. During continuous leaching at pH 1.5 Leptospirillum-like bacteria displaced Thiobacillus ferrooxidans. The iron leaching rate achieved by Leptospirillum-rich cultures was found to be up to 3.9 times higher than that by Leptospirillum-free cultures.     

Reduction of dichromate by Thiobacillus ferrooxidans

Chromium(VI) was reduced by Thiobacillus ferrooxidans grown with elemental sulphur as the sole energy source. Chromium(VI) reduction (as high as 2000 M), was due to the presence of sulphite and thiosulphate, among others with high reducing power which was generated during the sulphur oxidation by the bacteria. Therefore, Thiobacillus ferrooxidans could be used to treat chromium(VI)-containing industrial effluents.     

Antigenic determinants and specificity of antisera against acidophilic bacteria

Polyclonal antisera, produced against whole cells of Thiobacillus thiooxidans, T. ferrooxidans and Leptospirillum ferrooxidans, gave highly specific reactions when cross-reacted with 23 strains of acidophilic bacteria using an immunofluorescence (IF) staining technique. Strains of identical serotype exhibited maximum cross-reaction whereas strains of different serotype reacted only weakly. Lipopolysaccharides (LPS) examined by SDS-PAGE showed different, serotype-specific migration patterns indicating their rough or smooth character. LPS patterns may therefore be used for serological classification of acidophilic bacteria. Surface antigens of four strains were identified by immunoblot staining; LPS and some proteins were antigenic determinants with LPS the most specific.The authors are with the Universität Hamburg, Institut für Allgemeine Botanik, Abteilung für Mikrobiologie, Ohnhorststrasse 18, D-22609 Hamburg, Germany     

Flagella and Pili of Iron-Oxidizing Thiobacilli Isolated from a Uranium Mine in Northern Ontario, Canada

Five strains of Thiobacillus ferrooxidans, which included three recent isolates from a uranium mine, possessed flagella. Three of the strains had several pili per cell. The dimensions, fine structure, and orientation of the flagella were different. Both polar and peritrichous flagella were observed, indicating strain-dependent ultrastructural variation in acidophilic thiobacilli. Neither flagella nor pili were detected in eight other strains of T. ferrooxidans and two strains of Thiobacillus acidophilus by electron microscopy, although all of the cultures contained motile cells.     

Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using ³²P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T_d) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T_ds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.     

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.     

Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan

Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51?mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis. The analysis of the obtained sequences showed their similarity to clones recently detected in other moderately acidic mine drainages. Uncultured bacteria related to Ferrovum- and Gallionella-like clones were dominant in the microbial community. Analyses using specific primers for acidophilic iron- or sulfur-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum spp., Acidithiobacillus caldus, Acidithiobacillus thiooxidans, and Sulfobacillus spp. revealed the absence of these bacteria in the microbial community in ARD from the Yasumi-ishi tunnel. Clones affiliated with a member of the order Thermoplasmatales were detected as the dominant archaea in the ARD microbial population.     

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.     

In situ assessment of active Thiobacillus species in corroding concrete sewers using fluorescent RNA probes

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of concrete corrosion in sanitary sewers. Thiobacillus species are often considered the major representative of the acid-producing bacteria in these environments, and members of the genus Acidiphilium have been implicated to support their growth. Active populations of selected Thiobacillus, Leptospirillum, and Acidiphilium species were compared to total bacterial populations growing on the surfaces of corroding concrete using three oligonucleotide probes that have been confirmed to recognize unique sequences of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and Thiobacillus thiooxidans (probe: Thio820), Leptospirilium ferrooxidans (Probe: Lept581) and members of the genus Acidiphilium (probe: Acdp821). With these genetic probes, fluorescent in situ hybridizations (FISH) were used to identify and enumerate selected bacteria in homogenized biofilm samples taken from the corroding crowns of concrete sewer collection systems operating in Houston, Texas, USA. Direct epifluorescent microscopy demonstrated the ability of FISH to identify significant numbers of active acidophilic bacteria among concrete particles, products of concrete corrosion (e.g. CaSO₄), and other mineral debris. As judged by FISH analyses with the species-specific probe Thio820, and a domain-level probe that recognizes all Bacteria (Eub338), T. ferrooxidans and T. thiooxidans comprised between 12% and 42% of the total active Bacteria present in corroding concrete samples. Although both Acidiphilium and Leptospirillum have also been postulated to have ecological significance in acidic sulfur-oxidizing environments, neither genera was detected using genus-specific probes (Lept581 and Acdp821).     

