The Influence of Pressure on Fermentation

MEETING HELD ON FRIDAY, 11th FEBRUARY, 1898,
AT THE ROYAL VICTORIA STATION HOTEL, SHEFFIELD.

Mr. Harold Trinder, in the Chair.

The following paper was read and discussed:—

The Influence of Pressure on Fermentation
by R. E. Evans

I have selected for the subject of my paper the influence of pressure on fermentation, partly because some time back I had made some experiments in this direction, but chiefly because I thought that it might, perhaps, be the means of inducing someone having more time than myself to follow up certain lines which I believe my work suggests.

The circumstances which first directed my thoughts to this subject was the estimation of alcohol in some samples of fermented ginger beer. These samples were found to be under very great pressure; the amount of liquid that could be obtained after opening the bottles was found on distillation to contain about 12 per cent, of proof spirit, and it had an original gravity of 1090. As it is to be presumed that this ginger beer was bottled in the usual way, i.e., with about 1 per cent, of proof spirit present, we have an instance of great activity of a ferment under gradual and increasing pressure, in spite of unfavourable conditions—no proper yeast nourishment, and a sugar that required to be inverted before it could be fermented.

Having failed, on account of defective apparatus, to determine the maximum pressure which could be obtained by the fermentation of an ordinary wort, and not having time to repeat the experiment under other conditions, an attempt was made to estimate the pressure in bottled ales, &c. For this purpose several brands of bottled ales were obtained, and kept for some time in a vessel of water at about 45—50° F., so as to counteract the effect produced by any shaking they had received; after which they were connected with the apparatus now to be described. An ordinary cork borer is taken, and two small holes are drilled in the stem, at a distance of about 1¼ inches from the bottom, that is to say at a height from the bottom of the cork borer greater than the length of the cork. The top end is then connected by a piece of pressure tubing to a bottle containing mercury, from which a glass tube about 5 to 5½ feet projects. This tube must be supported in an upright position against a wall or plank, but should not be fixed too rigidly, or it is liable to be broken when connecting the bottle.

The cork borer is greased with a little vaseline and pushed through the cork by holding the borer very firmly (in pliers, if necessary), and slowly and gently revolving the bottle. Immediately the small holes reach the under surface of the cork, the internal pressure within the bottle causes the mercury to rise a certain height, which is measured and the temperature noted, if the water surrounding the bottle is then gradually warmed, the mercury slowly rises, but the temperature must be kept constant for some time before the mercury column becomes again stationary. When this is the case, the temperature may be increased to another point, and kept there till the pressure is stationary, and so on till the mercury gets near to the top of the tube.

The pressure of champagne is given as 3½ to 5 atmospheres, according to quality, i.e., about 52 to 75 lbs. per square inch, and this pressure is attained solely by the action of yeast in an already fermented liquid, which has been fined and further dosed with alcohol, whilst the saccharine priming added is, in nearly all cases, cane-sugar, which the minute residue of yeast must first invert before fermenting.

The fact that an organism of so delicate a structure as yeast can act and continue to act under such pressure is a rather curious phenomenon, and one that has hardly a parallel among more highly organised bodies. The life of plants and animals, even the lowest, is certainly not promoted by an increase in pressure, and is, in most cases, adversely influenced. In fact, the lower organisms show a remarkable resistance to various physical states, except an increase of temperature beyond a certain point. Yeast, for example, will stand an intense degree of cold. As an instance, a portion of pitched wort kept in ice and salt at 0° F. for a week, and when thawed out at the end of that time fermented in a perfectly normal manner, the cells being very strong and healthy, and the total attenuation quite equal to that of the same wort with the same yeast not frozen.

Sometime after the first experiment recorded, a few others were tried, with a view of determining the relative attenuation of a wort under pressure compared to one fermented in the normal way. In the first of these a wort of 1071·29° was pitched with an ordinary brewer’s yeast, and divided into two portions, one being fermented in a vessel so arranged that the exit tube for the carbon dioxide evolved dipped some 15 inches below the surface of mercury in a long tube, which would give a pressure in the vessel of about 7½ lbs. per square inch; the other was fermented in the usual manner.

