Lipid A with 2,3-diamino-2,3-dideoxy-glucose in lipopolysaccharides from slow-growing members of Rhizobiaceae and from “Pseudomonas carboxydovorans”

Lipid A's from two Bradyrhizobium species and from the phylogenetically closely related species Pseudomonas carboxydovorans were found to contain 2,3-diamino-2,3-dideoxy-glucose as lipid A backbone sugar. In contrast, three representatives of the genus Rhizobium, as well as the phylogenetically related species Agrobacterium tumefaciens, contain solely glucosamine as lipid A backbone sugar. These findings suppor independent studies on the phylogenetical relatedness based on 16S rRNA-data of the genus Bradyrhizobium with Pseudomonas carboxydovorans and Rhodopseudomonas palustris, which form a tight phylogenetical cluster and which all contain the 2,3-diamino-2,3-dideoxy-glucose-containing lipid A. The relatedness of these species to the glucosamine-containing species of the genus Rhizobium and to Agrobacterium tumefaciens is rather distant as documented by 16S rRNA studies.Abbreviations LPS lipopolysaccharide - KDO 2-keto-3-deoxyoctonic acid - GalA galacturonic acid - ld-heptose l-glycero-d-manno-heptose - dd-heptose d-glycero-d-manno-heptose - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - DAG 2,3-diamino-2,3-dideoxy-glucose     

Lipopolysaccharide of Providencia rettgeri

The chemical constitutional analysis of the lipopolysaccharide (LPS) isolated from Providencia rettgeri was carried out. Polyacrylamide gel electrophoresis using sodium dodecylsulfate or sodium deoxycholate showed that the lipopolysaccharide mostly consisted of short sugar chains. The lipid A was precipitated out after mild acid hydrolysis of LPS. From the supernatant degraded polysaccharide and unsubstituted core fractions were isolated. Compositional analysis of the core material revealed the presence of galacturonic acid, galactose, glucose, glucosamine, l-glycero-d-manno-heptose, 3-deoxy-d-manno-octulosonic acid, alanine and phosphorus. Methylation analysis of the core material indicated the presence of terminal units of glucose, galacturonic acid and glucosamine. The chemical structure of the lipid A was elucidated. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester and glycosidically-bond phosphate residues. The ester-bound phosphate was found to be substituted by a 4-amino-4-deoxy-l-arabinosyl residue. The amino groups of the backbone disaccharide are N-acylated by 3-O-(14:0)14:0 and 3-O-14:0.Two hydroxyl groups of the disaccharide are esterified by 3-O-(14:0)14:0 and 3-O-14:0. The taxonomical importance of these structural details will be discussed.Abbreviations LPS lipopolysaccharide - l-d-heptose l-glycero-d-manno-heptose - dOclA 3-deoxy-d-manno-octulosonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - PS degraded polysaccharide - glc-ms combined gas liquid chromatography-mass spectrometry     

The lipopolysaccharide of the phototrophic bacterium Ectothiorhodospira vacuolata

The lipopolysaccharide of Ectothiorhodospira vacuolata was obtained by the phenol-water procedure. It contained a 3-O-methyl-hexose, glucose, galacturonic and glucuronic acids. The finding of d-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate (tentatively identified) suggested a core-structure. The lipid fraction of the lipopolysaccharide contained phosphate and both, 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The major fatty acids were amine-bound 3-OH-10:0 and 3-OH-12:0 and esterbound 14:0 and 16:0 Sodium deoxycholate gel-electrophoresis, showing a single band only, indicated R-type character of the lipopolysaccharide of Ectothiorhodospira vacuolata.Abbreviations DOC sodium deoxycholate - GC/MS combined gasliquid chromatography - PAGE polyacrylamide gel-electrophoresis     

The lipopolysaccharides of Rhodospirillum rubrum,Rhodospirillum molischianum,and Rhodopila globiformis

