Ferrobacillus Ferrooxidans Research Articles

Review: Microbial Corrosion Of Metals

Biocorrosion processes of metal surfaces are associated with microorganisms, or the products of their metabolic activities including enzymes, exopolymers, organic and inorganic acids, as well as volatile compounds such as ammonia or hydrogen sulphide. These can affect cathodic and/or anodic reactions, thus altering electrochemistry at the biofilm/metal interface. Various mechanisms of biocorrosion, reflecting their variety of physiological activities carried out by different types of microorganisms, are identified and recent insights into these mechanisms reviewed. Many recent investigations have centred on the microbial-influenced corrosion of ferrous and copper alloys and of particular interest are Desulfurvibrio species, Thiobacillus species, Pseudomonas, Methanobacterium, Methanospirillum, Escherichia coli, and Ferrobacillus ferrooxidans. Some of the factors that influence biocorrosion include; pH, temperature, oxygen, moisture and the nature and types of metal. Key words: biocorrosion, sulphate reducing bacteria, enzymes, exopolymers, electrochemistry Ife Journal of Science Vol. 7(1) 2005: 21-25

Pyruvate Inhibition of Ferrobacillus ferrooxidans Reversed by Rhodotorula Yeast

Ferrobacillus ferrooxidans was isolated in this laboratory from uranium ores mined at Jaduguda, Bihar. Investigations on a laboratory scale indicated that this organism could leach uranium from ores. Pyruvic acid (10‐4 M) was found to inhibit growth of F. ferrooxidans as well as leaching of uranium ore by F. ferrooxidans. However, in the presence of a red pigmented Rhodotorula yeast isolated from the same ores, the pyruvate inhibition was found to be reversed.

Microbiological factor in acid mine drainage formation II. Further observations from a pilot plant study

Pilot plant studies examined the effect of chemoautotrophic organisms ( Ferrobacillus ferrooxidans, Ferrobacillus sulfooxidans and Thiobacillus thiooxidans) in the formation of acid mine drainage from pyritic mineral associated with coal mining. Discharges were measured for varying hydraulic flow-rates, air or nitrogen atmosphere and biological seeding or non-seeding. Under non-aerobic conditions, Fe(II) and S 2su−II mas release is constant and unaffected by organisms. Under aerobic conditions, acidity, ferrous and total iron and sulfate concentrations are zero order with respect to flow expressed in reciprocal time units, and much greater than under non-aerobic conditions. Total acidity is lower for seeded than for non-seeded aerobic conditions at higher retention times because microorganisms consume acid. Aerobic non-seeded release reaches a maximum that does not change with increasing flow. Aerobic seeded release reaches a maximum greater than that for aerobic non-seeded conditions but then decreases with increased flow. A dynamic equilibrium between pyrite dissolution and organism regeneration and flushout exists.

Recommendation that the Names Ferrobacillus ferrooxidans Leathen and Braley and Ferrobacillus sulfooxidans Kinsel Be Recognized as Synonyms of Thiobacillus ferrooxidans Temple and Colmer

The only characters currently used to distinguish Ferrobacillus ferrooxidans and F. sulfooxidans from Thiobacillus ferrooxidans are the inability of the first species to oxidize sulfur or thiosulfate and of the second to use thiosulfate. These differences are demonstrated to be invalid inasmuch as all three species can grow on either sulfur or thiosulfate. The names F. ferrooxidans and F. sulfooxidans are therefore regarded as later, subjective synonyms of Thiobacillus ferrooxidans.

Transition of chemolithotroph Ferrobacillus ferrooxidans to obligate organotrophy and metabolic capabilities of glucose-grown cells.

Transition of chemolithotrophic Ferrobacillus ferrooxidans to organotrophy occurred after 60 hr of incubation in an organic medium. Three distinct phases, based on metabolic activities of cells, were observed during the course of transition. Conversion of cellular nutrition to organotrophy resulted in a gradual loss of Fe(2+) oxidation and cessation of CO(2) fixation. These changes were concomitant with a rapid increase in uptake of glucose and phosphate during the latter part of transition period. The outcome of transition was governed by the pH of the medium, temperature of incubation, availability of oxygen, age of the chemolithotrophic cells, and the type of energy and carbon source available to the bacterium. Presence or absence of p-aminobenzoic acid and Fe(2+) ions did not influence transition of cells. A defined medium containing glucose, mineral salts, and p-aminobenzoic acid at pH 2.5 was found to be most suitable for transition and for culture of heterotrophic convertants. Maximum growth rate of the heterotrophic cells was attained with vigorous aeration at 35 C. The bacterium could be cultured on a variety of organic compounds, including complex organic media, provided they were used in low concentrations. Serological studies on autotrophic cells and the heterotrophic convertant have shown a definite antigenic relationship between the two cell types.

