Studies on Osmophilic Yeasts

Assimilation of galactose and maltose by Saccharomyces rouxii, which is a typical salt-tolerant yeast playing an important role in soy-brewing, was negligible or extremely poor in the medium containing 18% NaCl, although the assimilation in the ordinary medium was vigorous. The yeasts which were able to assimilate and ferment galactose, maltose and/or saccharose in the high-saline medium were limited to a few strains. From the studies on balances of fermentation products, it was revealed that ethanol yield based on sugar consumed in the 18% NaCl-medium was lower than that in the ordinary medium, suggesting that other fermentation products than ethanol should be accumulated. Analytical results of the fermented broths showed much polyalcohol production in the high-saline medium.     

Studies on Osmophilic Yeasts

A specific phenomenon of polyalcohol production by yeasts in the medium containing high concentration of sodium chloride was described. Pichia miso, an excellent polyalcohol producing yeast, produced only one type of polyalcohol, namely glycerol, in the medium containing sodium chloride at high concentration, although the yeast could produce three kinds of polyalcohols, glycerol, d-arabitol and erythritol, in the medium containing high concentration of sugar. It was also found that the various yeasts of non-glycerol producing type, could produce a considerable amount of glycerol in the highly saline medium. This phenomenon suggests that the metabolic pathways of yeasts may be markedly altered by the high concentrations of salts.     

Studies on Osmophilic Yeasts

Various conditions were studied which gave influences on polyalcohol production by Pichia miso. Pichia miso gave an excellent yield of polyalcohol showing good growth in the vitamin-free medium. Unlike Zygosaccharomyces high concentrations of phosphate such as 2% as KH₂PO₄ in the medium showed no detrimental effect on polyalcohol production. Remarkable reduction in polyalcohol yield was observed in the medium of high concentration of nitrogen sources, among which yeast extract showed the most striking effect. Polyalcohol fermentation was provoked in the medium of 0.1% yeast extract while the metabolic activity distinctly converted to ethanol fermentation when the organisms were incubated in the medium of 4.0% yeast extract. The fact that a large amount of ethanol more than 6% in the medium was produced aerobically by genus Pichia of oxidative dissimilation type seemed to be very interesting and noticeable. For the purpose of industrial production, it was shown that polyalcohol production in jar-fermenter scale was achieved with as good yield as that in shaking flask culture.     

Studies on Osmophilic Yeasts

In a medium containing high concentration of yeast extract was observed aerobic formation of a large amount of ethanol by various yeasts of non-fermenting or extremely poor fermenting type such as Torulopsis famata, Candida polymorpha and Pichia membranaefaciens etc. Acetaldehyde formed as a metabolic intermediate leading to ethanol formation was trapped by the addition of sulfite and was identified as 2,4-dinitrophenyl-hydrazone.     

Studies on Osmophilic Yeasts

The characteristic of salt-tolerance of the osmophilic soy yeasts was different from their sugar-tolerance in the points of viability of the individual cells in a high concentration of salts or sugars of the same osmotic pressure, and of the limiting osmotic pressure for their growth. It became much more apparent that growth of the osmophilic yeasts in the medium of a high concentration of sodium chloride involved a process of physiological adaptation. The order of the toxicity of the salts for the growth of Zygosaccharomyces major was as follows: K⁺<Na⁺<Mg⁺⁺<Ca⁺⁺<Li⁺ Cl^-<SO₄^--≒NO₃^-     

Studies on Osmophilic Yeasts

In order to ascertain the existence of polyol dehydrogenase which seemed to take a part in polyalcohol production by Pichia miso, the preparation and the partial purification of polyol dehydrogenase from the cells of Pichia miso were carried out. Some properties of this enzyme preparation and the identification of the products formed by this enzyme action were also described. This enzyme preparation was found to catalyze the following reactions:

D-arabitol+DPN⁺?D-Xylulose+DPNH+H⁺,Polyalcohol+DPN⁺?Ketose+DPNH+H⁺.     

Studies on the Antigenic Activity of Yeasts

In order to determine the determinant antigenic group of the mannan of Saccharomyces cerevisiae, a series of inhibition tests were carried out employing oligosaccharides which separated from the acetolyzate and the hydrolyzate of the mannan. Tetraose, Man α1→3 Man α1→2 Man α→2 Man², corresponding to the structure of the longer branching moieties of the mannan showed the strongest inhibition, while the isomer, Man α1→6 Man α1→6 Man, corresponding to the core moiety, produced only one-tenth the inhibition of the former. This provides evidence that the branching moieties of the mannan play important role in combining with antibody. The fact that the disaccharide, Man α→3, showed significantly stronger inhibition than those of the other disaccharides, Man α1→2 Man and Man α1→6 Man, indicates that the most important part of the determinant group of the mannan is α1→3 linked D-mannose residue. The antigenic inactivity of the periodate-oxidized mannan containing unoxidized mannose residues indicates that the presence of 3-O-substituted-D-mannose residues adjacent to the D-mannose residues and joined with α1→d2 linkages, are essential to fit the combining site of the antibody.     

