(1) All the DL-forming lactic acid bacteria used in this research, i.e., Lactobacillus plantarum (2 strains), L. arabinosus, L. japonicus, L. brevis, and Pediococcus hennebergi, demand nicotinic acid as an essential growth factor, while organic bases such as purines and pyrimidines are not essential for these strains. (2) If DL-formers are cultured in a medium which is limited in nicotinic acid, the resulting lactic acid reveals measurable optical activity. This is caused by the suppression of the formation of co-racemiase, DPN, by the insufficiency of its precursor. (3) By determining the optical forms of lactic acid thus formed, DL-formers can be divided into D- and L-fomers. This fact is considered fundamental from the standpoint of natural systematics.
(1) Natural variation in monospore lines of Koji molds (Asp. oryzae and Asp. sojae), isolated from commercial Koji material or soil and from laboratory stock cultures, has been observed. (2) We can divided the 58 strains of Koji molds investigated into two groups; one group consists of inconstant strains which are very liable to produce natural variants, and the other consists of strains which remain constant through successive single spore culture. (3) The inconstant strains develop colonies bearing various proportions of conidia and aerial mycelium (X-type). They generally form large conidia (Asp. oryzae var. magnasporus) but sometimes medium sized conidia (Asp. oryzae s. str.), which produce large conidia occasionally. The colonies of the constant strains show abundant conidial formation and smooth surfaces (C-type). The conidia are mostly small (Asp. oryzaevar. microsporus) but sometimes medium in size (Asp. oryzae s. str.). (4) The colony types of the variants are as follows: C (Conidial type, whole colony covered with conidia), M (mycelial type), R (restricted in growth rate), St (sterile type, little sporulation on all media tested), Nit (requiring reduced nitrate, very faint growth on Czapek's agar), and LS (semi lethal, growth cease immediately after germination). (5) Pedigree cultures of the 8 inconstant strains have been made, but no definite segregation ratios for each variant type have been recognized through successive generations. (6) The LS and N types commonly occur spontaneously from the M-type.
(1) The constant C- and M-types which were obtained from one of the "Wild Strains" (Asp. Oryzae aX) have been subjected to ultraviolet treatment. (2) M-type mutants have been obtained from the parent C-type, and C- and X-type mutants from the parent M-type. These mutants have proved to be the same as the natural variants mentioned above (1). (3) The mutants that have been obtained from the C-type are as follows: 1. Almost-normal 2. Restricted 3. Sterile 4. Reduced nitrate 5. Heavy 6. Olive 7. Brown 8. Albino 9. Mycelial 10. Yeast 11. Maroon-exudate. The mutants that have been obtained from the M-type are as follows: 1. Almost-normal 2. Restricted 3. Sterile 4. Reduced nitrate 5. C 6. CR 7. Brown 8. Yellow 9. X (4) The conidia of the C- and M-types which are natural variants are of comparatively uniform size. Among induced mutants from the C- and M-types, the conidia of Reduced nitrate, Brown, Mycelial, and X are generally found in a great variety of sizes. (5) Thus the morphology of Koji-molds has been remarkably changed by ultraviolet irradiation except for the structure of conidial and conidiophore walls.
1. The leucineless and histidineless mutant strains of Asp. Sojae which proved to be comparatively stable, were irradiated with ultraviolet light, and the following double auxotrophic mutants were isolated by total isolation and starvation techniques. From strain 10C his: requiring reduced nitrate (11 strains), arginineless (2), and methionineless (1). From strain 14C leu: requiring reduced nitrate (23), arginineless (1), histidineless (1), lysineless (1), methionineless (2), nicotinic-less (1), and adenine- or hypoxanthineless (1). 2. The starvation technique was effective for isolating double auxo trophs from some strains. 3. Among the mutant types selected by the starvation technique, the reduced nitrate strains were obtained in exceedingly high frequency (37 mutants out of 51 double auxotrophs). 4. In Koji-molds (Asp. Oryzae and Asp. Sojae) the most frequent deficient types have been found to be arginineless, histidineless, lysineless, and methionineless mutants. 5. Among single auxotrophs, lysineless, histidineless, and leucineless mutants were comparatively stable, but other mutants showed frequent spontaneous reversion. 6. Double auxotrophs proved to be more stable than single auxotrophs-in such genetic characters as colony type and nutritional requirement.
1.The causes of the difference in variability among strains of Koji-molds have been studied cytologically by examination of conidial size, number of nuclei in conidia and nuclear migration. 2. The constant strains of Koji-molds have conidia with comparatively uniform sizes (4-6μ). The inconstant strains, on the other hand, produce conidia in a variety of sizes including some with extraordinarily large diameters (6-10μ or more). 3. Feulgen-positive spherical structures are found at all growth stages in strains of Koji-molds. Mitotic-like pictures are frequently observed in germ-tubes, hyphal tips, or spore-producing tubes. 4. The conidia of the constant strains have been found to contain 1-4 nuclei; the majority contained two nuclei. The conidia of the inconstant strains, however, are multinucleate (8-20 or more). It is recognized that there is a close relationship between conidial size and the number of nuclei. 5. In the constant strains, one nucleus moves into conidium from apex of the sterigma at the beginning of the conidium formation, and is divided at once into 2-4 through what is probably mitotic division. In the inconstant strains, more than two nuclei migrate from sterigma into the newly formed conidium. The conidia become further multinucleate (8-20 or more) as a result of prompt independent nuclear divisions. 6. As concerns the migration of the nucleus from the vesicle into the sterigma, the authors have recognized that in the constant strains only one nucleus migrates into the sterigma, while in the inconstant strains at least 2-4 migrate simultaneously. 7. From the facts shown above it might be assumed that the variability of the inconstant strains of Koji-molds depends chiefly upon the multinucleate character of the conidia.
