Tsuyoshi Sugio

Isolation of iron-oxidizing bacteria from corroded concretes of sewage treatment plants

Thirty-six strains of iron-oxidizing bacteria were isolated from corroded concrete samples obtained at eight sewage treatment plants in Japan. All of the strains isolated grew autotrophically in ferrous sulfate (3.0%), elemental sulfur (1.0%) and FeS (1.0%) media (pH 1.5). Washed intact cells of the 36 isolates had activities to oxidize both ferrous iron and elemental sulfur. Strain SNA-5, a representative of the isolated strains, was a gram-negative, rod-shaped bacterium (0.5-0.6x0.9-1.5 microm). The mean G+C content of its DNA was 55.9 mol%. The pH and temperature optima for growth were 1.5 and 30 degrees C, and the bacterium had activity to assimilate 14CO2 into the cells when ferrous iron or elemental sulfur was used as a sole source of energy. These results suggest that SNA-5 is Thiobacillus ferrooxidans strain. The pHs and numbers of iron-oxidizing bacteria in corroded concrete samples obtained by boring to depths of 0-1, 1-3, and 3-5 cm below the concrete surface were respectively 1.4, 1.7, and 2.0, and 1.2 x 10(8), 5 x 10(7), and 5 x 10(6) cells/g concrete. The degree of corrosion in the sample obtained nearest to the surface was more severe than in the deeper samples. The findings indicated that the levels of acidification and corrosion of the concrete structure corresponded with the number of iron-oxidizing bacteria in a concrete sample. Sulfuric acid produced by the chemolithoautotrophic sulfur-oxidizing bacterium Thiobacillus thiooxidansis known to induce concrete corrosion. Since not only T. thiooxidans but also T. ferrooxidans can oxidize reduced sulfur compounds and produce sulfuric acid, the results strongly suggest that T. ferrooxidans as well as T. thiooxidans is involved in concrete corrosion.

Purification and some properties of sulfur reductase from the iron-oxidizing bacterium Thiobacillus ferrooxidans NASF-1

Thiobacillus ferrooxidans strain NASF-1 grown aerobically in an Fe2+ (3%)-medium produces hydrogen sulfide (H2S) from elemental sulfur under anaerobic conditions with argon gas at pH 7.5. Sulfur reductase, which catalyzes the reduction of elemental sulfur (S0) with NAD(P)H as an electron donor to produce hydrogen sulfide (H2S) under anaerobic conditions, was purified 69-fold after 35-65% ammonium sulfate precipitation and Q-Sepharose FF, Phenyl-Toyopearl 650 ML, and Blue Sepharose FF column chromatography, with a specific activity of 57.6 U (mg protein)(-1). The purified enzyme was quite labile under aerobic conditions, but comparatively stable in the presence of sodium hydrosulfite and under anaerobic conditions, especially under hydrogen gas conditions. The purified enzyme showed both sulfur reductase and hydrogenase activities. Both activities had an optimum pH of 9.0. Sulfur reductase has an apparent molecular weight of 120,000 Da, and is composed of three different subunits (M(r) 54,000 Da (alpha), 36,000 Da (beta), and 35,000 Da (gamma)), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first report on the purification of sulfur reductase from a mesophilic and obligate chemolithotrophic iron-oxidizing bacterium.

Isolation and Some Properties of a Strain of the Iron-Oxidizing Bacterium Thiobacillus ferrooxidans Resistant to 2, 4-Dinitrophenol

Abstract Among two hundred strains of iron-oxidizing bacteria isolated from the natural environment, only one, Thiobacillus ferroxidans NASF-1, grew in an Fe 2+ medium supplemented with 10 μM of 2,4-dinitrophenol (2,4-DNP). In contrast, growth of T. ferroxidans AP19-3, a strain representative of iron-oxidizing bacteria sensitive to 2,4-DNP, was strongly inhibited by 2,4-DNP at 1.0 μM, and completely repressed at 10 μM. Strain NASF-1 was also resistant to an other uncoupler, carbonylcyanide m -chlorophenylhydrazone (CCCP). The iron-oxidizing activities of strains AP19-3 and NASF-1 were respectively inhibited 69 and 23% by 10 μM 2,4-DNP, while the 14 CO 2 uptake activities of the two strains were inhibited 31 and 5% by 2,4-DNP at 1.0 μM, and 100 and 89% at 5.0 μM. When intact cells of strain AP19-3 were incubated with Fe 2+ and 1.0 μM 2,4-DNP for 10 min at pH 3.0, 49 pmol 2,4-DNP per mg of protein was incorporated into the cells. In contrast, strain NASF-1 did not incorporate 2,4-DNP under the same conditions, suggesting that, compared with strain NASF-1, strain AP19-3 has a plasma membrane through which 2,4-DNP is more easily incorporated in the cytosol.

