Further Observations on
Octosporus Yeast [Excerpted]

The following excerpts were published under the title 'Weitere Beobachtungen bei die Octosporus hefe' by M.W. Beijerinck in the German medical journal Centralblatt für Bakterilogie, Parasitenkunde und Infektionskrankheiten (Volume 3, Band II, pages 450-455 and 518-525) in 1897.

This NIH library translation (NIH-95-347) from German into English was provided by Ted Crump, translator. Copyright restrictions apply. This translation is for exclusive use of Frans Hochstenbach, and has been edited by Frans Hochstenbach and Norman Hu. It is displayed here with the explicit permission of Frans. Advice on scientific interpretation was given by David Yarrow, Centraalbureau voor Schimmelcultures, the Netherlands.

In this paper, Beijerinck describes the isolation of sporogenic and asporogenic strains of fission yeast Schizosaccharomyces octosporus from dried currants and figs. For their isolation, he developed a 'dry-heat' method in which the oriental fruits are rinsed with water to remove attached yeast cells, the rinsing water is dried down, and the remaining material is heated to kill vegetative yeast cells and enrich for heat-stable yeast ascospores, which subsequently are allowed to sporulate and grow.

When grown on wort agar, sporogenic strains can be distinguished from asporogenic strains on the basis of the color of the colonies: sporogenic strains of Schiz. octosporus are white, while asporogenic strains appear light-brown. He demonstrates that, when used for the formation of new colonies, light-brown (asporogenic) strains give rise to only asporogenic strains, while white (sporogenic) strains, in addition to fresh sporogenic strains, can give rise to asporogenic strains.

We now understand asporogenic strains to be either sporulation mutants that have lost the ability to generate ascospores, or heterothallic strains which contain cells of only one mating type. In contrast, sporogenic strains are homothallic strains generating cells of both the + and - mating types, which can mate pair-wise to form ascospore-containing asci. Homothallic strains can loose their ability to generate both mating types and thus become heterothallic, as demonstrated by Beijerinck in this paper.

Further Observations on Octosporus Yeast [Excerpted]

(With illustrations)

by M. W. Beijerinck

At the present time, the genus Schizosaccharomyces encompasses three different species, namely S. octosporus, S. pombe (footnote 1) and S. asporus (arrack yeast). When I published the discovery of the first of these three in 1894, I was still unaware of S. pombe through my own analysis, and, on the basis of the descriptions available, believed that S. pombe and S. asporus could be identical. Subsequently, certain observations have led me to doubt this, and I became more familiar with pombe yeast thanks to the kindness of Herr Lindner. I immediately found that Herr Eijkman's arrack yeast is very different [from S. pombe]. For example, many S. pombe cells constantly produce four ascospores (which stain weak blue with iodine), while S. asporus is completely asporogenic. There are many other differences as well.

Certain questions about the variability of cultured wild organisms prompted me to resume my earlier, somewhat fragmentary study of the octosporus yeast, because this seemed to provide extraordinarily suitable material for experiments on cell variability. My expectations were not disappointed, and the now often-observed phenomena, especially the origin of an asporogenic strain, could lead to the hypothesis that both Schizosaccharomyces pombe and Sch. asporus could have originated as profoundly altered culture forms. In any event, I can demonstrate that during the relatively brief time that I have had the octosporus yeast in culture, profound strain changes have become so noticeable without selection, that if they were found in nature, no taxonomist would hesitate to attribute them to species differences. To strictly demonstrate this, it was necessary to compare the old strain with a freshly isolated strain; therefore, I had to find a method for repeated pure cultures of my yeast from natural raw materials. After many failed attempts, I found just such a method, which could be termed the 'dry method,' and which is based on the different behavior of vegetative cells and ascospores during drying at high temperatures.

The direct causes of the formation of the asporogenic yeast species are still obscure, as is common where questions of variability are involved. Two circumstances, which in laboratory experiments apparently have a direct effect, are unilateral exhaustion of certain nutritive substances and continued contact with moist or liquid medium, that is, the irregular alternation between growing and drying. Whether these rather complicated conditions can be broken down into simpler factors must await further experiments.

A phenomenon that is generally widespread in alcohol yeasts, but appears with particular clarity in the octosporus yeast, is proteolysis. Thus far we have only the sparse information that Herr H. Will has compiled. The clear relationships in the octosporus yeast also shed more light on that question. Namely, it was shown that the separation of the enzyme [from cells into the medium] is linked to the slow dying of the cell content, and that the enzyme itself belongs to the typsins and not to pepsins. Indications suggest naming it yeast trypsin, because it is not completely identical to pancreatic typsin. The same result could be determined for other alcohol yeasts examined.

