AUTOFERMENTATIOX AND LIQUEFACTION OF PRESSED YEAST. 1227 CXXX-Auto fermeiztation and Liyue faction of Pressed Yeast. By ARTHUE HARDEN acd SYDNEY ROWLAND. IF a sample of washed and pressed yeast be kept in an open vessel, it will, in course of time, be observed to become darker in colour, and to change its dry, powdery condition, until, passing through all stages of pastiness, it finally becomes a thick liquid. In the course of a series of experiments on expressed yeust juice, it was found advisable to study this phenomenon, especially with regard to the influence of temperature upon it and upon the accompanying phenomena of evolution of carbon dioxide and absorption of oxygen, points which have not hitherto received attention from the authors1228 HARDEN AND ROWLAND : AUTOFERMENTATION AND who have described the liquefaction of yeast, or have studied the absorption of oxygen and evolution of carbon dioxide by yeast suspended in water, PFeparatiorz of Yeast.-The yeast employed in the following experi- ments was top yeast obtained from an English brewery.It was freed from wort by means of a small filter press, and washed with water until the washings were quite colourless. The pasty mass was then removed from the filter press and submitted to pressure in a hydraulic press. I n this WAY, the yeast was obtained as a friable, white mass, containing about 30 per cent. of solid matter at looo, and capable of being crumbled between the fingers. In$zcence of Temperature on Rate of Lipuefccction (Autoplasmolysis). -The rapidity with which liquefaction is accomplished varies greatly with the temperature, and also depends on the condition of the yeast, the length of time for which it has been kept, and the temperature to which it has been exposed.The following numbers apply to a sample of yeast which was skimmed on February 15th, washed and pressed on February 19th, and then a t once employed for the experiment. I n this case, liquefaction a t the ordinary te-mperature was delayed for more than two weeks, whilst a t 50' it occurred within so short a time as 1-14 hours, intermediate times being required at 26" and 39O. Temperature. Time of liquefaction. 14O 16 days. 26 53 hours. 60 1.25 hours, 39 5 9 ) These experiments were carried out in an atmosphere of carbon dioxide, so as to avoid any complication due to absorption of oxygen and consequent rise of temperature (see p.1231). Evolution of Carbon Dioxide (Autofermentation of Glycogen).-When yeast is preserved, the glycogen which is present in the cells gradually undergoes fermentation with production of carbon dioxide and alcohol. This phenomenon occurs both in the presence and in the absence of air, but in the former case it is accompanied by an oxidation process in which oxygen is absorbed. In order to ascertain the inffuence of temperature on the evolution of carbon dioxide alone, and to measure the extent of this evolution, the yeast was examined in an atmosphere of carbon dioxide or nitrogen, the nature of the indifferent gas being found to be immaterial. For this purpose, the washed and pressed yeast was rubbed into a fine powder and placed in a flask closed by an indiarubber stopper carrying two tubee, through which a current of carbon dioxide was passed to displace the air.The flask was then placed in an incubator a t the desired temperature and connected withLIQUEFACTION OF PRESSED YEAST. 1229 20 minutes .................. 4 0 , , .................. 1 hour ........................ 1 ,, 20 minutes ......... 2 hours ..................... 3 ,, ..................... an apparatus for collecting and measuring the evolved gases. This consisted simply of a bottle containing brine covered with a layer of oil, and connected by a syphon tube wibh a second graduated bottle, in which the brine displaced by the gas was collected and measured a t regular intervals of time.The following table and the curves on p. 1230 show the characteristic results thus obtained a t four different temperatures (50°, 39*, 2 6 O , and 14') with equal portions of a sample of yeast which contained 31.45 per cent, of solid matter a t 100'. One hundred grams of yeast :were taken in each case, and the gas was measured at atmospheric temperature and pressure over brine. 190 450 790 950 950 960 Table showing the rate of evolution of cadon dioxide by yeast exposed The time to dzferent temperatures in an indzyevmt atmosphere. of Eipwfaction is shown by the lcwger type. -____ 39". 