Some properties of a novel obligately autotrophic iron-oxidizing bacterium isolated from seawater

Abstract An iron-oxidizing bacterium obligately requiring NaCl for growth was isolated from seawater in Seto Inland Sea, Japan, and designated as strain KU2-11. Strain KU2-11 was a Gram negative, non-spore-forming and rod-shaped bacterium, and can grow autotrophically by using ferrous ion and elemental sulfur as sole energy sources. The optimum growth pH and temperature were 2 and 30°C, respectively. The G+C content of the DNA was 59 mol%. The bacterium could not grow in the medium without NaCl and obligately required NaCl for growth. The optimum NaCl concentration for the growth was 2%. Strain KU2-11 seemed to be identified as a member of Thiobacillus ferrooxidans on the basis of morphological and physiological properties. However, the phylogenetic analysis based on 16S rDNA gene sequences indicated that strain KU2-11 is distinct from T. ferrooxidans . Iron- and sulfur-oxidizing activity of the bacterium were not detected in a reaction mixture without NaCl, and depended on the presence of NaCl. The optimum NaCl concentrations for the iron- and sulfur-oxidizing activities were 2–4% and 1%, respectively. On the basis of the phenotypic characteristics of strain KU2-11 and its phylogenetic position, we suggested that this bacterium should be placed in a new species of the genus Thiobacillus .

Purification and Some Properties of Ubiquinol Oxidase from Obligately Chemolithotrophic Iron-oxidizing Bacterium, Thiobacillus ferrooxidans NASF-1

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.

Isolation of a Sulfur-oxidizing Bacterium That can Grow under Alkaline pH, from Corroded Concrete

To study the early stages of concrete corrosion by bacteria, sulfur-oxidizing bacterium strain RO-1, which grows in an alkaline thiosulfate medium (pH 10.0) was isolated from corroded concreate and characterized. Strain RO-1 was a Gram negative, rod-shaped bacterium (0.5-0.6×0.9-1.5 μm). The mean G+C content of the DNA of strain RO-1 was 65.0 mol%. Optimum pH and temperature for growth were 8.0. and 30-37°C, respectively. When grown in thiosulfate medium with pH 10.0, growth rate of the strain was 48% of that observed at the optimum pH for growth. Strain RO-1 used sulfide, thiosulfate, and glucose, but not elemental sulfur or tetrathionate, as a sole energy source. Strain RO-1 grew under anaerobic conditions in pepton-NO3 (-) medium containing sodium nitrate as an electron acceptor, and had enzyme activities that oxidized sulfide, elemental sulfur, thiosulfate, sulfite, and glucose, but not tetrathionate. The bacterium had an activity to assimilate (14)CO2 into the cells when thiosulfate was used as an energy source. These results suggest that strain RO-1 is Thiobacillus versutus. Strain RO-1 exuded Ca(2+) from concrete blocks added to thiosulfate medium with pH 9.0 and the pH of the medium decreased from 9.0 to 5.5 after 22 days of cultivation. In contrast, Thiobacillus thiooxidans strain NB1-3 could not exude Ca(2+) in the same thiosulfate medium, suggesting that strain RO-1, but not T. thiooxidans NB1-3, is involved in the early stage of concrete corrosion because concrete structures just after construction contain calcium hydroxide and have a pH of 12-13.

Production of hydrogen sulfide from tetrathionate by the iron-oxidizing bacterium Thiobacillus ferrooxidans NASF-1

When incubated under anaerobic conditions, five strains of Thiobacillus ferrooxidans tested produced hydrogen sulfide (H2S) from elemental sulfur at pH 1.5. However, among the strains, T. ferrooxidans NASF-1 and AP19-3 were able to use both elemental sulfur and tetrathionate as electron acceptors for H2S production at pH 1.5. The mechanism of H2S production from tetrathionate was studied with intact cells of strain NASF-1. Strain NASF-1 was unable to use dithionate, trithionate, or pentathionate as an electron acceptor. After 12 h of incubation under anaerobic conditions at 30 degrees C, 1.3 micromol of tetrathionate in the reaction mixture was decomposed, and 0.78 micromol of H2S and 0.6 micromol of trithionate were produced. Thiosulfate and sulfite were not detected in the reaction mixture. From these results, we propose that H2S is produced at pH 1.5 from tetrathionate by T. ferrooxidans NASF-1, via the following two-step reaction, in which AH2 represents an unknown electron donor in NASF-1 cells. Namely, tetrathionate is decomposed by tetrathionate-decomposing enzyme to give trithionate and elemental sulfur (S4O6(2-)-->S3O6(2-) + S(o), Eq. 1), and the elemental sulfur thus produced is reduced by sulfur reductase using electrons from AH2 to give H2S (S(o) + AH2-->H2S + A, Eq. 2). The optimum pH and temperature for H2S production from tetrathionate under argon gas were 1.5 and 30 degrees C, respectively. Under argon gas, the H2S production from tetrathionate stopped after 1 d of incubation, producing a total of 2.5 micromol of H2S/5 mg protein. In contrast, under H2 conditions, H2S production continued for 6 d, producing a total of 10.0 micromol of H2S/5 mg protein. These results suggest that electrons from H2 were used to reduce elemental sulfur produced as an intermediate to give H2S. Potassium cyanide at 0.5 mM slightly inhibited H2S production from tetrathionate, but increased that from elemental sulfur 3-fold. 2,4-Dinitrophenol at 0.05 mM, carbonylcyanide-m-chlorophenyl- hydrazone at 0.01 mM, mercury chloride at 0.05 mM, and sodium selenate at 1.0 mM almost completely inhibited H2S production from tetrathionate, but not from elemental sulfur.