Generation of acid mine drainage around the Karaerik copper mine (Espiye,Giresun, NE Turkey): implications from the bacterial population in the Acısu effluent

The Karaerik Cu mine is a worked-out deposit with large volumes of tailings and slags which were left around the mine site without any protection. Natural feeding of these material and run-off water from the mineralised zones into the Ac?su effluent causes a serious environmental degradation and creation of acid mine drainage (AMD) along its entire length. This research aims at modelling the formation of AMD with a specific attempt on the characterisation of the bacterial population in association with AMD and their role on its occurrence. Based on 16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community was determined to consist of Acidithiobacillus ferrivorans, Ferrovum myxofaciens, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans as iron-oxidising bacteria, Acidocella facilis, Acidocella aluminiidurans, Acidiphilium cryptum and Acidiphilium multivorum as iron-reducing bacteria, and Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidiphilium cryptum as sulphur-oxidising bacteria. This association of bacteria with varying roles was interpreted as evidence of a concomitant occurrence of sulphur and iron cycles during the generation of AMD along the Ac?su effluent draining the Karaerik mine.     

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.     

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.     

Phagotrophy and NH4+ regeneration in a three-member microbial food loop

In a series of batch experiments we compared the efficiencyof nitrogen regeneration of a two- and three-member microbialfood loop consisting of a mixed bacterial assemblage, a small(3–5 µm) heterotrophic flagellate (Paraphysomonassp.), and a large (7–12 µm) heterotrophic flagellate(Paraphysomonas imperforata). In the two-member system the nitrogenregeneration efficiency for NH₄⁺ (R_n) was 41% and the grossgrowth efficiency (GGE) was 57% during active grazing by thesmall flagellate on bacteria. Regeneration of NH₄⁺ continuedduring the stationary phase so that R_n was 75% after 6 daysincubation. When the larger flagellate was introduced at theend of exponential growth of the smaller grazer in the three-membersystem, initially there was rapid regrowth of bacteria, tyingup 15% of the nitrogen originally in the bacteria. The largerflagellate grazed the smaller one with a GGE of 55%. Total nitrogenregeneration efficiency through exponential growth of the largerflagellate was 73%. Because microbial food loops in naturalwaters are far more complicated and with more grazing stepsthan portrayed in this study, we would expect the bulk of nutrientswithin these systems to be recycled with little transfer tohigher trophic levels.     

Morphological Response of Microcystis aeruginosa to Grazing by Different Sorts of Zooplankton

In the experiment we investigated the effect of grazing by different sorts of zooplankton on the induction of defensive morphology in the cyanobacterium Microcystis aeruginosa. The results showed that protozoan flagellate Ochromonas sp. grazing could induce colony formation in M. aeruginosa, whereas M. aeruginosa populations in the control and the grazing treatments of copepod Eudiaptomus graciloides, cladoceran Daphnia magna, and rotifer Brachionus calyciflorus were still strongly dominated by unicells and paired cells and no colony forma occurred. In the protozoan grazing treatment, the proportion of unicells reduced from 83.2% to 15.7%, while the proportion of cells in colonial form increased from 0% to 68.7% of the population at the end of the experiment. The occurrence of a majority of colonial M. aeruginosa being in the treatment with flagellates, indicated that flagellate grazing on solitary cells could induce colony formation in M. aeruginosa. The colonies could effectively deter flagellate from further grazing and thus increase the survival of M. aeruginosa. The colony formation in M. aeruginosa may be considered as an inducible defense against flagellate grazing under the conditions that toxin cannot deter flagellate from grazing effectively.     