110 210 330 475 905 1880 228 0 2400 2590 2590 26". 90 165 225 290 400 530 680 1505 2500 2695 2720 2730 14". 80 115 140 173 210 250 275 390 530 610 775 880 1320 1840 2128 2128 ~ ~~ I C.C.of carbon dioxide evolved at Time. I ____-- 1 50". ..................... .................... .................... ..................... ..................... ..................... ..................... ..................... ..................... ...................... These numbers and curves show, in the first place, that a t 50' the evolution of carbon dioxide commences with great rapidity, but is sud- denly interrupted by the liquefaction of the yeast, although micro- scopical examination reveals the presence of abundance of glycogen in the cells. I n the three other cases, however, the total volume of gas evolved does not differ very greatly, although there is a great difference between the rates a t which it is evolved. The maximum volume is produced at 2 6 O , and amounts to 2730 c.c., which corresponds to about 5.3 per cent.of the weight of the pressed yeast, or 16.8 per cent'. of the dry yeast present. It is to be noted that the evolution of gas ceases some time before liquefaction occurs. A t 3 9 O , the rate of1230 HARDEN AND ROWLAND : AUTOFERMENTATION AND evolution is considerably greater, but the total volume is slightly less, the shape of the curve suggesting that in this case, as a t 50°, the auto- fermentation is interrupted by the liquefaction of the mass. At 14", the rate of evolution is extremely slow, and the total volume only about 75 per cent. of that evolved at 2 6 O . This may possibly be due to the gradual exhaustion of the fermentative power of the cells. In every case, the yeast after liquefaction was capable OF growth in wort, although the samples which had been exposed to the higher temperatures commenced to develop only after a considerable time.3200 2800 2400 2000 %, 2 % 1600 1200 800 400 0 1 2 3 4 5 7 8 9 Time in days. The arrowhead shows time of liqzctfaction. It will be noted that the processes of liquefaction and evolution of carbon dioxide are interdependent in so far as evolution of gas never proceeds after liquefaction has occurred, although, on the other hand, the cessation of gas evolution does not necessarily imply immediate liquefaction. Relation of Alcoho2 to Carbon Dioxide produced by Autofemmntation of Glycogen.-The production of carbon dioxide by yeast kept in an atmosphere free from oxygen appears to be due t o a true alcoholic fermentation, inasmuch as alcohol is -produced in the characteristicLIQUEFACTION OF PRESSED YEAST. 1231 proportion to the carbon dioxide.Thus 100 grams of yeast containing 31.7 per cent. of solid matter gave, a t 37', 2160 C.C. of carbon dioxide a t 18' and 766.5 mm. pressure, or 4.01 grams, together with 3.61 grams of alcohol, allowance being made for the amount of alcohol originally present in the yeast. Hence the ratio of alcohol to carbon dioxide is 1 : 1.1, which agrees closely with the ratio usually found in the ordinary alcoholic fermentation, namely, 1 : 0.96. The slight deficit of alcohol is probably due to the over-estimation of the original alcohol of the yeast, which would include the amount formed during the heat- ing up of the water with which it was distilled.Absoiptivn of Oxygen.-The fact that yeast when exposed to the air absorbs oxygen is a well-established one, and the relation of this phenomenon t o the production of carbon dioxide by autofermentation has been studied by Schutzenberger (" Les Fermentations "), and by Grbhant and Quinquaud (Art%. sc. nat. Botccnique, 1889, p. 269, quoted by Duclaux, Trucit5 de Microbiologie, 3, 231), who examined the yeast suspended in pure water. The general result of these researches was that the amount of oxygen absorbed increases with the tempera- ture, the maximum obtained being 9.6 C.C. per gram of dry yeast per hour at 50°. The ratio of carbon dioxide to oxygen (respiratory quotient) was also found by Grbhant and Quinquaud to increase from 1.06 at 1 4 O to 4.5 at 46'.As these experiments had all been carried out with yeast suspended in water, it was thought advisable to re-examine the phenomenon as i t occurs in pressed yeast, since in this condition each cell is entirely dependent on the material contained within its walls, and complica- tions due to alimentary functions are eliminated. For this purpose, a current of air or oxygen was passed over the yeast at a known uniform rate, and the resulting mixture