1. Occurrences in Nature

In my first report on Schizosaccharomyces octosporus (footnote 2) I expressed the view that this yeast certainly must be widespread (footnote 3). I based this view on the microscope pictures of some fermentations obtained with currants in malt wort. [In those fermentations], despite an overproliferation of other alcohol yeasts, which hindered the isolation, some of the extremely characteristic cell pairs of our plant were found. To renew the isolation, I repeated the aforementioned fermentation experiment several times, and in fact obtained the result that I found unequivocally in a number of cases with Saccharomyces on currants in microscopical preparations (of unknown origin, but certainly from Greece, Asia Minor, and Turkey). In such fermentations, there were only vegetative cells and no asci; isolation was not possible in these instances because of massive overproliferation of ordinary yeast, so that even I could not say with any certainty whether the cells observed under the microscope are actually completely identical to the yeast I isolated in 1892; it is only determined that the vegetative states correspond very closely to each other, and that in any event a closely related form must be present.

With these renewed experiments I extended my observations to other oriental fruits that are marketed in a dry condition. Thereby it emerged that our yeast also occurs on figs, and in fact on the better quality from Smyrna. On the other hand, dates and raisins have always given a negative result, although I have conducted many experiments, especially with raisins. Direct isolation was not possible from fig fermentations, again, because of the overproliferation of other yeast species. Therefore, I had to search for a better isolation method.

In fermentation experiments using fruits of several northern regions, the Schizosaccharomyces cells were always completely absent. This is clearly linked to the fact that this genus only noticeably grows above 20C, and does not proliferate below 15C. Therefore, we are dealing with an inhabitant of warmer climates, and although natural heat sources in our regions, such as manure, certainly meet the requirements of many thermophilic bacteria, they do not meet those of alcohol yeasts.

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2. Isolation Method: Separation of the Ascospores from the Vegetative Yeast Cells

As I became convinced by experience gained in several fermentations that the octosporus yeast is a universally-occurring species, I took pains to demonstrate it in the rinsing waters of the aforementioned oriental fruits. I suspected that the eight-spore asci, which are so characteristic that they certainly cannot be hidden by soil and dirt, would have to be present. I found this demonstration also to be successful in the rinsing waters of a certain type of currant. As I furthermore became convinced that the octosporus yeast also occurs in raw material in an ascospore state, the separation of these ascospores from other yeasts, which at least generally are found only as vegetative cells, became possible.

I then asked myself: Is it possible to kill the vegetative cells of yeasts without thereby damaging the ascospores? The octosporus yeast itself proved to be particularly suited as an experimental material, because it had produced an asporogenic strain, and on the other hand offered massive spore material. I will not describe here the various experiments which were inconclusive, but rather outline directly those which led to success: The ascospores could be heated in a dry state up to 105, 110, even 115C (and perhaps even higher under absolute water-free conditions), without dying off. The vegetative cells, on the other hand, completely die off in a dry state even at much lower temperatures, namely at about 80C, while even at 56C a massive dying-off begins (footnote 4).

As I expected, and this became the principle of the separation method, the vegetative cells of the foreign yeast also begin to die massively at about 56C when heated in a dry state. Through these means, an enrichment of the ascospores relative to the vegetative cells of other yeasts in any material can be achieved; this is the one thing that is essential for a successful isolation.

This finding then became the basis for the following separation method: Currants soiled with much adherent dirt (footnote 5) and also figs were rinsed off with a small quantity of water; this was allowed to stand for a short time (so as not to extract too much sugar), poured onto a glass plate, and then slowly dried in incubators at 30C. The plates were then placed in a drying room and very slowly heated to 56C. At this temperature, some material was scratched off with a knife, or it was weighed, and the material either was used for cultivation on malt wort gelatins (whereby, however, octosporus colonies were obtained only in very isolated cases), or [the material] was used in fermentation experiments, in which it was placed in liquid wort of 10 saccharometer degrees, and then brought to the acid titer of 6-9% of normal acidity with lactic acid. Notably the last experimental procedure yielded an interesting and almost constant result. When set at 30C, most flasks remained unchanged for three days. Then (in addition to mycelia of Aspergillus niger, which could be easily removed with a platinum needle) a white precipitate of tetrads and octads clearly formed in the pure culture. As this proliferated vigorously, a common Saccharomyces also developed, which on the sixth day achieved single-handed control, so-to-speak, while the octosporus yeast regressed. Naturally, on the fourth day, when the foreign yeast was still barely detectable, I established a wort gelatin plate, which, with the 'surface cultivation' that I had always practiced, yielded a wonderful mixture of colonies of snow-white, drier, and somewhat roughly dotted octosporus colonies, and at least three more yellowish-stained common yeast species. These latter three had become so lethargic from drying at 56C, that they required several days for germinating, while octosporus clearly had already germinated. Wine yeast was absent from the preparations used for this experiment. If it had been present, I would have used a higher temperature, because it has a higher lethal temperature.