Studies on the Antigenic Activities of Yeasts

In order to determine the antigenic determinant groups of the mannan of Candida albicans by the precipitation-inhibition test, several oligosaccharides were prepared by acetolysis of the polysaccharide. The manno-oligosaccharides, from biose to heptaose were separated by a charcoal-Celite chromatography and a subsequent cellulose column chromatography. The oligosaccharides thus separated were examined on the degrees of polymerization and the mode of the linkages, and evidence was obtained that the biose and triose were joined through α1→2 linkage only, while the tetraose, pentaose and hexaose contained α1→3 linkage in addition to α1→2 linkages. Heptaose was joined entirely through α1→2 linkage. In the precipitation-inhibition test, the inhibitory power of the oligosaccharides of acetolysis product was found to be the following order: hexaose>heptaose>pentaose>tetraose>triose>biose, and the amount for the 50% inhibitions were 0.025, 0.09, 0.12, 0.60, 3.96 and 5.84 μmoles respectively. On the other hand, the biose, triose and tetraose, which were isolated from the acid-hydrolysate of the mannan of Saccharomyces cerevisiae and joined through α1→6 linkage, showed poor or nearly no inhibitory power. The above facts provide an evidence that the consecutive α1→6 linkages were not located in a position that is responsible for antigenic specificity of the mannan of C. albicans.     

Studies on the Antigenic Activities of Yeasts

In order to provide further information on the chemical nature of the antigenideterminants of the mannan of Saccharomyces cerevisiae, the mannan was digested by Arthrobacter α -mannosidase, and 9, 21, 35, 59 and 62%-partially degraded mannans were prepared in the present study. Acetolysis of each degraded mannan showed that only a small amount of the tetrasaccharide was detectable in the 35%-digested mannan, whereas the predominant product of the 59 and 62%-digested mannan was mannose. The result of a quantitative precipitation reaction with the degraded mannans showed that the precipitation activities were partially or completely destroyed by the action of the enzyme. The lack of the tetrasaccharide moieties of the mannan were noticeable by a decrease in the precipitating ability. It was observed that the decreasing ratio of either the maximum amount of the antibody N precipitable by the mannan or per cent degradation of the mannan were essentially equal and yielded nearly a straight relationship between 0 and 2.0 hr digestion. However, the 59 and 62%-digested mannans, containing trace amounts of di- and trisaccharides in the branching parts, showed no significant antigenic activities. Furthermore, the molar ratio of the tetrasaccharide relative to the trisaccharide also gradually decreased. These observations confirm that the tetrasaccharide moiety, Man α1→3Man α1→2Manα1→2Man, plays an important role as the antigenic determinant. The core mannan moiety completely lost both the precipitating ability and inhibitory activity in ranges employed up to 1500 μg. These findings offer a direct proof that the core mannan moiety of mannan is not responsible for antigenic activity, and functions merely as the “carrier” of the antigenic determinants which dominate the immunological specificity.     

Studies on the Antigenic Activities of Yeasts

The antigenic mannan of Candida albicans was degraded by acid-hydrolysis and the resultant oligosaecharides were fractionated by a carbon-Celite and a subsequent cellulose-powder chromatography to yield four oligosaecharides, pentaose, hexaose, heptaose and octaose, which involved 2,6-di-0- and 6-0-substituted mannopyranosyl residues as the common. feature. These oligosaccharides showed lower precipitation-inhibition activity than that of the hexaose of acetolysate, the strongest inhibitor among the oligosaccharides described in the preceding study. The order of inhibitory powers of oligosaccharides was as follows: hexaose of acetolysate>heptaose>pentaosez=octaose>hexaose. The μmoles requiring for 50%-inhibition were 0.025, 0.15, 0.20, 0.20 and 0.50 respectively. The results clearly indicate that the determinant groups of the mannan of C. albicans employed this study are the hexaose moieties which constitute the branching parts of polysaccharide.     

Studies on Utilization of Hydrocarbons by Yeasts

The production of microbial cell substances from hydrocarbons has been attracting attention of people for many years. Production of bacterial cell from hydrocarbons is disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell production. The effect of medium composition on yeast growth and the utilization of individual hydrocarbon by yeast, strain Y-3, were investigated.

