458 



NA TURE 



[September 9, 1897 



In 1883, Hansen, of Copenhagen, taking advantage of the 

 strict methods of culture introduced and improved by De Bary, 

 Brefeld, Klebs, and other botanists, had shown that by cultivat- 

 ing yeast on solid media from a single spore it was possible to 

 obtain constant types of pure yeasts, each with its own peculiar 

 properties. 



One consequence of Hansen's labours was that it now became 

 possible for every brewer to work with a yeast of uniform type 

 instead of with haphazard mixtures, in which serious disease 

 forms might predominate and injure the beer. Another con- 

 sequence soon appeared in Hansen's accurate diagnosis of the 

 specific or varietal characters of each form of yeast, and among 

 other things he showed that a true yeast may have a mycelial 

 stage of development. The question of the nucleus of the yeast- 

 cell, on which Mr. Wager will enlighten us, has also occupied 

 much attention, as have also the details of spore formation. 



Meanwhile, a question of very general theoretical interest had 

 arisen. 



Reess, Zopf, and Brefeld had shown that many higher fungi 

 can assume a yeast-like stage of development if submerged in 

 fluids. Various species of Mucor, Ustilago, Exoascus, and as 

 we now know, numerous Ascomycetes and Basidiomycetes as 

 well, can form budding cells, and it was natural to conclude 

 that probably the yeasts of alcoholic fermentation are merely 

 reduced forms of these higher fungi, which have become 

 habituated to the budding condition — a conclusion apparently 

 supported by Hansen's own discovery that a true Saccharomyces 

 can develop a feeble but unmistakable mycelium. 



With many ups and downs this question has been debated, 

 but as yet we do not know that the yeasts of alcoholic ferment- 

 ations can be developed from higher fungi. 



During the last two years it appeared as if the question would 

 be settled. Takamine stated that the Aspergillus used by the 

 Japanese in brewing sake from rice develops yeast-like cells 

 which ferment the sugar derived from the rice. JUhler and 

 Jorgensen then extended these researches and claimed to have 

 found yeast-cells on other forms of fungi on the surface of fruits, 

 and to have established that they develop endogenous spores — 

 an indispensable character in the modern definition of the genus 

 Saccharomyces — and cause alcoholic fermentation. 



Klocker and Schionning have this last year published the 

 results of their very ingenious and thorough experimental 

 inquiry into this question, and find, partly by pure cultures of 

 the separate forms, and partly by means of excellently devised 

 cultures on ripening fruits still attached to the plant, but im- 

 prisoned in sterilised glass vessels, that the yeasts and the moulds 

 are separate forms, not genetically connected, but merely as- 

 sociated in nature, as are so many other forms of yeasts, bacteria 

 and moulds. 



It is interesting to notice how here, as elsewhere, the lessons 

 taught by pure cultures are found to bear fruit, and how Hansen's 

 work justifies the specialist's laboratory. 



Among the most astonishing results that have come to us 

 from such researches are Hansen's discoveries that several of 

 the yeasts furnish quite distinct races or varieties in different 

 breweries in various parts of the world, and it seems impossible 

 to avoid the conclusion that their race characteristics have been 

 impressed on the cells by the continued action of the conditions 

 of culture to which they have so long been exposed — they are, 

 in fact, domestic races. 



Much work is now being done on the action of the environ- 

 ment on yeasts, and several interesting results have been ob- 

 tained. One of the most striking examples is the fact observed 

 by Sauer, who found that a given variety of yeast, whose ac- 

 tivity is normally inhibited when the alcohol attains a certain 

 degree of concentration in the liquid, can be induced to go on 

 fermenting until a considerably higher proportion of alcohol is 

 formed if a certain lactic-acid bacterium is added to the fer- 

 menting liquor. The bacterium, in fact, prepares the way for 

 the yeast. Experiments have shown that much damage may 

 be done to beers and wines by foreign or weed germs gaining 

 access with the yeasts, and Hansen has proved that several 

 yeasts are inimical to the action of the required fermentation. 

 But not all pure fermentations give the desired results : partly 

 because the race-varieties of even the approved yeasts differ in 

 their action, and partly, as it appears, on account of causes as 

 yet unknown. 