Thiobacillus plumbophilus spec. nov., a novel galena and hydrogen oxidizer

From an uranium mine three strains of rodshaped, mesophilic, chemolithoautotrophic bacteria were isolated. They grow by oxidation of H₂S, galena (PbS) and H₂. Anglesite (PbSO₄) is formed from galena. No ferrous iron is oxidized by the isolates. They grow between pH 4 and 6.5 at temperatures of about 9 to 41°C (optimum around 27°C). The G+C content of the DNA is around 66 mol %. Based on their ability to oxidize sulfur compounds, the new organisms belong to the genus Thiobacillus. No significant homology with Thiobacillus ferrooxidans and Thiobacillus cuprinus was detected by DNA-DNA hybridization. Therefore the new isolates represent a new species within the genus Thiobacillus. Based on the unusual growth on galena, we name the new species Thiobacillus plumbophilus (type strain Gro 7; DSM 6690).     

An indirect fluorescent antibody staining technique for determining population levels ofThiobacillus ferrooxidans in acid mine drainage waters

Thiobacillus ferrooxidans is believed to be responsible for the oxidation of ferrous ion at low pH, the rate-limiting step in the oxidation of pyrite ores and subsequent formation of acid mine drainage (AMD). It has been suggested that efforts to control this environmental problem include procedures that would inhibit this bacterium. At present, a most probable number (MPN) procedure requiring a minimum of 10 days is used to enumerate this microorganism in natural waters. If control of AMD through inhibition ofT. ferrooxidans is to be feasible, it will be necessary to develop a more rapid method to determine population levels to facilitate application of control measures.An indirect fluorescent antibody (FA) staining technique was developed for this purpose which provided reliable estimates within a few hours. Artificial samples containing approximated numbers ofT. ferrooxidans were analyzed using the FA and MPN procedures, and the FA technique more closely approximated expected numbers of cells. The MPN method was excessively conservative, detecting only 3% to 21% of the cells enumerated by the FA procedure.     

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.     

Ferric Iron Reduction by Acidophilic Heterotrophic Bacteria

Fifty mesophilic and five moderately thermophilic strains of acidophilic heterotrophic bacteria were tested for the ability to reduce ferric iron in liquid and solid media under aerobic conditions; about 40% of the mesophiles (but none of the moderate thermophiles) displayed at least some capacity to reduce iron. Both rates and extents of ferric iron reduction were highly strain dependent. No acidophilic heterotroph reduced nitrate or sulfate, and (limited) reduction of manganese(IV) was noted in only one strain (Acidiphilium facilis), an acidophile which did not reduce iron. Insoluble forms of ferric iron, both amorphous and crystalline, were reduced, as well as soluble iron. There was evidence that, in at least some acidophilic heterotrophs, iron reduction was enzymically mediated and that ferric iron could act as a terminal electron acceptor. In anaerobically incubated cultures, bacterial biomass increased with increasing concentrations of ferric but not ferrous iron. Mixed cultures of Thiobacillus ferrooxidans or Leptospirillum ferrooxidans and an acidophilic heterotroph (SJH) produced sequences of iron cycling in ferrous iron-glucose media.     

Ferrous ion oxidation by Thiobacillus ferrooxidans immobilized in calcium alginate

Summary Thiobacillus ferrooxidans was immobilized by entrapment into calcium alginate matrix. The immobilized bacteria were used in packed-bed column reactors for the continuous oxidation of ferrous ion at pH 1.5. The presence of mineral salts resulted in a shorter lag period before a steady-state of about 95% iron oxidation was achieved. Parallel shake flask experiments were used to evaluate pH, mineral salts, and alginate toxicity as factors influencing biological iron oxidation. Manometric experiments indicated that the previous growth history of T. ferrooxidans was important in determining the rate of iron oxidation. Scanning electron microscopy and energy dispersive analysis of X-rays were used to characterize bacteria entrapped in calcium alginate and the enrichment of iron in the matrix.