a portion of our demonstrated knowledge, and ether waves from a few feet to thousands of miles in length are believed to have been recognised.

All this may well fill us with the belief, not only that Nature's fertility of resource is inexhaustible, but that Nature scarcely places limits to the possibilities of scientific discovery, or to the width and depth of human knowledge.

SUB-SECTION B.

BACTERIOLOGY AND FERMENTATION.

THE

MICRO-ORGANISMS AND THEIR
APPLICATIONS IN THE INDUSTRIES.

By R. GREIG SMITH, M.Sc., Macleay Bacteriologist to the
Linnean Society of New South Wales.

INDUSTRIAL bacteriology or mycology treats chiefly of. the changes that are effected in such carbohydrates as starch and sugar by various yeasts, moulds, and bacteria. In less degree, it deals with the fermentation of albuminoids, the growth of yeasts, moulds, and bacteria for the production of trade cultures, the preparation of toxines, and the manufacture of antitoxines and protective sera.

Ferments and Fermentation.

Before proceeding to consider in detail the various applications of bacteriology, it will be well to understand the meaning of fermentation. The old idea of formed and formless ferments has had its day, and the discovery of zymase, the alcoholic ferment, showed the folly of attributing fermentative action to protoplasm simply because we did not know how to separate its active products, the enzymes. Protoplasm produces the enzymes which do the work. As a rule, the fermentation of any simple or complex organic chemical substance is referred to the microorganisms themselves, instead of to the enzymes which they secrete. This is done only for the sake of simplicity; and if we should so refer to the yeasts or bacteria, let us not forget that fermentation is a change brought about by the action of enzymes, and that the term "ferment" now applies to the enzyme, and not to the micro-organism. The organic

ferments or enzymes are bodies of unknown more or less complicated constitution. Zymase, the alcoholic ferment, approaches nearer to protoplasm in its reactions than the

older known enzymes, such as rennet, diastase, and pepsin. Indeed, at one time, after the discovery of zymase, there was much discussion as to whether alcoholic fermentation was the work of an enzyme or of diffused protoplasm; it is now accepted that it is really done by the former. The microorganisms may secrete many ferments; for example, yeast has been shown to produce at least five. It can convert starch to maltose by means of a diastase; it can invert canesugar by its invertase; maltose is changed to glucose by glucase; glucose is fermented to alcohol by zymase; and, finally, albuminoids are digested by a proteolase.

Moulds in the Alcoholic Fermentation.

Undoubtedly the most important fermentation from an economic point of view is the alcoholic; and perhaps the oldest fermentation of all, the lactic, comes next. In the Western world, the yeast-plant as a producer of alcohol holds sway; but within the last few years moulds from the East have been gradually introduced, in some cases to aid the yeast, in others to displace it; and, in all cases, chiefly to dispense with the expensive malt.

It is a matter of common knowledge that the steps in the process of making alcohol are starch, maltose, glucose, alcohol, and sometimes saccharose, glucose, alcohol. The active "-ases in effecting these changes are diastase, glucase, zymase, and invertase, pymase. Instead of using malt or a mixture of malt and grains, such as rye, for the production of fermentable sugar, a cheaper cereal, rice, is sometimes infected with the mycelia of Aspergillus oryzae, a mould that occurs along with a yeast in Japanese koji, the grains of which are used by the Japanese in making a strong ricewine. Aspergillus oryzae has a strong diastatic action, and furnishes taka diastase, a trade product.

But another mould or group of moulds, the Mucors, have found more favour with European distillers. The most important species is Mucor (or Amylomyces) Rouci, which produces practically a straight fermentation from starch to alcohol, the saccharification and fermentation proceeding simultaneously. The process was first used in Europe at Lille, in France, and the method consisted in sterilising a mash of brewer's settlings by heating under a pressure of two atmospheres. This was followed by cooling and infecting with the Mucor. After 20 hours. when the mycelia had penetrated uniformly throughout the mass, a few cubic centimetres of yeast were added, and the fermentation allowed to proceed. When the process was finished, 97 5

per cent. of the theoretical yield of alcohol was obtained. The method is now used in the Belgian distilleries; but, instead of Rouxia, a more energetic variety-Mucor B, or Belgian koji is employed. One gramme (15 grains) of

Mucor suffices for 25 tons of grain, thus replacing three tons of malt, and saving a loss of six cwts. of starch, which is equivalent to 44 gallons of pure alcohol.

The yeasts and moulds, however, are not the only microorganisms that can saccharify and ferment. Some bacteria ferment starch to alcohol, and others ferment sugar. An example of the latter is Bact. coli commune, a common intestinal bacterium, which ferments one-sixth of the glucose in the medium to common alcohol.

Yeasts and Yeasts.

