Growth response of a strain of Micrococcus glutamicus to ferrichrome or related compounds, a large amount of ascorbic acid and iron salt were further studied with the consideration about the relationship between these substances, and following results were obtained. (1) Rutin and 8-hydroxyquinoline supported the growth at very low concentrations, and the effectiveness of some other chelating agents was confirmed. (2) The growth of the bacterium was supported also by autoclaving glucose in the medium, and the active factor produced in the medium was speculated to be a chelating agent in view of other literature reports. (3) The effect of iron salt is specific and could not be replaced by other metals. (4) A very minute amount of maganese specifically competed with iron in supporting the growth of the bacterium. (5) Synergistic action between iron and rutin was noticed, while the mutual action between manganese and rutin was obscure. From these results, the mode of the action of these growth supporting compounds were suggested as follows. In the basal medium, manganese competes the iron and retards the utilization of iron by the bacterium. The iron in the medium can be utilized by chelation with some chelating agent. The chelating agent also accelerates the utilization of iron by excluding the harmful action of some metals (for example, manganese, copper and zinc) by chelating them. Autoclaving glucose in the medium produces a chelating agent which acts in the same manner as described above. A large amount of iron salt overcomes the action of manganese and supports the growth of the bacterium. The effect of iron salt or some chelating agent or autoclaving glucose in the medium was also confirmed in some other coryneform bacteria. From these results, the singnificance of chelating agents in the growth of microorganisms was discussed.
Thirteen phenolic inhibitors were tested for their effectiveness in inhibiting the growth of Staphylococcus aureus in nutrient broth and nutrient agar. The presence of agar in the test medium interfered with the inhibitory activity of nine of the compounds studied. It was found that washing the agar prior to use removed the antagonistic material and completely restored the inhibitory activities of seven of the inhibitors. The effectiveness of three of the compounds was only partially restored by washing the agar.
Hydrocarbon-utilizing bacteria were isolated from oil-brine, soils etc. sampled in oil fields in Japan during 1956, and the following species were identified: Corynebacterium hydrocarboclastus nov. sp., 11 strains; Pseudomonas nitroreducens nov. sp., 1 strain; Pseudomonas maltophila HUGH and RYSCHENKOW, 5 strains: Brevibacterium lipolyticum (HUSS) BREED, 2 strains; Pseudomonas desmolytica GRAY and THORNTON, 5 strains; Flavobacterium ferrugineum SICKLES and SHAW, 1 strain; and Alcaligenesfaecalis CHASTELLANI and CHALMERS, 1 strain. One difference between Gram-negative bacteria and Gram-positive bacteria was described on the basis of the ability of assimilating hydrocarbons.
Determinative studies were carried out with the pseudomonads isolated from oil-brines and the other related materials obtained in petroleum zones in Japan, and the following species were identified: Pseudomonas iners nov. sp., 2 strains; Pseudomonas stutzeri (LEHMANN and NEUMANN) KLUYVER, 10 strains; Pseudomonas putrefaciens (DERBY and HAMMER) LONG and HAMMER, 2 strains; Pseudomonas azotoformans IIZUKA and KOMAGATA, 7 strains; Pseudomonas nitroreducens IIZUKA and KOMAGATA, 3 strains; Pseudomonas schuylkilliensis CHESTER, 4 strains; and Pseudomonasaeruginosa (SCHROETER) MIGULA, 4 strains. From the physiological and biochemical characteristics of these pseudomonads, the authors discussed the reason for their occurrence in oil-brines.
Peptone, urea, ammonium sulphate and sodium nitrate were added, in different amounts, to a nutrient medium which was inoculated with several strains of Beijerinckia indica from soils in different parts of India. The medium was incubated at 30° for fourteen days after which the nitrogen contents of the culture liquids were determined. It was observed that addition of nitrogen through these substances had an inhibitory effect on nitrogen fixation. The highest rate of depression of nitrogen fixation was observed in the case of sodium nitrate and the lowest in the case of urea.