The cell wall lipopolysaccharides from three phototrophic species of the alpha₁-group of Proteobacteria, Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis were isolated and chemically characterized. Sodium deoxycholate polyacrylamide gel electrophoresis patterns revealed that the lipopolysaccharides of all three species possess O-chains. They are composed of repeating units only in R. molischianum and R. globiformis. The presence of l-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate indicated core structures in all three lipopolysaccharides. Glucosamine was found as backbone amino sugar in lipid A of R. molischianum and R. rubrum, while R. globiformis has 2,3-diaminoglucose as backbone amino sugar. The latter species also differed from the two former ones in its content of hydroxy fatty acids (3-OH-14:0, 3-OH-16:0 in R. rubrum and R. molischianum and 3-OH-14:0, 3-OH-18:0 and 3-OH-19:0 (possibly iso- or anteisobranched) in R. globiformis).Abbreviations DOC-PAGE sodium deoxycholate polyacrylamide gel electrophoresis - GC/MS combined gas-liquid chromatography/mass spectrometry - KDO 2-keto-3-deoxyoctonate     

The lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum

The cell wall lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum was obtained by the phenol-chloroform-petroleum ether and the hot phenol-water methods, respectively. It contained mannose, glucose, galacturonic acid, glucosamine, glycine, and small amounts of rhamnose, galactose and glucuronic acid. In addition to d-glycero-d-mannoheptose, the corespecific constituents 2-keto-3-deoxyoctonate and l-glycero-d-mannoheptose were found. Polyacrylamide gel-electrophoresis in the presence of sodium deoxycholate gave no indication for the presence of O-specific repeating units. Degradation of the lipopolysaccharide required 10% acetic acid (100° C, 2 h). The lipid A moiety contained the total of glucosamine of the lipopolysaccharide as well as small amounts of 2,3-diamino-2,3-dideoxy-glucose. It was phosphate-free. The fatty acid spectrum comprised 3-OH-14:0, 3-OH-16:0, and iso-3-OH-18:0 besides little 12:0, 14:0 and 16:0. Hydroxylaminolysis and sodium methylate treatment revealed all of the three hydroxy fatty acids to be amidebound.Abbreviations DOC sodium deoxycholate - PAGE polyacrylamide gel-electrophoresis     

Characterization of arsenopyrite oxidizing Thiobacillus. Tolerance to arsenite,arsenate, ferrous and ferric iron

Two strains of Thiobacillus, T. ferrooxidans and T. thiooxidans, have been isolated from a bacterial inoculum cultivated during a one-year period in a 1001 continuous laboratory pilot for treatment of an arsenopyrite/pyrite concentrate. The optimum pH for the growth of both strains has been found to be between 1.7 and 2.5. Because of the high metal toxicity in bioleach pulps, the tolerance of T. ferrooxidans and T. thiooxidans with respect to iron and arsenic has been studied. The growth of both strains is inhibited with 10 g/l of ferric ion, 5 g/l of arsenite and 40 g/l of arsenate. 20 g/l of ferrous iron is toxic to T. ferrooxidans but 30 g/l is necessary to impede the growth of T. thiooxidans.     

Chemical composition of the lipopolysaccharides of Ectothiorhodospira shaposhnikovii,Ectothiorhodospira mobilis,and Ectothiorhodospira halophila

Lipopolysaccharides were isolated from the moderate halophilic Ectothiorhodospira shaposhnikovii slight to and Ectothiorhodospira mobilis and from the extremely halophilic Ectothiorhodospira halophila by the hot phenol-water and purified by the phenol-chloroform-petroleum ether methods. The isolated lipopolysaccharides of all three species contained 3-deoxy-d-manno-octulosonic acid and d-glycero-d-mannoheptose indicating the existence of a core. They contained additionally glucose and uronic acids (E. shaposhnikovii and E. mobilis) or glucose, uronic acids and threonine (E. halophila). Sodium deoxycholate gel-electrophoresis of the three lipopolysaccharides, each showing only one major band, indicated R-type character of the lipopolysaccharides of the three Ectothiorhodospira species.The lipid A fractions of the lipopolysaccharides from E. shaposhnikovii and E. mobilis represented phosphorylated mixed lipid A types with both 2,3-diamino-2,3-dideoxy-d-glucose and d-glucosamine. The lipid A from E. halophila contained also phosphate and 2,3-diamino-2,3-dideoxy-d-glucose but only traces of d-glucosamine, which would indicated lipid A_DAG. The fatty acid spectra were characterized by amide-bound 3-OH-10:0 and 3-OH-12:0 (E. shaposhnikovii), 3-OH-10:0 (E. mobilis), or 3-OH-10:0,3-OH-14:0, and 3-oxo-14-0 (E. halophila). The predominant ester-bound fatty acids were 14:0 and 16:0 (E. shaposhnikovii and E. mobilis), or 12:0 and 14:1 (E. halophila).Abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 3-deoxy-d-manno-octulosonic acid - GlcA glucuronic acid - GalA galacturonic acid - GC-MS combined gas liquid chromatographymass spectrometry - GlcN Glucosamine - DOC sodium deoxycholate - LPS lipopolysaccharide - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Taxonomic relationships ofThiobacillus halophilus,T. aquaesulis,and other species ofThiobacillus,as determined using 16S rDNA sequencing