The first, i.e., with pressure attenuated to 1013·73; the other with no pressure to 1024·12, in 60 hours, giving a gain in the attenuation of 10·39° to that with the pressure.

This experiment was repeated in the same manner with a wort of original gravity 1065, maintained at 73° F. for three and a half days. In this case the wort under pressure went down to 1016·9, the other with no pressure to 1021·4, giving a gain of 4·5° for that fermented under pressure, which was the same as in the former case, i.e., 7½ lbs. per square inch. In neither of these cases were the acidities before or after noted, but it is to be particularly remarked that these experiments were conducted with carefully sterilized worts pitched with a very pure yeast; the difference will be seen later.

A third experiment was made to see if by fermentation under pressure there was any perceptible reduction in the amount of the nitrogenous matters in the resulting beers, or, in other words, if the yeast acted in a normal manner. For this purpose a wort of 1041·5° was taken, pitched, divided into two portions, and allowed to ferment, as in the two former experiments, the total nitrogen in the resulting beers being estimated by Kjeidahl’s method.

The result was that the wort under pressure attenuated to 1012·2, 10 c.c., treated according to Kjeldahl’s method, required 12·4 c.c. N/10 HCl; that under no pressure attenuated to 1015·82, and 10 c.c. of it required 13 c.c. N/10 HCl.

It will thus be seen that there is a gain of attenuation of 3·80° for that fermented under pressure, but the amount of residual nitrogen is practically the same in both cases. From the results of the above experiments, it will he seen that under pressure a slight increase of attenuation, or, in other words, an increased activity of yeast, took place, which is the more remarkable, as it does not at all accord with the generally accepted theory of fermentation. If fermentation takes place inside the yeast cell, the decomposition of the sugar would produce alcohol and carbon dioxide, which must then be regarded as excretory products of the cell, and the confining of these in solution should exercise an injurious effect on the development of the yeast. This, I think, may be taken as true of all organisms, that the products excreted are poisonous or harmful to their life, and their immediate removal is very beneficial. On the other hand, if the fermentation takes place outside the cell, the products cannot be regarded as excretory, and their removal becomes more a matter of indifference, whilst if the actual medium of fermentation he an enzyme secreted by the cell, then pressure should favour its action, as it is well known to do in the case of other enzymes—diastase, invertase, &c.

I should like to draw your attention to some recent experiments by E. Buchner, which, though hardly directly connected to the subject, yet have a very great interest to all those engaged in fermentation industries.

In these experiments a quantity of yeast was taken and ground up with some kieselguhr and quartz sand so as to rupture the yeast cells as far as possible. The mass was then placed under a pressure of 3,000 lbs. per square inch, by which means a certain quantity of fluid was expressed. This was now filtered through a Berkefeld filter to remove any organisms and added to some sterilised wort, with the result that fermentation took place, although at the end the liquid was still sterile, and no cells of any kind could be detected under the microscope.

Now, assuming there was no possibility of any mistake being made, the only conclusion to be drawn is that fermentation is not a direct result of the life of the yeast cell, but of an enzyme it secrets, and which is capable of decomposing certain saccharine matter without the necessity of living organisms being present.

I may say that these results have been denied by M. Stavenhagen, who, however, employed a Chamberland filter for preparing the sterilised extract of the yeast, so that the point cannot be considered settled as yet, it being only necessary to add that it is just possible that a highly complicated body 6uch as the enzyme must be, if it exists, might pass through one porous medium when it would not pass through the other. Buchner has, however, since replied to Stavenhagen’s criticism.

If it should ever be accepted that the actual decomposition of sugar is effected by the action of an enzyme, the total destruction of a part of the sugar and the production of succinic acid and glycerin, which as is well known occurs in normal fermentations, may have a new significance.

