The Nitrogen Requirements of Yeast

COMMUNICATION
THE NITROGEN REQUIREMENTS OF YEAST
by A. A. D. Comrie, B.Sc.

Many brewers have by now conceived a dislike to the word nitrogen. They have been told that it is probably the most important factor in brewing, and, almost in the same breath, the thing about which we know least. On the score of yeast nutrition it was not of such importance in the days when beer was stronger than it is now; the yeast was reasonably certain of having more than enough for its needs, with care in the choice of malt and in the brewing. Circumstances have altered, and very little disturbing influence is sufficient to reduce the available nitrogenous food below the necessary minimum. Add to this the tendency to be chary with the use of nitrogen as something full of dangerous possibilities and we have arising the full risk of underfeeding the yeast, which has been aptly described as the pivot of all brewing operations.

“Nitrogen,” in brewing technology, very seldom means the elemental gas, but substances containing nitrogen as part of their chemical constitution, and especially those in which it occurs in amine or ammonium combination. At the bottom of the scale come the inorganic ammonium compounds, followed by amides, amines, and amino acids; next in order of complexity come the peptides, in which two or more amides or amino acids have become linked together, and then the albumoses and peptones, which are themselves the first products of disintegration of the true proteins. All these substances are present in varying proportions in brewery wort as it comes from the mash tun. Within limits, these proportions are decided by two things, the malt and the conditions of mashing. The state of combination of the nitrogen in the malt—which may for brevity be called its nitrogenous state—is dependent on the nitrogenous state of the barley from which it has been made and on the way it has been malted. Recently L. R. Bishop has been shedding light on the changes that occur in the nitrogenous state of barley during malting (this Journ., 1928, 101-118; and 1929, 316-338), and every maltster knows that the forcing of malt will produce a higher percentage of soluble nitrogen than is normal. In the mash tun the nitrogenous bodies of the malt are further changed by the action of the malt proteases and the extent of this action is determined by the temperature, reaction, composition of the brewing liquor, and duration of the mash. Finally, a part of the nitrogenous matter is removed from the wort during boiling and in the cooling process, so that the nitrogenous state of the wort as it enters the fermenting vessel is the resultant of many forces. Granted that we are beginning to understand something of the nature of this resultant, and of the forces which produce it, it is still to be decided what its desirable and undesirable points are, its’ necessary and unnecessary components.

Broadly speaking, the nitrogen in a fermenting wort may (1) impart flavour, fulness, and head-retention properties to the beer, (2) provide food for the yeast, (3) provide food for other micro-organisms, such as bacteria, and (4) ultimately be precipitated as a haze to the annoyance of the brewer. It is not the intention of this article to deal with other than what comes under the second heading, food for the yeast, a quantity which at its maximum does not much exceed 40 per cent, of the total wort nitrogen under brewery conditions.

We are indebted to F. Hayduck (ibid, 1906, 282-5) for a resume of some of the work done in the latter part of last century on the role of nitrogen in the life of yeast. M. Hayduck, in 1881, stated that the fermentative power of a yeast was proportional to its nitrogen content, and that this value increased to a maximum with increase in available nitrogen in the medium (Zeits. Spiritusind, 1881, 85 and 173). M. Delbriick, discussing some experiments by O. Reinke two years later, came to the conclusion that yield of yeast was not restricted by lack of available nitrogen but by lack of fermentable sugar (Korres. Vereiiis, Versucha-und Lehranslalt, Brau, 1883, 35). In the following year Lintner (Woch. Brau., 1884, 3) stated that amino compounds were more assimilable than peptones, where excess of nitrogenous nutriment was present, and that both were more assimilable than proteins, while M. Hayduck (ibid, 1884, 177 and 345) made the observation that brewery yeast by continued use became saturated with nitrogen and could remove no further nitrogen from the worts.