Comparison of the kinetics of thiosulfate oxidation by three iron-sulfur oxidizers.

The sulfur metabolism of Thiobacillus ferrooxidans, Ferrobacillus ferrooxidans, and Ferrobacillus sulfooxidans was found to differ in the rate of utilization of thiosulfate and tetrathionate, with tetrathionate showing the most significant difference between the three organisms. The apparent Km values of the three organisms ranged from 2–5 × 10−2 M for T. ferrooxidans and F. sulfooxidans to 5–7 × 10−3 M for F. ferrooxidans. Increasing the temperature caused a greater effect on thiosulfate oxidation by F. ferrooxidans than with the other two as shown by the apparent Ea values (Ea′ for F. ferrooxidans, 18 000; F. sulfooxidans, 11 400; T. ferrooxidans, 11 000).The initial pH of the medium determined the rate of thiosulfate oxidation. If the initial pH was 4.0–4.5, a sharp break in oxygen uptake was shown by T. ferrooxidans and F. sulfooxidans after 20–30 min, but not with F. ferrooxidans.When the disappearance of thiosulfate and tetrathionate from the medium was used as an indicator of the affinity of the three test organisms for the sulfur compounds, F. ferrooxidans demonstrated the highest net loss of both compounds from the medium.

Tolerance of Thiobacillus ferrooxidans to some metals.

During iron oxidation,Thiobacillus ferrooxidans (Ferrobacillus ferrooxidans) was able to tolerate high concentrations of Zn, Ni, Cu, Co, Mn and Al (more than 10 g/litre). Silver and anions of tellurium, arsenic and selenium were toxic in concentrations of 50–100 mg/litre. Molybdenum (as molybdate), at concentrations above 5 mg/litre, was lethal toT. ferrooxidans. During thiosulphate oxidation, the tolerance to Zn, Ni and Co was greatly reduced, cobalt now being at least 2000 times more toxic, and the inhibitory levels of Zn and Ni being 600 mg Zn/litre and 150 mg Ni/litre. During sulphur oxidation, the tolerance to heavy metals extended to concentrations above 5 g/litre. Adaptation to Zn, Ni or Cu during iron oxidation was found to result in increased tolerance to some of the other metals also.

Inorganic pyrophosphatase from Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans).

An inorganic pyrophosphatase was isolated from Ferrobacillus ferrooxidans and purified 21-fold. The cation Mg2+ was required for maximum activity; Mn2+, Zn2+, and Co2+ supported less than 10% of the activity with Mg2+. The pH optimum of the enzyme was between 7.5 and 8.5, using a magnesium to pyrophosphate ratio of one. The purified enzyme was unable to hydrolyze adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, or various other monophosphates; p-nitrophenyl-phosphate, the substrate for alkaline pyrophosphatase, was not hydrolyzed. Sulfhydryl binding agents did not inhibit enzyme activity, and the enzyme, in the presence of Mg2+, was heat-stable.

Oxidation of elemental sulfur and sulfur compounds and CO2 fixation by Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans).

The characteristics of growth of Ferrobacillus ferrooxidans on elemental sulfur are reported. This organism is not able to oxidize thiocyanate, but can oxidize elemental sulfur, sulfite, dithionite, thiosulfate, tetrathionate, and sulfide. Sulfide is only partially oxidized. All compounds that can be oxidized support CO2 fixation.

Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. 3. Properties of thiosulfate-oxidizing enzyme and proposed pathway of thiosulfate oxidation.