It may be that only a portion of the sugar passes through the cell where it is decomposed, the energy derived from the breaking down of the sugar molecule inducing through the medium of the enzyme the decomposition of the main bulk. To draw a rough analogy, the small portion of sugar decomposed may act as a fulminate cap does in inducing the explosion of the bulk of the charge.

Whether the employment of pressure during fermentation on the practical scale would be beneficial is rather doubtful, and such a, system would have to show enormous advantages to compensate for the great cost of the vessels in which it had to be conducted. These would have to be of great strength to resist the pressure and prevent leakage, whilst there is the question of flavour, reproduction of yeast, &c, to be considered, all of which cannot be settled in the laboratory. Certainly in the union system of fermentation the wort is under a little pressure, depending on the length and bore of the swan neck, and beers produced on this system are greatly esteemed for their flavour and stability, but it does not follow that if the pressure were increased any further advantages would be obtained.

There must, however, have been some good reasons for the adoption of such a system of which not only demands so much extra labour, but which also involves a large percentage of waste, and it may have been a special stability and purity of yeast crop justified its adoption.

Even if a greatly increased pressure did not tend to improve the yeast, or did not give the desired flavour, there are some industries, such as distilling, in which these points are not of so much importance, and in these cases it might be beneficial to utilise this property of fermentation under pressure. In a great many industries carbon dioxide is required under more or less pressure, and seeing that it is a waste product, so to speak, of the decomposition of sugar, it might pay to produce it in this manner instead of paying for acid and chalk to prepare it. The alcohol produced at the same time would pay all expenses and leave perhaps a small margin for profit. It would have the advantage of being pure, i.e., free from other gases, some of which are very objectionable under certain conditions, of being easily sterilised by passing through a filter of cotton wool, of being evolved and delivered under certain pressure without the complication of pumps, &c, of being cheap and doing away with the handling of dangerous acids or cylinders of compressed gases.

For example, it would be interesting to employ it for the carbonating of finished beers by connecting the cylinder directly to a vat in which wort was being fermented under a fair pressure. The gas would certainly be accompanied by the vapours of alcohol, but this would not be any disadvantage. In many industries the CO₂ required might be produced in like manner, the resulting liquid being in most cases run through a still to recover the alcohol, for which a ready sale could always be found.

Of course in a country like this where every obstacle is put in the way of the production or use of alcohol, the thing is impossible at present; but some day when it is forced on the powers that be that the technical application of organic chemistry, the dyes, the preparation of alkaloids and pharmaceutical products, &c., has been driven from this country by the restrictions on the use of alcohol, a considerable change of opinion may occur.

The amount of carbon dioxide produced by the brewing industry alone in a single year is enormous, nearly all of which goes to waste. It is the most important by-product of the brewer, who is in this respect more fortunate than most manufacturers, and being a compound so largely found in nature, it is perhaps doubtful if it would pay to collect.

Taking an average of 36 million barrels brewed every year of an original gravity of 1055 and attenuated to normal extent, this would give about 19 lbs. of carbon dioxide per barrel, or a total of 35,357 tons, all of which is let loose into the atmosphere to be again absorbed by various plants. This amount of gas is capable of giving out a certain amount of energy if liberated under pressure. One gram of carbon dioxide, liberated at a pressure of 75 lbs, per square inch and allowed to expand to atmospheric pressure, will give 56·404 foot pounds of energy, and will then occupy a space of (at 20° C.) 544·4 c.c. Now, as 19 lbs. equal 8664 grams, a barrel of fermenting wort will give energy amounting to 488,649·6 foot pounds, or, expressed in another way, sufficient force to lift 218·1 tons one foot.

Multiplying this by the total number of barrels brewed we obtain a potential energy capable of lifting 78,516,000 tons to the height of 100 feet, which would raise 12 pyramids the size of the great Pyramid of Cheops, which weighs over 6,000,000 tons, to the height of 100 feet. This amount of power directly and actually represents part (and only a small part) of the energy derived from the heat of the sun shining down on the green leaves of the barley, &c., in various parts of the world, but vast as this seems, it becomes small compared with the enormous quantity stored up by the sun in ages past in the form of coal, for the whole of the above amount of energy -would be only equivalent to the consumption of 16,350 tons of coal, reckoning 4 lbs. of coal per indicated horse power hour.

