Yasuji Minoda

Hydrogenobacter thermophilus gen. nov., sp. nov., an extremely thermophilic aerobic, hydrogen-oxidizing bacterium

Six strains of strictly thermophilic, obligately chemolithotrophic, hydrogen-oxidizing bacteria were isolated from hot springs located in Izu and Kyushu, Japan. The bacterial strains which we tested were gram negative, nonmotile, nonsporeforming, long, straight, and rod shaped. The cell size was 0.3 to 0.5 by 2.0 to 3.0 μm. The deoxyribonucleic acid guanine-plus-cytosine contents of the six strains were between 43.5 and 43.9 mol%. The optimal temperature for autotrophic growth on H2-O2-CO2 was between 70 and 75°C. None of the strains grew at 37 or 80°C. The neutral pH range was suitable for growth. No strain showed heterotrophic growth at the expense of 48 organic compounds or on complex media, in contrast to all other known aerobic hydrogen-oxidizing bacteria which are facultative autotrophs. The major cellular fatty acids were a straight-chain saturated C18:0 acid and a straight-chain unsaturated C20:1 acid with one double bond. C16:0 and C18:1 fatty acids and a C21:0 cyclopropane acid were minor components. Cytochromes b and c were found in all of the strains. The polyacrylamide gel electrophoretic patterns of the total soluble proteins of all of the strains were essentially the same. The name Hydrogenobacter thermophilus gen. nov., sp. nov., is proposed for the six new isolates, and type strain TK-6 has been deposited with the culture collection of the Institute of Applied Microbiology, University of Tokyo, as strain IAM 12695.

The CO2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus

The incorporation of 14CO2 by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with 14CO2, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO2 fixation.In cell-free extracts of Hydrogenobacter thermophilus, the two key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase could not be detected. The key enzymes of the reductive tricarboxylic acid cycle, fumarate reductase and ATP citrate lyase were present. Activities of phosphoenolpyruvate synthetase and pyruvate carboxylase were also detected. The referse reactions (dehydrogenase reactions) of α-ketoglutarate synthase and pyruvate synthase could be detected by using methyl viologen as an electron acceptor.These findings strongly suggested that a new type of the reductive tricarboxylic acid cycle operated as the CO2 fixation pathway in Hydrogenobacter thermophilus.

Isolation and Identification of Alkaline Lipase Producing Microorganisms, Cultural Conditions and Some Properties of Crude Enzymes

Two bacterial strains 26.IB (I) and 22.39B (II) were isolated from soil as alkaline lipase producing microorganisms. Strain 26. IB (I) was identified as Pseudomonas nitroreducens nov. var. thermotolerans, and strain 22.39B (II) was identified as Ps. fragi. When they were cultivated aerobically in a 20-liter jar fermentor in medium containing 2.0% soybean meal, they secreted a large amount of alkaline lipases. The enzymes were recovered efficiently as precipitates by adjusting the pH of the culture fluids to 4.0 with HC1. The enzymatic characteristics included: optimum pH, 9.5 (I,II); stable pH range, 5~11 (I,II); optimum temperatures, 50°C (I) and 75~80°C (II); and more than 95% of the enzyme activity remained after incubated at 70°C for 20 min (I,II). Lipase activities were inhibited remarkably in the presence of anionic surfactants (I,II) and bile salts (I,II).

Studies on Tannin Acyl Hydrolase of Microorganisms:Part III. Purification of the Enzyme and Some Properties of It

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.The molecular weight of the enzyme estimated by gel filtration method was about 200,000.The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.

Studies on Tannin Acyl Hydrolase of Microorganisms: Part II. A New Method Determining the Enzyme Activity Using the Change of Ultra Violet Absorption

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the err...

Growth and Taxonomy of Thermophilic Hydrogen Bacteria

Growth characteristics of thermophilic hydrogen bacteria TH–1 and TH–4 were exarnined and taxonomic studies of these organisms were performed. Both strains assimilated various organic acids but not the tested sugars. Autotrophic growth utilizing hydrogen, oxygen and carbon dioxide was more rapid than heterotrophic growth on organic acid media. With pyruvate as the sole carbon source, TH–1 grew more rapidly under oxy-hydrogen atmosphere (H2: O2=4: 1) than in air. Neither TH-1 nor TH–4 fit the description of any previously described species; and they are thus proposed as new species, Pseudomonas hydrogenothermophila nov. sp. Goto, Kodama and Minoda and Flavobacterium autothermophilum nov. sp. Goto, Kodama and Minoda, respectively.