of gases was collected at equal intervals of time, measured, and then passed through potash and again measured. A record was thus obtained for successive equal intervals of (1) the volume of oxygen supplied, (2) the carbon dioxide evolved, and (3) the oxygen absorbed. I n order to effect this, oxygen was maintained at a constant pressure by means of a hydro- statically balanced valve in a gas-holder containing dilute caustic soda solution.The gas was then pumped through the yeast, contained in a glass cylinder, by means of two oscillating glass cylinders working in mercury, from which the gas was alternately discharged to the yeast and admitted from the reservoir, constancy of direction in the stream of gas being attained by a three-way tap changed over a t the end of stroke by a gravity trip gear. The varying pressure of the brine column contained in the collecting vessel was counterbalanced by means of a mercury valve arranged in such a manner that the pres- sure on the gas in the delivery tube of the pump remained constant,1232 HARDEN AND ROWLAND : AUTOFERMENTATION AND so that each stroke of the pump delivered exactly the same volume of gas.Under these circumstances, the phenomena observed were of the same general character as those described by previous observers. It was found almost impossible t o keep the temperature of the yeast constant during the course of such an experiment, owing to the large amount of heat evolved by the oxidation process, and in most cases, therefore, no attempt was made to do this, but the rise of temperature was observed by a thermometer inserted in the mass of yeast. I n a current of nitrogen at the temperature of the air, on the other hand, no rise of temperature was observable, the autofermentation of the glycogen being very slow at this temperature and there being no oxidation, to which the rise appears to be mainly due.CURVE 11. 6 12 18 94 30 36 42 48 54 Equal times ; pedods of Jive minutes. Nom.--In the m e of the curve showing the temperature efect, the ordinates represent degrees of temperature. The typical course of such an experiment, carried out with freshly pressed yeast in a current of oxygen, is shown by the accompanying curve (Curve II), which applies to 69-1 grams of yeast containing 27 per cent. of solid matter. The temperature gradually rose from 16.5' to 41*35O, and then slowly fell t o 22-45', at which point the experiment was discontinued, 4.6 hours after its commencement. During this time, 1103 C.C. of oxygen mere absorbed and 2148 C.C. of carbon--dioxide evolved, this being an average of 3.47 C.C.of oxygen and 6.76 C.C. of carbon dioxide per gram of fresh yeast per hour, or 12.85 C.C. of oxygen and 25.03 C.C. of carbon dioxide per gram of dry yeast per hour. This amount of oxygen, which represents the average of the whole experiment, is con-LIQUEFACTION OF PRESSED YEAST. 1233 siderably greater than the maximum amount observed by Schutzen- berger, which amounted to 9.6 C.C. and mas observed a t 50'. The respiratory quotient was 2-25 a t the highest temperature observed, and gradually fell to 1, the average being 1.94. The great ease with which gases penetrate the cell walls of yeast in this condition and come into equilibrium with the cell contents is illustrated by a phenomenon which was observed during these experi- ments. When yeast is allowed to stand for a short time, the cell contents become saturated with carbon dioxide.If now a measured volume of some indifferent gas be passed in, the partial pressure of the carbon dioxide being thus lowered, a large amount of the dissolved gas is at once liberated, so that there appears to be a sudden evolution of gas. Thus on passing 51 C.C. of nitrogen into 87 grams of pressed yeast contained in a cylinder connected to two gas burettes, no less than 90 C.C. of gas were at once obtained. When the yeast has been freed from carbon dioxide by a current of air or nitrogen, and carbon dioxide is then passed in, a very rapid absorption occurs, and in two or three minutes the yeast is again saturated. As the autofermentation of the glycogen in the absence of air had been found to correspond with normal alcoholic fermentation, a com- parison was instituted between the amount of carbon dioxide evolved in the presence of oxygen and in its absence, in order to obtain, if possible, some information as to the chemical action OF the oxygen, (1) One hundred grams of fresh yeast at 39' in absence of oxygen gave 1730 C.C.of carbon dioxide. One hundred grams of the same sample in a current of oxygen at 39' gave 2090 C.C. of carbon dioxide, 844 C.C. of oxygen being absorbed. Ratio of excess of carbon dioxide evolved in presence of oxygen to the oxygen absorbed = 2090 - 1730 = o,43. 