Total base sequences of the 16S rRNA genes ofThiobacillus halophilus andThiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative ofT. halophilus isThiobacillus hydrothermalis (with 98.7% similarity), and the closest relative ofT. aquaesulis isThiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genusThiobacillus are clearly so diverse that they need reclassification into several genera. The type species,T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus nameThiobacillus be restricted to the chemolithoautotrophic species falling into that group.T. aquaesulis andT. thioparus may thus be regarded as true species ofThiobacillus. The relatively large number of obligately chemolithoautotrophicThiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.     

Chemical composition of lipopolysaccharides from Legionella bozemanii and Legionella longbeachae

Lipopolysaccharides (LPS) from Legionella bozemanii serogroup 1 and Legionella longbeachae serogroup 1 were subjected to chemical analyses. The lipid A part of both LPSs contained 2,3-dideoxy-2,3-diamino-d-glucose as major constituents and d-glucosamine and glycerol as minor constituents of the sugar backbone structure. Both LPSs exhibited a very complex fatty acid composition. Twenty amide-linked 3-hydroxy fatty acids were detected in LPS of L. longbeachae, whereas seventeen were encountered in LPS of L. bozemanii. Both LPSs contained nine ester-linked nonhydroxy fatty acids and the unique long-chain fatty acids 27-oxo-octacosanoic acid, 29-oxotriacontanoic acid, heptacosane-1,27-dioic acid and nonacosane-1,29-dioic acid. SDS-PAGE showed that L. bozemanii produced smooth-form LPS, whereas L. longbeachae LPS was mainly of the R-type. Composition analyses were in accordance with these electrophoretic patterns. d-Quinovosamine and l-fucosamine constituted 80 mol% of the polysaccharide part of L. bozemanii LPS. Other sugars identified were d-glucosamine, d-mannose, d-glucose, l-rhamnose, d-glycero-d-manno-heptose, l-glycero-d-mannoheptose, 2-keto-3-deoxy-octonic acid and glycerol. The polysaccharide chain from LPS of L. longbeachae appeared to be shorter, but composed of the same sugars except l-fucosamine. Both LPSs contained glycerol phosphate and glucosamine phosphate and L. longbeachae LPS contained in addition glucose phosphate.Abbreviations EI Electron impact - GlcN3N 2,3-Diamino-2,3-dideoxy-d-glucose - HPAEC High pH anion-exchange chromatography - Kdo 2-Keto-3-deoxy-octonic acid - LPS Lipopolysaccharide - PCP Phenol/chloroform/petroleum ether solvent - PED Pulsed electrochemical detection - PS Polysaccharide - TFA Trifluoroacetyl - TMS Trimethylsilyl     

Identification of a 2,3-diamino-2,3-dideoxyhexose in the lipid a component of lipopolysaccharides of Rhodopseudomonas viridis and Rhodopseudomonas palustris