Thiosulfate-oxidizing enzyme was purified from Thiobacillus thioparus extracts 120- to 160-fold and the properties were studied. The enzyme had a molecular weight of 115 000 and contained 2 moles of non-heme iron. Ferricyanide was a much better electron acceptor than cytochrome c, but with cytochrome c the Km for thiosulfate was lowered from 0.1 mM to 5 μM and the pH response of the enzyme changed. Sulfite was a very strong inhibitor destroying 50% of the activity at 5 μM. The inhibition was time-dependent and essentially irreversible. Properties of the T. thioparus enzyme were compared to those of thiosulfate-oxidizing enzyme isolated from Thiobacillus neapolitanus and Ferrobacillus ferrooxidans. A pathway of thiosulfate oxidation is proposed, and metabolic roles of various enzymes studied in T. thioparus are discussed.

The rhodanese enzyme of Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans).

Extract of sulfur-grown Ferrobacillus ferrooxidans contains the thiosulfate-splitting enzyme rhodanese. Rhodanese was purified about 40-fold and was shown to have a wide pH optimum (pH 7.5–9.0). The Km for thiosulfate and cyanide was 5.8 × 10−4 M and 1.1 × 10−2 M, respectively. The enzyme was stable at 25 °C and was inhibited by the thiol-alkylating agents iodoacetamide and N-ethylmaleimide.

Kinetic studies of iron oxidation by whole cells of Ferrobacillus ferrooxidans.

A colorimetric assay was developed for studying the kinetics of iron oxidation with whole cells of the chemoautotroph, Ferrobacillus ferrooxidans. The assay was more advantageous than the conventional method of Warburg manometry because of its simplicity, rapidity, and the small amount of cells required. The assay measured Fe(3+) as a chloride complex which absorbs at 410 nm. Kinetic analysis showed the apparent K(m) for iron oxidation to be 5.4 x 10(-3)m in an unbuffered system and 2.2 x 10(-3)m in the presence of beta-alanine-SO(4) (2-) buffer. Glycine and beta-alanine buffers were used in the measurement of the pH optimum for iron oxidation; the optimum ranged from 2.5 to 3.8. The effect of pH was primarily on the V(max) while the K(m) remained constant. Added SO(4) (2-) was found to stimulate iron oxidation by increasing the V(max) of iron oxidation by whole cells, but it did not affect the K(m). Results of assays of iron oxidation in systems containing various mole percentages of SO(4) (2-) and Cl(-) indicated that Cl(-) did not inhibit iron oxidation but that SO(4) (2-) was required. Sulfate could be partially replaced by HPO(4) (2-) and HAsO(4) (2-) but not by BO(3) (-), MoO(4) (2-), NO(3) (-), or Cl(-); formate and MoO(4) (2-) inhibited iron oxidation.

Extracellular complex from the culture filtrate of Ferrobacillus ferrooxidans.

An extracellular complex was isolated from the culture filtrate of Ferrobacillus ferrooxidans grown on either iron or sulfur. The complex isolated from the culture filtrate of iron-grown cells was red with an absorption maxima at 500–510 mμ. This complex consisted of approximately 60% non-phosphorus lipid. Ferrous iron was exclusively associated with a lipopolysaccharide–phospholipid fraction of the complex. The sulfur complex unlike the iron complex contained approximately 70% phospholipid and was yellowish. On the basis of hexosamine and heptose values, less lipopolysaccharide appeared to be associated with the phospholipid fraction of the complex from sulfur-grown cells than from the iron-grown cells.

Poly-beta-hydroxybutyrate in the chemolithotrophic bacterium Ferrobacillus ferrooxidans.

Poly-beta-hydroxybutyrate in the chemolithotrophic bacterium Ferrobacillus ferrooxidans.

Enzymatic studies of the iron-oxidizing bacterium, Ferrobacillus ferrooxidans: evidence for a glycolytic pathway and Krebs cycle.

Whole cells and cell-free extracts of the chemolithotroph, Ferrobacillus ferrooxidans, were used to investigate certain enzymatic reactions related to the glycolytic pathway and Krebs cycle measuring oxygen uptake manometrically; most glycolytic pathway and Krebs cycle intermediates when used as substrates were oxidized. With the use of specific enzyme assays, most of the enzymes of the glycolytic pathway and Krebs cycle were detected in crude cell-free extracts. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, enzymes of the hexose monophosphate pathway, were not found.

