Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation.Germination and outgrowth are developmental stages in the transition of a spore to a vegetative cell (4, 5). Both stages consist of simultaneous chemical and physical alterations that are reflected in the appearance of the spore. In this report, germination is regarded as a change from a light-refractile spore to a nonrefractile cell. Outgrowth designates the sequence of development after germination that culminates with cell division. Previously, germination and outgrowth of bacterial spores have been examined with a scanning electron microscope (16). The three-dimensional images produced by the scanning microscope revealed distinctive changes in spore surface and overall anatomy during the developmental process.In this investigation, we examined germination and outgrowth of single spores of Saccharomyces cerevisiae by scanning electron and phase-contrast microscopy. The only other work involving microscopy reported on the germination of this organism includes thin sections of resting and germinated spores contained within an ascus (7) and hand-drawn representations of different morphological ' Present address: stages observed with a light microscope (6,12). In an effort to distinguish morphological features as well as certain chemical and physical modifications that occur during the developmental phases, we also determined changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOHsoluble carbohydrates during germination and outgrowth.MATERIALS AND METHODS Organism and cultural conditions. A diploid S. cerevisiae Y-55 (20) was used in this study. Details concerning its growth and sporulation and the techniques for preparation of single spores were described earlier (15). Spores were germinated in a 0.2% succinic acid synthetic medium designated SSM (17); glucose and Tween 80 (15) were added aseptically before inoculation to give a final concentration of 1% for each component. Inoculated flasks were aerated by rotary agitation at 250 rev/min :30 C, in an incubator shaker. Samples were removed from the flasks at 30-min intervals and quickly cooled. Cells then were separated from the cult...

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.

Conditions are described that led to the isolation of NRRL B-2309M, a strain of Bacillus popilliae which sporulates regularly in laboratory culture. Colonies grown on a medium formulated with yeast extract and the ingredients of Mueller-Hinton with phosphate, trehalose, and agar, produced 20% spores in 10 to 12 days. The quantity and kind of yeast extract determine the extent of sporulation, although there are other requirements for optimal growth and sporulation. Spore inocula free of viable vegetative cells are necessary to maintain sporogenicity since asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain, NRRL B-2309N, is also asporogenic in larvae, but lethal, owing to vigorous vegetative growth. Strain B-2309M is infective when vegetative cells or spores are injected into Japanese beetle larvae but fewer spores are formed in vivo than when infections are caused by NRRL B-2309. The characteristics of four related strains of B. popilliae are tabulated.

Oxidation of pyruvate, acetate, succinate, and glutamate was compared in Bacillus thuringiensis, B. alvei, B. lentimorbus, and B. popilliae. Cells of B. thuringiensis and B. alvei in transition from vegetative growth to sporulation oxidized these substrates by tricarboxylic acid (TCA) cycle reactions. No TCA cycle activity was exhibited by B. lentimorbus and B. popilliae cells that do not sporulate. B. popilliae decarboxylated C-1 of pyruvate and glutamate; B. lentimorbus, C-1 of pyruvate only. B. thuringiensis and B. alvei oxidized pyruvate and acetate at a much higher rate in the absence of amino acids and related compounds than when these nutrients were exogenously supplied; in contrast, there was no appreciable increase in C-1 decarboxylation of pyruvate by B. lentimorbus and B. popilliae. No nutrient effect was observed on succinate and glutamate oxidation in any of these four organisms.