Production of hydrogen sulfide by a moderately thermophilic iron- oxidizing bacterium strain TI-1

Abstract A moderately thermophilic iron-oxidizing bacterium strain TI-1 has been shown to have a unique enzyme system that produces hydrogen sulfide (H2S) extracellularly under acidic conditions. The optimal culture conditions for H2S production were determined to apply the bacterium to the treatment of acidic wastewater containing heavy metals. The production of H2S by the cells was dependent on l -glutamic acid, ferrous sulfate and elemental sulfur in the medium. The optimal culture medium for H2S production contained 0.3% l -glutamic acid, 1.7% ferrous sulfate, and 1.0% elemental sulfur and the optimal pH and temperature were 4.0 and 55°C, respectively. Under the optimal culture conditions, strain TI-1 produced H2S continuously at a rate of 0.2 μmol/ml/d for 14 d.

Existence of Two Kinds of Sulfur-reducing Systems in Iron-oxidizing Bacterium Thiobacillus ferrooxidans

Intact cells of Thiobacillus ferrooxidans NASF-1 incubated under anaerobic conditions in a reaction mixture containing 0.5% colloidal sulfur produced hydrogen sulfide (H2S) extracellularly. The amount of H2S produced by cells increased corresponding to the cell amounts and colloidal sulfur. Two activity peaks of H2S production were observed at pH 1.5 and 7.5. We tentatively called the enzyme activities pH 1.5- and pH 7.5-sulfur reducing systems, respectively. Seven strains of T. ferrooxidans tested had both the activities of pH 1.5- and pH 7.5-sulfur reducing systems, but at different levels. T. ferrooxidans NASF-1 showed the highest activity of the pH 1.5-sulfur reducing system and strain 13598 from ATCC showed the highest activity of the pH 7.5-sulfur reducing system. Further characteristics of H2S production were studied with intact cells of NASF-1. The optimum temperatures for pH 1.5- and pH 7.5-sulfur reducing systems of NASF-1 were 40°C. Hydrogen sulfide production continued for 8 days and total amounts of H2S produced at pH 7.5 and 1.5 were 832 and 620 nmol/mg protein, respectively. The pH 7.5-sulfur reducing system used only colloidal sulfur as the electron acceptor. However, the pH 1.5-sulfur reducing system used both colloidal sulfur and tetrathionate. Thiosulfate, dithionate, and sulfite could not be used as the electron acceptor for both of the sulfur reducing systems. Potassium cyanide activated by 3- fold the pH 1.5-sulfur reducing system activity at 0.5 mM but did not affect the activity of the pH 7.5-sulfur reducing system. An inhibitor of sulfite reductase, p-chloromercuribenzene sulfonic acid, did not affect either enzyme activity. Sodium molybdate and monoiodoacetic acid strongly inhibited the activity of the pH 1.5-sulfur reducing system at 1.0 mM, but not the activity of pH 7.5-sulfur reducing system.

Isolation and characterization of a marine iron-oxidizing bacterium requiring NaC1 for growth

An iron-oxidizing bacterium was isolated from seawater, and designated strain KU2-11. The strain KU2-11 was a Gram negative, non-spore-forming and rod-shaped bacterium. Strain KU2-11 can grow autotrophically by using ferrous iron and elemental sulfur as sole energy sources. The bacterium obligately required NaC1 for growth.The optimum NaC1 concentration was 2%. Optimum growth pH and temperature were 2 and 30°C, respectively. G+C content of the DNA was 59 mol%. An iron-oxidizing activity of the bacterium also depended on the NaC1 concentration. The role of NaC1 for this bacterium was investigated. The maintenance of cell morphology is necessary for both the growth and iron-oxidizing activity.NaC1 played an important role in maintaining the morphology of this bacterium.