(1) Effect of variation of day-length, day- and night-temperatures and of intensity of daylight upon the growth rate of Chlorella was investigated using combinations of conditions which were varied as follows: Day-length: 6hours (18 hours dark), 12 hours (12 hours dark), 18 hours (6 hours dark), and 24 hours (no darkness). Day-and night-temperatures: 25°, 15° and 7°C. Intensity of daylight: 50, 10, 2, and 0.4 kilolux. (2) In general the rate of growth was affected by changes of day-temperature by far more profoundly than by changes of night-temperature. In the temperature range studied, the higher the day-temperature, the greater was the growth rate. Higher night-temperatures had no recognizably favorable effect except when the day-temperature was as low as 7°. (3) In general, the growth rate was directly proportional to the day-length ("day-limited growth") at shorter day-lengths. Such a proportionality extended to longer day-lengths, the lower the daylight intensity; and the intensity of daylight, under which a day-limited growth occurred markedly, was higher, the higher the day-temperature. At longer day-lengths, and especially under stronger daylight, the growth rate tended to become independent of day-length (the phenomenon of "day-saturation"), or to become rather smaller with the increase of day-length (the phenomenon of "day-oversaturation"). The latter phenomenon was accompanied by a bleaching of algal cells. Both the day-saturation and -oversaturation occurred ceteris paribus more markedly at lower day-temperatures. The day-oversaturation occurred most profoundly when both the day and night temperatures were 7°, but ceased to occur when the day-temperature was 7° and the night-temperature was 25°. (4) Although the growth rate increased, in general, with the increase of day-length, the favorable effect of long-day condition decreased with the decrease of day-temperature. At 7°, and especially when the daylight intensity was high, there was almost no difference in the growth rates under short-day and long-day conditions. It was also observed that the temperature-dependence of growth rate decreased and eventually tended to disappear with the decrease of daylight intensity and with the shortening of day-length, and that the daylight intensity, under which the growth rate became light-saturated, was lower at lower day-temperatures. All these facts indicate that the short-day condition and weaker daylight become relatively less disadvantageous with the decrease of day-temperature. (5) The phenomena of day-limited and day-saturated growths as they were conditioned by temperature, intensity of daylight and day-length were explained on the basis of observations made earlier by Tamiya et al., who, by determining the relative rates of light-dependent and light-independent phases in the growth process of algae, have found that the light-independent phase has a considerably greater temperature-coefficient than the light-dependent phase. (6) The phenomenon of "day-oversaturation", which occurred at lower temperatures and under stronger daylight, was explained as being due to the injurious effect of strong light upon the so-called "light cells" which have been shown by Tamiya et al, to become abundant in cultures when temperature was low and light was strong.
That α-Ketoglutaric acid fermentation is widely distributed in microorganisms was confirmed by experiments with shaken cultures of Pseudomonasfluorescens, Serratia marcescens, Bacillus megatherium, Bacillus natto (a variant of Bacillus subtilis), Bacterium succinicum, Gluconoacetobactercerinum and a new Bacterium strain No. 84C (temporary name), isolated from soil by the authors. The maximum yield of α-Ketoglutaric acid among the species examined was obtained with Bacterium strain No. 84C which produced up to 56% of the acid for total glucose supplied in a 10% glucose concentration after 72 hours' incubation. A description of the morphological and physiological properties of this bacterium is presented. With regard to the constituents of the fermentation medium, various nitrogen and carbon sources and their optimum concentrations, as well as the additive influences of metals, arsenite and vitamins, were examined, especially in relation to α-Ketoglutaric acid fermentation in Serratia marcescens and Bacterium strain No. 84C, both of which are considered to have value for industrial application. α-Ketoglutaric acid was formed from D-glucose, D-mannose, D-galactose, D-fructose, D-xylose, L-arabinose, L-rhamnose, K-gluconate and K-2-ketogluconate in Serratia marcescens No. 18. In the case of Bacterium strain No. 84C, α-Ketoglutaric acid formation was tested with limited carbon sources involving of D-glucose, D-galactose, D-fructose, sucrose, maltose, and Ca-gluconate, and the results indicate that all substrates were utilized for acid formation. The optimum range of glucose concentration in the medium was found to lie at 3-4% for the former strain; in the latter it was somewhat at a higher, 5-7%, and in this strain complete fermentation was possible even glucose concentration of 10%, provided that the medium was balanced in other components and an excess of CaCO3 was added. Among inorganic nitrogen sources, ammonium salts such as (NH4)2SO4, NH4H2PO4, or a mixture of the two, were found to be suitable, and a rather sharp peak seemed to exist in the range of limited concentration, 0.15-0.17% in the case of (NH4)2SO4 in medium containing 7% glucose. The highest yield was obtained by using Bacterium strain No. 84C and a medium containing 0.135g (NH4)2SO4 and 0.135g NH4H2PO4 with a 10% concentration of glucose. The addition of iron to the medium increased the acid yield in Serratiamarcescens; the optimum quantity was found to be 0.1-0.6ppm Fe. Remarkably interesting were the effects of adding arsenite and thiamin; these gave in optimum concentrations a marked increase in acid production in Serratia, while in Bacterium strain No. 84C no stimulative effect was observed. From the investigation of chemical changes during fermentation and from the isolation of intermediates of the initial stages of fermentation, it may be said that α-Ketoglutaric acid formation in Pseudononas fluorescens No. 33F, Serratia marcescens No. 18 and Gluconoacetobacter cerinum most probably proceeds via a direct oxidative pathway as has been proposed by Koepsell et al. In Bacterium strain No. 84C, however, the Emden-Meyerhof scheme, or some alternative pathway involving a direct oxidation not involving 2-ketogluconate as an intermediate, are considered possible.
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