Finally, I have to record a few experiments made to see if any improvement could be effected in an impure yeast by a series of cultivations under pressure. In other words, to see which was most affected by pressure, the yeast proper, or the wild yeasts and bacteria liable to accompany it. A quantity of wort was prepared and put up in flasks, plugged with cotton wool and sterilised so that the same wort could be used for ouch series of experiments. A portion of this wort was now pitched with a very impure yeast, being in fact partly derived from the deposit of a very bad beer and partly from some ordinary yeast that had been kept a long time. This was done so as to make the conditions as unfavourable as possible to accentuate the differences, for if this be not done, these experiments on so small a scale do not give any very definite results. The wort now, after well shaking, was quickly divided into three portions. The first was fermented under a pressure of 15 inches mercury, the second in a closed vessel with the tube just dipping one-eighth of an inch below the surface, and the third in a vessel merely plugged with cotton wool; these three constituted the first experiment of the series.

In this case the one under pressure seemed to be done first, and most of the yeast came to the top, in the second most of the yeast went to the bottom with a little on the top, while the third, with no pressure, was most unsatisfactory, with a greasy film on top, and remained quite thick.

It will be seen that the difference in pressure has not made a corresponding difference in attenuation, which is contrary to the former experiments, but there was, of course, a vast difference in the yeasts, which might account for it. The deposits from each were now examined under the microscope.

No. 1. Cells round and regular, with few wild yeasts and very few bacteria.
No. 2. Not so good, cells more irregular; about the same wild yeasts, but more bacteria.
No. 3. Bad, cells irregular; more wild yeasts and much more bacteria.

To the remainder of the yeast left in the vessels more sterilized wort was added, and they were put back to ferment under the same conditions as before. The general appearance was the same, and after three days they were taken off and examined.

The yeast was examined as before, and it was noticed that while No. 1 showed an improvement, No. 3 was very much worse. The quality of No. 2, i.e., that under very light pressure, was much nearer No. 1 than No. 3.

The remaining yeast was again employed to ferment a fresh lot of wort under the same conditions as before, the results being—

Unfortunately the acidity of this series was not done, so that the comparison cannot be made as before, hut it may be noted that while No. 1 and No. 2 were palatable, No. 3 was most strongly acid. The condition of the yeast was very good in No. 1, fair in No. 2, and very bad in No. 3. Also, it will be seen that though the same wort (kept sterilised) was used for each series of experiment, the amount of attenuation of those under pressure gradually increased, showing that the yeast was becoming more and more active.

The result of these experiments shows most clearly the influence of air in promoting acidity and the growth of harmful organisms, but the difference of those under pressure is not so marked, there being little to choose between them either in attenuation or acidity. The greatest difference shown is in the general appearance of the yeast under the microscope, which is impossible to express in figures, and hard to do in writing. If it had been possible, I should have liked to have laid before you photographs representing each stage of the series, but the weather has been unfavourable, as I have to rely on the sun as a source of illumination. The general appearance all through was in favour of that from the wort under 15 inch pressure, both for regular size of cells and the gradually diminishing quantity of wild yeasts and bacteria.

Discussion
Mr. Evans, in reply to certain questions by Messrs. J. B. Moody and George Pearson, said that in the experiments he had carried out the acidity of the fermented wort gradually diminished as the pressure increased. He was not aware that fermentation under pressure was carried out in practice to any greater extent than in the Burton Union system. If the latter, as he had mentioned in the paper, the wort was under somewhat increased pressure during the greater part of the fermentation. The actual pressure had not been estimated, but, judging from the “rush” of yeast from the swan-necks, it must be considerable. To obtain pressure in a fermentation vat there

must be something more than an ordinary cover; the vessel must be closed air-tight, and there must be only one opening for the gas to pass off, the pressure being regulated by a valve.

A vote of thanks was then passed, to which Mr. Evans suitably replied.