I cannot differentiate octosporus yeast isolated from Smyrna figs from the seemingly identical yeast isolated from currants.

Since my primary intention was achieved, it is not necessary to examine any further the consequences of additional heating of the dried yeast material at this point.

With respect to experimental procedure, I would also like to note that fermentation could occur more rapidly if the wort is not acidified to 6-9% of normal acidity, but rather to a lower acidity. However, since the octosporus yeast tolerates acid well, and considering the numerous bacterial spores that were introduced, I found it advisable to keep the acid titer high. All other observations arising from my experiment must be precluded at this time (footnote 6).

3. Formation of an Asporogenic Strain in Octosporus Yeast

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Next, let me define 'certain cultural conditions' to mean the placement of colony cultures on thick wort agar plates. This is generally required in order to have a certain distinguishing feature for selection; on wort gelatins, the difference between the two [asporigenic and sporogenic] forms cannot be observed macroscopically. On the other hand, it became clear that the colony cultures that were placed on wort agar plates, or in test tubes in wort agar, when completely mature (that is, after depletion of the nutrient medium) are of a three-fold nature: The first kind are white, and consist only of asci and ascospores; The second are light brown, and contain only vegetative cells and asci-like, but ascospore-free cells; The third are very light brown, 'mixed' with all three elements. These subtle color differences led me to discover the strain formation.

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In the inoculum area the light-brown colonies developed as constantly and hereditarily asporogenic. In the inoculum of the 'white' colonies, in addition to the many white forms, some 'brown' forms developed, from which, as before, the asporogenic form can be derived as a constant strain, and numerous 'mixed' colonies as well.

If new colony inoculums are established from the very light-brown 'mixed colonies,' or on wort agar, then they also break down into 'white' and 'brown' forms, but in a different ratio than in the inoculum of the white forms, from which they only slightly differ.

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The asporogenic strain has the following characteristics: The majority of the cells are almost exactly round and only somewhat ellipsoidally elongated just before division (see plate VIII, fig. 3). The proliferation first takes place in the colonies on wort gelatins through the usual pattern with 'conjugate formation' (plate VII, fig. 1); on the other hand, as the culture becomes older, it occurs only by very regular division into two [daughter cells], wherein the partial products for the most part separate immediately without producing the peculiar 'conjugates,' which are so characteristic for an asci-forming strain (plate VII, fig. 2). If one examines cultures of the vegetative strain that have aged on the wort gelatins when the growth has halted because of nutrient depletion, they appear as follows: Numerous cells are noticeably swollen, though not as strongly as in asci formation, and the swelling in other cells is accompanied by a renewed cell division, often with walls arranged perpendicularly to each other, such that a sarciniform appearance is formed. Such cell groups, the most part however, only contain 4, 5, or 6 cells. The large swollen cells exhibit, with extraordinary clarity, a large flattened, spherical, often doubled cell nucleus in the protoplasm. If cultivated in liquid wort, there is much less difference between the two strains, since in both the spore-forming and asporogenic strains not only two-cellular conjugates appear, but also cell strands of 3-4 cells, as well as the tetrads and octads so peculiar to Schizosaccharomyces. The only constant variation, I believe, exists in the fact that the cells of an asporogenic fermentation, on average, are somewhat smaller than those of a sporogenic fermentation; however, this difference is very small.

The differences are still not pronounced in the young colonies on wort gelatins and agar, so that fig. 1, which illustrates a very young colony culture of the asporogenic form, would not be different if colonies of the same developmental stage originating from ascospores were photographed.

Although strain formation (or polymorphism) also occurs very markedly in several other yeast species, and is expressed both in the general body build as well as in ascospore formation, I am still not aware, despite much experience in this area, of an example where, in addition to such a profound morphological difference between the strains, such noteworthy physiologic differences have been developed as here.

The first example of these I could name is the strong regression of the trypsin formation in the asporogenic strain, while this function, under conditions to be discussed in detail later, is very strongly developed in the asci-forming strain. I also recall a very clear color difference, which can be noted even in the sediments of the fermentations, that results from the decidedly different color of the vegetative strain. It should be noted that blue staining with iodine can be achieved only on the spore walls, and likewise cannot occur in the vegetative strain. Clear differences are noticeable in growth of the colonies on nutrient gelatins . It is also characteristic in the not-inconsiderable acid formation, which is characteristic for all three Schizosaccharomyces species known at the present time. It is higher in the vegetative strains than in the sporogenic.