There are facts which lead to the suspicion that the search 

 for the best possible variety of yeast may not yield the desired 

 results, if this particular form is used as a pure culture. The 



NO. 1454, VOL. 56] 



researches of Hansen, Rothenbach, Delbriick, Van Laer, and 

 others, suggest that associated yeasts may ferment better than 

 any single yeast cultivated pure, and cases are cited where such 

 a symbiotic union of two yeasts of high fermenting power has 

 given better results than either alone. 



If these statements are confirmed, they enhance the theoretical 

 importance of some investigations I had made several years 

 previously. English ginger-beer contains a curious symbiotic 

 association of two organisms — a true yeast and a true bacterium 

 — so closely united that the yeast-cells imprisoned in the 

 gelatinous meshes of the bacterium remind one of the gonidia 

 of a lichen entangled in the hyphoe of the fungus, except that 

 there is no chlorophyll. Now it is a singular fact that this 

 symbiotic union of yeast and bacterium ferments the saccharine 

 liquid far more energetically than does either yeast or bacterium 

 alone, and results in a different product, large quantities of 

 lactic and carbonic acids being formed, and little or no alcohol. 



In the kephir used in Europe for fermenting milk, we find 

 another symbiotic association of a yeast and a bacterium ; in- 

 deed, Freudenreich declares that four distinct organisms arc 

 here symbiotically active and necessary, a result not confirmed 

 by my as yet incomplete investigation. I know of at least one 

 other case which may turn out to be different from either of the 

 above. Moreover, examples of these symbiotic fermentations 

 are increasing in other directions. 



Kosai, Yabe, and others have lately shown that in the fer- 

 mentations of rice to produce sake, the rice is first acted on 

 by an Aspergillus, which converts the starch into sugars, and 

 an associated yeast — hitherto regarded as a yeast-form of the 

 Aspergillus, but, as already said, now shown to be a distinct 

 fungus symbiotically associated with it— then ferments the sugar, 

 and other similar cases are on record. 



Starting from the demonstrated fact that the constitution of 

 the medium profoundly affects the physiological action of the 

 fungus, there can be nothing surprising in the discovery that 

 the fungus is more active in a medium which has been favour- 

 ably altered by an associated organism, whether the latter aids 

 the fungus by directly altering the medium, or by ridding it of 

 products of excretion, or by adding some gas or other body. 

 This granted, it is not difficult to see that natural selection will 

 aid in the perpetuation of the symbiosis, and in cases like that 

 of the ginger-beer plant it is extremely difficult to get the two 

 organisms apart, reminding us of the similar difficulty in the 

 case of the soredia of Lichens. Moreover, experiments show 

 that the question of relative abundance of each constituent 

 affects the matter. 



I must now return for a moment to Buchner's discovery that 

 by means of extremely great pressures a something can be ex- 

 pressed from yeast which at once decomposes sugar into alcohol 

 and carbon-dioxide, and concerning which Dr. Green will in- 

 form us more fully. This something is regarded by Buchner 

 as a sort of incomplete protoplasm — a body composed of pro- 

 teid, and in a structural condition somewhere between that 

 of true soluble enzymes like invertin and complete living 

 protoplasm. 



If this is true, and Buchner's zymase turns out to be a really 

 soluble enzyme, the present theory of alcoholic fermentation 

 will have to be modified, and a reversion made towards Traube's 

 views of 1858, a reversion for which we are in a measure pre- 

 pared by Miquel's proof m 1890 that Urase, a similar body 

 extracted from the urea-bacteria, is the agent in the ferment- 

 ation of urea. At present, however, we are not sufficiently 

 assured that the body extracted by Buchner is really soluble, 

 and I am told that very serious difficulties still face us as to 

 what solution is. The enormous pressures required, and the 

 fact that the "solution" coagulates as a whole, might suggest 

 that he was dealing with expressed protoplasm, still alive, but 

 devoid of its cell-wall ; against this, however, must be urged 

 the facts that the " solution " can be forced through porcelain 

 and still act, and this even in the presence of chloroform. 



We may fairly expect that the further investigation of 

 Buchner's " zymase," Miquel's " urase," and the similar body 

 obtained by E. Fischer and Lindner from Monilia Candida will 

 help in deciding the question as to the emulsion theory of 

 protoplasm itself. 



In any case, soluble or not, these enzymes are probably to 

 be regarded as bits off the protoplasm, as it were, and so the 

 essentials of the theory of fermentation remain, the immediate 

 machinery' being not that of protoplasm itself, but of something 

 made by or broken off from it. Enzymes, or similar bodies, 