As a nitrogen source, urea was more effective than ammonium nitrate. When a very smal! amount of corn steep liquor was added, yeast growth was very improved. Aliphatic series of hydrocarbon lower than C₉ were not or very slightly assimilated by this yeast.

Generally speaking, series of even-number hydrocarbons were more effective than those of odd-number hydrocarbons.

We found that the yeast Y-3 strain reported in the previous paper¹⁾ has a diterminal oxidation system of hydrocarbon.

This yeast capable of growing in mineral-salts solution with hydrocarbons as sole source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was also isolated from n-decane cultures.

Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating it with resting cells of this yeast.

1,11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric acid by the same treatment as described above.     

Studies on the Antigenic Activities of Yeasts

In order to identify the antigenic determinant groups of the mannan of C. albicans serotype A, six kinds of manno-oligosaccharides of up to 7 units in chain-length connected by α1→2 linkages were prepared from the partial acetolysate of the parent mannan. In the precipitation-inhibition test of anti-C. albicans serotype A serum with its homologous mannan, inhibitory power of the oligosaccharides was of the following order: heptaose→: hexaose>pentaose>tetraose>triose> biose, and the amounts for 50%-inhibition of the former four oligosaccharides were 0.08, 0.10, 0.50 and 3.0 μmole respectively, and the inhibitory power of the latter two oligomers at 0.5 μmole were 8 and 5% respectively. On the other hand, the cross-inhibition test of anti-C. albicans serotype A serum with the heterologous mannan of C. albicans serotype B afforded the result that the order of inhibitory activities was hexaose>heptaose>pentaose>tetraose>triose> biose, and that the amounts for 50%-inhibition were 0.05, 0.08, 0.1, 0.45, 0.50 and 3.0μmole respectively. Furthermore, the results of inhibition test on the anti-C. albicans serotype A serum absorbed with the mannan of C. albicans serotype B revealed that the biose, triose and tetraose did not show significant inhibitory power in the range employed, whereas the pentaose, hexaose and heptaose did not significantly affect the inhibitory activities. Thus, it was concluded that the antigenic determinants of the mannan of C. albicans serotype A are α1→2 linked hexaose or heptaose moieties. Based on the above facts, the serological differences between two antigenic mannans of C. albicans serotype A and B may reside at least in the length of the antigenic determinants in which the former is longer than the latter considering the length of the α-D-manno-pyranosyl residue.     

Studies on the Antigenic Activities of Yeasts

In order to provide further information about the immunochemical differences between two mannans of Candida albicans serotype A and serotype B, quantitative precipitation-inhibition tests of anti-C. albicans serotype B serum were carried out in the present study. Oligosaccharides were prepared by acetolysis of a homologous mannan, and a1→2 linked di-, tri-, tetra-, penta- and hexasaccharide were separated in chromatographically homogeneous states. The latter two oligomers contained a small amount of an isomer containing a1→2 and a1→3 linkages. In the precipitation-inhibition tests of anti-C. albicans serotype B serum with its homologous mannan and heterologous mannan of C. albicans serotype A, the inhibitory power of the oligomers was of the following order; hexa-> penta-> tetra-> tri-> disaccharide, and the amounts for 50%-inhibition of the former 4 oligomers were 0.02–0.03, 0.05–0.07, 0.1–0.2 and 0.3–0.4 μmoles respectively, whereas disaccharide was very poor inhibitor. The lower oligomers, a1→2 linked tri- and tetrasaccharide, showed considerably strong inhibitory activities. The results obtained in the present study confirmed that the antigenic determinants of the mannan of C. albicans serotype B is the hexasaccharide moiety corresponding to the longest branched chains of mannan, and moreover, the a1→2 linked tri- and tetrasaccharide moieties play an important factor in dominating immunochemical specificity.     

Studies on the Utilization of Hydrocarbons by Yeasts

The growth of M. japonica in hexadecane or decane medium was markedly improved by dialysis culture and there existed a “growth-inhibitory factor” in the dialyzable material. This material inhibited the growth in hydrocarbon medium but not in glucose containing medium. Free fatty acids excreted extracellularly by dialysis culture in hexadecane medium were mostly palmitic, myristic and lauric acids in the culturing chamber, but almost exclusively lauric acid was found in the dialyzable material. These acids, assimilated themselves by the organism, inhibited the growth in hexadecane medium but did so only weakly in glucose. It was concluded that lauric acid was at least partly an active principle of “growth-inhibitory factor” in the dialyzable material in hexadecane medium.