Also peculiar is that in the cultures of the asporogenic form so many shed cell walls appear, which are absent in the sporogenic strains, whereby they are certainly replaced by the walls of the emptied asci.

The best method to determine the types of sugar suited for the fermentation is to dissolve about 10% of the material to be examined in acidified yeast water (that is, 10-20 g compressed yeast boiled in 100 g water, and filtered clear) and sow the yeast in this liquid in fermentation flasks (footnote 7).

Examined in this manner, I could only confirm my earlier statements, so that I do not need to go into further detail here (footnote 8).

Moreover, in the course of the alcohol fermentation, small differences were unmistakable; the fermentation proceeds most rapidly in the asporogenic form, and the main fermentations, on the other hand, proceed most rapidly in the sporogenic form.

I was not able to resolve to my complete satisfaction the question of whether the origin of the asporogenic strain is based on unknown conditions, and, as can be derived from the experiment, on 'germ variability,' or can be brought about by 'adjustment' to certain living conditions, or artificially in another way; perhaps, I will have an opportunity to return to it later. I would simply like to emphasize here that my octosporus yeast, from a morphological and cultural standpoint, represents a particularly suitable material for the study of such questions in general, and I point this out to those biologists who have taken up the important physiological processes of cell variability.

Before I conclude this paragraph, I would still like to say a word about strain formation in other yeasts.

Once I became familiar with it, I could find, without effort, 'brown' and 'white' colonies in the agar cultures of Schizosaccharomyces asporus, which clearly corresponded to the white and 'mixed' colonies in Sch. octosporus. This was all the more noteworthy, since Sch. asporus does not produce any ascospores. It turns out that the white-cell colonies consist only of thick and short cells, while the brown [colonies] consist of thick and short, as well as long and thin cells. The white cells in the exhausted cultures produce much more markedly and irregularly swollen cells than do the brown, and seem to correspond to the asci-bearing strain in S. octosporus. The strains obtained in this manner have thus far remained constant.

In Sch. pombe I could likewise find 'brown' and 'white' colonies, and, at the same time, achieve a further division into two strains, of which one produces many more spores than the other.

Peculiarly, I found a division into 'brown' and 'white' colonies in the very first wine yeast I examined in this regard; nevertheless, the relationship to ascospore formation could not be so clearly determined here, so that the phenomenon perhaps cannot be attributed to the preceding data; as for a theoretical observation, of which there are plenty available at this time, I still have not made sufficient observations to say anything of real interest and conclusiveness.

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Laboratory of Technical Botany at Delft, July 10, 1897.

Legends to figures in plates VII and VIII

Everything belongs to Schizosaccharomyces octosporus

Fig. 2. The sporogenic strain, matured, with many 'cell conjugates' involved in ascus formation (Return to main text).

Fig. 3. The asporogenic strain under the same conditions as Fig. 2, matured (Return to main text).

Footnotes

(1) If the unfortunate name 'pombe' did not have priority, its replacement with 'tetrasporus' would be very desirable. A good name is not a matter of indifference, especially when, as in this case, we are dealing with an organism of high scientific importance (Return to main text).

(2) Centralbl Bakt Parasitenkd Inf 1894;16:49 (Return to main text).

(3) In accord with my expectations expressed at that time, the octosporus yeast has allegedly been observed by other investigators, such as Herr Bouin in Nancy and Schlionning in Copenhagen (Return to main text).

(4) I will report later how other alcohol yeast behave in this regard (Return to main text).

(5) Turkish currants were particularly soiled and bore much octosporus yeast (Return to main text).

(6) I wish only to note one thing. To substantiate his view that the alcohol fermentation does not necessarily require the presence of living protoplasm, E. Buchner states (Ber Dtsch Chem Ges 1897;30:1110; see also H. Will, Z Brauwesen 1897;20:362) that yeast dried at 100C (under certain circumstances) is still capable of fermenting raw sugar. As my experiments show, I see no proof that the protoplasm of the yeast cell used for the ascospores can also be heated to 100C without dying. But even the fact that a yeast cell can no longer be induced to grow does not simply mean that all its constituents are dead. I would be able to present all sorts of analogies from the plant and animal kingdom that show that certain parts of cells, though no longer capable of growing, nevertheless can still be alive (Return to main text).

(7) My fermentation flasks hold about 25 cm3 in the closed leg and are sealed with ground-in glass caps (Return to main text).

(8) I would like to note here that while the octosporus yeast does not ferment cane sugar, the Schizosaccharomyces asporus found by Eijkman in the arrack fermentations in Java certainly does, and with rather high intensity (Return to main text).

Updated last on July 9, 1997, moved to La Jolla April 2003 and to Los Angeles April 2004