The thiosulfate-oxidizing enzyme of Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans).

The thiosulfate-oxidizing enzyme tetrathionase was purified about 250-fold from sulfur-grown Ferrobacillus ferrooxidans. It reduced 1 mole of ferricyanide per mole of thiosulfate oxidized, with the production of 0.5 mole of tetrathionate. The pH optimum was 5.0 with acetate buffer, and the Km for thiosulfate was determined to be 9 × 10−4 M. Inhibition studies showed that enzyme activity is not reliant on metal ions or sulfhydryl groups, and no cofactor requirements were found. Tetrathionate was slightly inhibitory to the reaction. A possible role of this enzyme in the oxidation of sulfur and thiosulfate is discussed.

Sulfur-oxidizing enzyme of Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans).

The sulfur-oxidizing enzyme was purified about 15-fold from sulfur-grown Ferrobacillus ferrooxidans. The enzyme has a pH optimum of 7.8 and requires both elemental sulfur and reduced glutathione (GSH); however, a glutathione–polysulfide complex could also serve as substrate. The Km for GSH was determined to be 2 × 10−3 M. Non-heme iron and labile sulfide were present in the enzyme preparation, and sulfite was found to be the end product of the reaction.

Carbon dioxide fixation and phosphoenolpyruvate carboxylase in Ferrobacillus ferrooxidans.

Carbon dioxide fixation was studied in intact cells and cell-free extracts of Ferrobacillus ferrooxidans. The major pathways of fixation were found to be the carboxydismutase system and the phosphoenolpyruvate (PEP) carboxylase system. PEP carboxylase activity was shown to be under metabolic regulation, similar to the regulation established in heterotrophic microorganisms.Acetyl-CoA stimulated PEP carboxylase activity, while aspartate was inhibitory. The F. ferrooxidans enzyme appeared to have a neutral or acidic pH optimum, in contrast to the same enzyme isolated from heterotrophs.

Phospholipids from the chemoautotroph [formula omitted

The chemoautotroph Ferrobacillus ferrooxidans like other gram-negative bacteria possesses a multi-layered cell envelope ( Remsen & Lundgren 1966) which is believed to be involved in Fe ++ ion oxidation-the primary energy yielding reaction of the organism ( Dugan and Lundgren, 1965). This report presents a phase of our study concerning the characterization of phospholipids in membranes of F. ferrooxidans . Reports of phospholipids in other chemoautotrophs have been published (Schaeffer and Umbreit, 1962; Jones and Benson, 1965; Hagen, Goldfine, and Williams, 1966).

Ferrous iron oxidation by Ferrobacillus ferrooxidans. Purification and properties of Fe++-cytochrome c reductase.

The electron transport chain and Fe++-cytochrome c reductase of Ferrobacillus ferrooxidans were studied to elucidate the mechanism of iron oxidation by this autotrophic bacterium.The iron oxidation involved the cytochrome c and a type cytochrome of F. ferrooxidans in the electron transport system. The initial enzyme of the oxidation system was found to be Fe++-cytochrome c reductase. The iron oxidase system was labile to freezing or sonication; either treatment disrupted some link between the cellular cytochromes c and a. Fe++-cytochrome c reductase and cytochrome oxidase retained their individual activities after either treatment.Fe++-cytochrome c reductase was purified 60-fold to 70-fold. The enzyme was judged to be approximately 90% pure by disc electrophoresis, sedimentation, and DEAE-cellulose chromatography. A suitable assay system with a veronal–acetate buffer was developed for the determination of enzyme activity. The effects of inhibitors and potential activators were studied. No specific inhibitor or cofactor was found, although the enzyme was inhibited by various ionic compounds.Fe++-cytochrome c reductase was dissociated into two subunits, one protein and the other ribonucleic acid (RNA). Neither of the subunits had enzymatic activity and efforts to reconstitute the holoenzyme from the two subunits were unsuccessful. The molecular weights of the holoenzyme, protein subunit, and RNA subunit were determined as 100,000–110,000, 27,000–30,000, and 315,000–330,000, respectively. The protein subunit contained one non-heme iron atom per protein molecule. It was concluded that RNA is an essential component of the enzyme and the failure to recover the activity from subunits is due to the aggregation of RNA after dissociation.