A hitherto unknown amino sugar (Compound A), detected in acid hydrolyzates of lipopolysaccharides of Rhodopsuedomonas viridis and Rhodopseudomonas palustris, is present in the Lipid A component but not in the O-specific part of the lipopolysaccharides. 2-Amino-2-deoxy-D-glucose is lacking in the purified Lipid A of both strains. Compound A, characterized by a very high migration in paper electrophoresis was obtained in a pure state by ion-exchange chromatography and shown by m.s of the alditol acetate to be a 2,3-diamino-2,3-dideoxyhexose. G.I.c. and periodate oxidation excluded all possible stereoisomers with the exception of 2,3-diamino-2,3-dideoxyglucose and 2,3-diamino-2,3-dideoxyidose. G.I.c. of the alditol acetates of Compound A and of the glucose derivative suggests that Compound A is 2,3-diamino-2,3-dideoxyglucose. The significance of the occurrence of this new aminodeoxy sugar in the lipid A component of Rhodopsuedomonas viridis and Rhodopseudomonas palustris O-antigens for the biological properties of the respective lipopolysaccharides and for the taxonomy of the Rhodospirillaceae family is discussed.     

The ultrastructure of chemolithoautotrophic Gallionella ferruginea and Thiobacillus ferrooxidans as revealed by chemical fixation and freeze-etching

By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly--hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

A simple apparatus for measuring hardness of agar and Gelrite plates

A simple apparatus for measuring hardness of Gelrite and agar plates, that can be easily fabricated, is described. It gives reproducible results comparable with those obtained with expensive commercial equipment. It has been successfully used to improve enumeration ofThiobacillus ferrooxidans andT. thiooxidans on solid media plates.     

The role of exopolymers in the bioleaching of a non-ferrous metal sulphide

Exocellular polysaccharides were extracted from Thiobacillus ferrooxidans cells grown in the presence of iron. Cells without these compounds could not adhere to covellite. The loss of the layer of exocellular polysaccharides also affected the direct mechanism of bioleaching of covellite in a negative way. This ability to attach to and leach covellite was restored within a few hours when exopolymeric material was produced again. The addition of exocellular compounds to cells stripped of exocellular polymers also restored their ability to the same level as that of untreated cells. Thiobacillus thiooxidans was not able to attach to and leach covellite even when exocellular compounds from Thiobacillus ferrooxidans were added. Received 2 June 1998/ Accepted in revised form 8 January 1999     

Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60°C. The molecular weight was 100000 to 107000. The K _m-values were 3.3 mM for D(-)-butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)-butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH 9 and for reduction pH 4.5. The optimum temperature was 32–36°C. The molecular weight was 100000 to 107000. The K _m-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.Abbreviations Bud 2,3-butanediol - DH dehydrogenase     

Microbial desulfurization of coal and oxidation of pure pyrite byThiobacillus ferrooxidans andAcidianus brierleyi

Summary Thiobacillus ferrooxidans andAcidianus brierleyi were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. First order reactions were assumed in the kinetic analysis performed. For oxidation of pure pyrite the rate constant was higher forA. brierleyi than forT. ferrooxidans. For sulfur removal from coal the values of the rate constants were comparable for the two microorganisms.     

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).     

Biodesulphurization of coal: rate enhancement by sulphur-grown cells

Pyritic sulphur was removed from coal by growing Thiobacillus ferrooxidans in a 250 ml batch bioreactor. Thiobacillus ferrooxidansgrown on sulphur and which was added 5 days after initial inoculation, enhanced the iron solubilization rate by 35% as compared to control (without addition of sulphur-grown cells). About 93% pyritic sulphur was removed in presence of sulphur-grown cells as compared to 77% in the control.     

Enhancement of Copper Dissolution from a Sulfide Ore by Using Thiobacillus thiooxidans

Copper dissolution from a sulfide ore (with covellite as the main copper phase) was investigated in cultures of Thiobacillus ferrooxidans or Thiobacillus thiooxidans and in abiotic controls. In unsupplemented media, T. ferrooxidans was more efficient than T. thiooxidans. In the presence of ferric iron, the dissolution of covellite was not significantly different in cultures inoculated with T. ferrooxidans or T. thiooxidans. However, the most extraction was found in T. thiooxidans cultures supplemented with ferrous sulfate. The first results were explained by the mechanism proposed by Schippers and Sand (Appl Envir Microbiol 65:319-321, 1999), which involves polysulfides and sulfur as intermediates. This mechanism was extended to explain the behavior of T. thiooxidans culture supplemented with ferrous iron.