In the wine industry, moulds are also used, but this is done unwittingly, and much bad wine could be traced to their agency. Those fermented beverages which contain the original juice of the fruit, or extract of the grain, and in the fermentation of which it has been the custom to employ. yeast, cannot have the yeast replaced either partly or entirely by moulds, because these produce substances which are disagreeable to the palate. I use the word yeast with all due caution, because there are yeasts and yeasts, some of which are as objectionable as moulds. It is a common idea that

all yeasts ferment, and therefore any yeast is good enough to produce alcohol. That is quite right, and also quite wrong. There are species of yeasts and of these species there are varieties, and of these there are races-and there is as much difference between the races of yeasts as between the races of mankind. If we take, for example, the culti vated yeast of the brewer (Saccharomyces cerevisiae), we find that it can be divided into top and bottom varieties, the former rising to the top of the wort during fermentation, and the latter remaining at the bottom of the fermenting vat. These, again, can be subdivided into races, each of which will have a different effect upon the finished beer. There are possibly as many races of Sacch. cerevisiae as there are breweries..

The Routine of Brewing.

The brewer is a happy mortal. He kills the bacteria in his mash by boiling, and adds hops. the essential oil and extractive matters of which hinder the subsequent growth of bacteria. He can, if he desire, add to his wort a pure culture of yeast, so that he has everything under control. He knows the composition of his wort, and can attemperate

it to any degree. He is scientific in his methods, and can repeat the process day after day with the utmost regularity, and with the sense of security engendered by the knowledge that his beer a year hence will be the same as it is today.

Yeast and Wine.

The vigneron is by no means so happily placed. He does not crush his grapes every day, and he cannot boil his must. Many a vigneron does not know the benefit that can be derived from using pure cultures of yeast. A spontaneous fermentation is allowed to set in, with the result that yeasts and moulds of every creed and denomination strive for a mastery; so that a wine is produced whose future career is wrapped in doubt. There is only one partial safeguard the vigneron has, and that is the acidity of the must, which favours the wine-yeasts, and checks other yeasts and bacteria. A check, however, is not a prevention, and some Australian wines are prone to develop turbidity, and to undergo the mannitic fermentation, both of which diseases are caused by bacteria. But the acidity and the sugar are varying quantities, and it is easy to believe that the winemaker must be an expert, since he has to contend with so many difficulties. These may be to some extent overcome by the use of pure cultures of selected yeasts. In the selection of a yeast, one microscopic cell is taken, and cultivated, and the action of the culture upon must has to be tested chemically, and with the trained palate. The temperature and the acidity best suited to its growth must also be ascertained. Perhaps the temperature is the most difficult condition the vigneron has to regulate; but it is quite possible that a native race of the wine-yeast, Saccharomyces ellipsoideus, might be found that would ferment out at an Australian summer temperature the sweet Australian musts.

Although a previous pasteurisation of the must is theoretically advantageous prior to the addition of a pure culture, yet, by using a large quantity of starter-yeast, the action of the natural yeasts and moulds can be kept under. This is the principle of mass effect when 25 to 100 yeast-cells are added for every yeast or mould that may be on the surface of the grapes. The added yeasts of the starter are more numerous and more vigorous, and, generally speaking, finish the fermentation before the others have had time to begin. The advantages of using pure cultures in the wine-fermentation may be summarised thus:-A quicker fermentation, a better clearing, a cleaner taste, and an improvement in flavour and bouquet. At present European yeasts are

used in a few isolated cases, where the vigneron has realised the importance of leaving nothing to chance in this world of competition.

Distillery Yeasts.

I

Vignerons, however, are not the only men who are ignorant or apathetic regarding the properties of yeasts. have heard of a distillery where they use a brewery yeast for pitching their wort. It is possible that this may be correct, but it is more than probable that it is not. Distillery yeasts and brewery yeasts are two different things as different from one another as Gorgonzola is from Cheddar cheese. To the distiller, an incomplete fermentation is a loss, but to the brewer it may not be. The distiller requires a straight fermentation, while the top-fermentation brewer relies upon an after-fermentation in the barrels.

Malt Wine.

Wines are presumably derived from the grape; but within the last few years, wines have been made from malt. The sweet wort is infected with lactic bacteria, and after the requisite acidity has developed various wine-yeasts are added, according to the kind of malt-wine desired. The fermentation is accelerated by the addition of raw sugar. After the fermentation, the only thing that remains to be done, and that the most important, is to eliminate the malt flavour. This is accomplished by keeping the wine at a temperature of 50° C., and continually renewing the air in contact with the surface for some weeks. The malt odour entirely disappears, and a pleasant aroma takes its place. By using different yeasts, sherry, Madeira, and other malt wines can be made, which compare favourably with the average quality of wine usually sold.

The production of different malt wines from the same wort by specific yeasts infers that the yeast is entirely responsible for the flavour and bouquet. This is a matter that is open to doubt, and, although the different yeaste have some influence in producing distinctly flavouring substances, yet it can scarcely be denied that the grape is of the most importance. It is interesting to note in this relation that the sixth Austrian Wine Congress concluded that pure yeast must be carefully chosen; that the distinction caused by its use is most marked in young wines, and less in mature wine; and that the effect of the yeast on the bouquets of well-made wines is hardly noticeable.