For the preparation of clean bacterial spore samples, cultural conditions were examined under which the least possibility of contamination of vegetative cells and of non-refractile spores could be attained. The cultivation of bacilli on the nutrient agar of 5mm in thickness for 3 days was found to be favorable for this purpose. For the purification of spores, an improved method of SACKS and ALDERTON was adopted. After analyzing the composition of the upper layer of their Y-system, a single layer system of 25% Carbowax 4000 solution in 0.5M phosphate buffer (pH 7.1) was found to be favorable solvent for the separation of spores and vegetative cells. When impure sample was mixed vigorously with this solvent and centrifuged at 600×g for 30min, spores remained homogeneously dispersed in the supernatant, while vegetative cells sedimented at the bottom. Recommended procedure for the purification of bacterial spores is described.
The empirical formulas reported previously in the prediction of hindered settling velocities of fine particles in suspension were applied to the determination of equivalent size of specific microbial cells in gravitational as well as in centrifugal field. The agreement between the size determined so far from the velocities in hindered settling and that measured directly with a microscope seemed to be satisfactory in the case of yeast cells (Saccharomyces cerevisiae) and bacterial cells (Serratia marcescens) studied. The determination was also made using a strain of Actinomyces (Streptomyces griseus) and an activated sludge, both of which are usually complicated enough in shape compared with bacteria and yeast so that no quantitative studies have ever been made to assess their shape. If an attempt is made on transfer problems from/to microbial cells, the application of this sort of study will be useful.
The purification of Rhizopus delemar lipase was undertaken to compare its enzymatic properties with those of the crystalline Aspergillus niger lipase (1). From culture filtrate of Rhizopus delemar grown by shaking culture, the enzyme was purified by ammonium sulfate fractionation, Duolite A2 resin treatment, SE-Sephadex column chromatography, acetone fractionation and gel-filtration by Sephadex G-75. The specific activity of purified enzyme thus obtained was 2, 600-fold higher than that of the starting culture filtrate. Olive oil hydrolyzing activity of the enzyme was maximum at pH 5.6 and at 35°. The enzyme was stable at the range of pH 4 to 7 and at temperatures lower than 45°, but quickly inactivated above 50°. A weak esterase activity found in the enzyme preparation would be considered to be shown by the activity of the lipase itself. Remarkable differences of the enzymatic properties of Rhizopus and Aspergillus lipases are found in their isoelectric points, turnover numbers and reaction conditions.
A vitamin B12-complex has been isolated from Lactobacillus delbrueckii No. 1 walls by treatment with 0.2N HCl. This was purified by means of repeated CM-cellulose column chromatography. The complex was obtained as red powder with a yield of 0.43mg per gram of cells (dry wt.). Ultracentrifugal analysis showed a single peak with the sedimentation coefficient of 1 S20, w. From the absorption at 361mμ and the dry weight of the complex and consideration of the sedimentation coefficient, the molecular weight of the complex is deduced to be approximately 15, 000. Absorption spectra and chemical analysis showed that the complex is composed of B12 and peptide. Using excess cyanide ions in alkaline pH, B12-moiety is suggested to combine with peptide at the position of cobalt which is ordinarily co-ordinated with the N 3 of benzimidazole-moiety The intact complex can be used as well as heat- and/or KCN-treated ones for the growth of the organism.
The B12-complex and several wall preparations with various treatments have been analyzed for the amino acid compositions. The complex consists of lysine 30, threonine 15, valine 10, alanine 7, aspartic acid 7, leucine 7, glycine 5, glutamic acid 4, phenylalanine 4, isoleucine 3, serine 3, tyrosine 3, and vitamin B12 1. The major amino acids of the wall preparations are aspartic acid, glutamic acid, alanine, and lysine with the molar ratio of 1:1.2:2.1:1.2. The reason of difference of amino acid compositions of the complex and the wall preparations is discussed as a consequence of isolating a minute part of the wall for the binding of B12 which may be specific in the B12-requiring Lactobacillus.