844 (2) One hundred grams of fresh yeast at 39' in absence of oxygen gave 2160 C.C. of carbon dioxide, and in a current of oxygen gave 2370 C.C. of carbon dioxide, 635 C.C.of oxygen being absorbed. Ratio of excess of carbon dioxide to oxygen absorbed = 2370 - 2160 = o,33. 635 I f the oxygen oxidised the glycogen completely to carbon dioxide and water according to the equation C,H,,O, + 60, = 600, + 5H20, the amount of carbon dioxide evolved by the oxidation process would be three times that evolved by the normal fermentation process from the same weight of glycogen according to the equation C,H,,O, + H20 = 2C0, + 2U2H60, and hence the difference between the volumes of carbon dioxide1234 AUTOFERMENTATION AND LIQUEFACTION OF PRESSED YEAST. evolved in presence, and in absence, of oxygen should be two-thirds of the volume of the oxygen absorbed. As, however, the ratio of excess of carbon dioxide to oxygen absorbed was invariably found t o be considerably less than two-thirds, it appears probable that carbon dioxide and water are not the sole products of the oxidation.In view of the large amount of carbon dioxide evolved by yeast when it is exposed to a current of oxygen, an experiment was made to ascertain the limit of this evolution. Twenty-five grams of yeast, containing 31.1 per cent. of solid matter, were exposed to a current of oxygen at the temperature of the air, and the carbon dioxide absorbed in caustic potash and weighed. In 12 days, 3.7422 grams of carbon dioxide were collected, so that 1 gram of dry yeast gave 0.4812 gram of carbon dioxide=0.131 gram of carbon. Since dry yeast contains about 50 per cent. of carbon, it follows that in this case 26 per cent.of the carbon of the yeast was evolved as carbon dioxide. Microscopical Appearunce.-Coincident with the series of changes culminating in the liquefaction of the yeast, the following series of structural changes was observed microscopically. The freshly pressed yeast consists of large cells, with a small vacuole and granular proto- plasm, staining a deep brown with iodine. As the evolution of carbon dioxide proceeds, the vacuole increases in size, the brown stain obtained with iodine diminishes, and just before liquefaction there is usually no glycogen left in the cell. After liquefaction, the cells have no vacuole and are shrunken, the cell walls being crumpled and the cell substance highly granulated and contracted to a centrally aggregated mass, floating in a small amount of a clear fluid.No brown reaction with iodine is, as a rule, obtainable, and although in the case of yeast liquefied at SO", the brown stain is obtained, the cell does not in other respects differ from the normal char act er. It therefore seems probable that the liquefaction of the yeast is due to the discharge of the contents of the vacuole, and that the progressive increase in the size of the vacuole results from the accumulation of some substance produced along with carbon dioxide from the glycogen. O'Snllivan and Tompson (Trans., 1890, 57, 872) have described the liquefaction of yeast, but did not attribute the liquefaction of the mass to the discharge of the vacuolar fluid. On the contrary, they state that '' A microscopical examination at this stage shows that the yeast cells have shrivelled up into a comparatively small bulk, whilst their outline is irregular and ill-defined. The cell wall has almost entirely disappeared, but the large majority of the cells are unbroken. The vacuole occupies practically the whole of the interior of the cell, This point, however, requires Further investigation.SUEOXIDE OF PHOSPRORUS. PART 11. 1235 and the granulations are very marked, being, in fact, by far the most distinct feature of it.” I n all the numerous samples which have come under our observation, however, the phenomenon occurs as we have described it above, and the whole progress of the change, terminating in the extrusion of the contents of the vacuole has been watched on the hot stage. The cell wall, moreover, can be quite readily distinguished throughout the process. Our thanks are due to Mr. W. J. Young for some assistance in the experimental portion of the work. JENNZR INSTITUTE OF PREVENTIVE MEDICINE.