Microbial transformation of α-pinene

SummaryA bacterium capable of utilizing α-pinene as a sole carbon and energy source was isolated from soil. This strain, named strain S201-1, which was identified as Pseudomonas maltophilia on the basis of its taxonomical properties, accumulated limonene, borneol, camphor, perillic acid, and 2-(4-methyl-3-cyclohexenylidene) propionic acid from α-pinene in the culture broth. It was demonstrated that α-pinene, β-pinene, borneol, camphor, and a number of p-menthane derivatives were oxidized by this strain. Relations between the protonation of α-pinene and the formation of the products by the microbe are discussed.

Microbial Conversion of Petro-sulfur Compounds:Part II. Culture Conditions of Dibenzothiophene-Utilizing Bacteria

In order to enhance the efficiency converting dibenzothiophene (DBT) into water-soluble compounds by DBT-utilizing bacteria, the following subjects were studied: comparison of activities of isolated strains, their mixed cultivation, DBT dosing, nitrogen sources, substrate concentration, pH, temperature, and oxygen supply. Light oil solution of DBT (5%) was used as substrate. The conversion efficiency was calculated by the amount of sulfur accumulated in the aqueous layer of culture broth.The conversion ratio of 40% was attained by the mixed culture of Pseudomonas abikonensis and Ps. jianii under the following conditions; medium, DBT 4.6 g dissolved in 87.8 g of light oil, meat extract 4.0 g, Na2HPO4·12H2O 9.5 g, KH2PO4 1.4 g, MgCl2·6H2O 0.2 g, distilled water 1000 ml; pH, 6.9 to 7.3 (m/30 phosphate buffer); oxygen supply, 50 ml broth 500 ml flask, shake culture at 220 rpm; inoculum size, 10%; temperature, 28°C; and period of cultivation, 3 days.

The Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas sp.

To clarify biodegradation pathways of isoalkyl substituted aromatic hydrocarbons, oxidation products of isopropylbenzene and isobutylbenzene by Ps. desmolytica S449B1 and Ps. convexa S107B1 were examined.Oxidation products from isopropylbenzene were determined to be 3-isopropylcatechol and (+)-2-hydroxy-7-methyI-6-oxooctanoic acid. Isobutylbenzene was also oxidized to 3-isobutylcatechol and (+)-2-hydroxy-8-methyl-6-oxononanoic acid by the same strains.From these results, the existence of an unknown reductive step in the degradation of these isoalkyl substituted aromatic hydrocarbons and the initial oxidation of these aromatic hydrocarbons by the strains were made clear. The degradation pathways of isopropylbenzene and isobutylbenzene by these strains were discussed.

Application of Feeder Layer Method for Improved Colony Formation of Grape Cells and Protoplasts at Low Cell Density

derived from protoplasts), had some difficulties for practical application, Red-colored grape cells plated at less than 1 x 105 cells/ml could not form colonies. We examined the effects of numerouscomponentsof culture mediaon cell division of grape protoplasts from red-colored cells using the multiple-drop-array (MDA) technique.1] The frequency of cell division was 35%at the maximum, but these protoplasts stopped proliferation after dividing a few times and wefailed to obtain colonies from these grape protoplasts by combining may components of media. We had already reported the grape protoplast culture of a cell line that did not produce anthocyanin pigments using the feeder layer method.2) Wefurther examined the effects of conditioned medium (filtrate of cultured broth) and the feeder layer method in order to overcome the difficulties mentioned above with cell suspensions of a red-colored cell line of grape as a test object. Here we report the diminution of plating cell densities of the red-colored grape cells and the colony formation of grape protoplasts derived from the red-colored cell line by application of the feeder layer method. Tobacco cells also could be used as feeder cells for colony formation of red-colored grape cells.