E. Ehrich, in 1896, drew a line of distinction between the proteins and the remaining nitrogenous bodies in a wort (this Journ., 1896, 291-3). The former, he said, were of little use as yeast nutrients; peptones and amides were of much greater and approximately equal value. This statement was supported a year later by C. G. Matthews (ibid., 1897, 369-400) who referred to the work of A. Mayer and Nageli. The former had found urea, guanin, allantoin, asparagine and uric acid to be assimilable, the peptones of value in the order of their diffusibility, and the true proteins of little use. Creatin, creatinin and nitrates were valueless. Nageli had found peptones, leucine, asparagine, and ammonium tartrate to be assimilable. That amino nitrogen is almost the only form of nitrogen removed during fermentation, and that it goes directly to maintain the life of the yeast, is the statement Wahl and Hanke made at this time (Amer. Brew. Rev., 7, 32), and G. Heinzelmann also awarded to an amide, asparagine, the premier place as yeast nutrient, with peptone second and diastase third (this Journ., 1898, 427-8).

Using asparagine, A. L. Stern, in 1898, made a quantitative examination of the nitrogen required by yeast and studied the factors affecting its assimilation (ibid, 1898, 654-5). He found that the equivalent of .025 grm. nitrogen per 100 cc. was the optimal concentration, and that amounts in excess of this did not increase the assimilation, the nitrogen in the yeast, the yeast weight, or the sugar fermented. Sulphur was essential, and hydrogen sulphide always appeared to be evolved. A distinction between peptone and asparagine of a purely physical character had previously been demonstrated by Kusserow (Brennerei Zeit., 1897) who discovered that the improved settling properties of yeasts fed on peptone were due to precipitation of the latter in the form of flakes with consequent entrainment of the yeast. This observation was corroborated by H. Lange (this Journ., 1899, 318-9). Three years later P. Thomas reached conclusions similar to those of Stern in regard to the quantitative feeding of yeast (ibid., 1902, 160-1). Using urea instead of asparagine, fermentation and yield of yeast were good in 20 per cent, sugar solutions, but poor in 10 per cent, solutions. This is interesting as an indication of the influence that the other members of a nutrient mixture can have on their nitrogenous associate. As in Stern’s experiments with asparagine, the yield of yeast and the nitrogen assimilation reached a maximum with a certain optimal concentration of urea. When ammonium carbonate was substituted the same results were obtained, only with a different maximum and optimal point. Thomas added that complications were introduced if more than one nutrient nitrogen compound were present, and showed that one source of nitrogen was made available by the presence of another, as in the case of acetamide. This compound is useless alone, but becomes assimilable with the addition of ammonium acetate. In the same year T. Bokorny confirmed the earlier statements of A. Mayer that the nitrogen of nitrates was not available for yeast (ibid, 1902, 758-9). He also stated that meat albumose was of no value and that peptone took precedence of the amino acids as a yeast food. This finding was expressed in the general statement that the food value to yeast of protein degradation products varied inversely as the extent of the degradation—a reversal of the idea that had hitherto been accepted.

In 1907 H. Pringsheim announced the discovery that for yeast to carry on its normal fermentation functions the nitrogenous food supplied to it had to contain the group NH. CO. (ibid 1907, 189-190). In addition he found that, using peptone, fermentation decreased with decreasing nitrogen in solution, while with leucine, asparagine, and ammonium sulphate the fermentation decreased as the nitrogen in the solution increased. Nitrogen assimilation and the production of nitrogen compounds increased with increase in the nitrogen supplied. (Ibid 1907, 382).

H. T. Brown was the first to stress the importance of the proportions of assimilable and non-assimilable nitrogen in brewery wort, and his figure for assimilable nitrogen (expressed as percentage of total wort nitrogen) is 42 per cent, as a maximum, although only 20 to 36 per cent, is removed from the wort during the average brewery fermentation. (Ibid 1907, 394-457). Brown also distinguished between readily assimilable nitrogen and total assimilable nitrogen, and his experience with malt culms as a yeast food went to prove, in his opinion, that with normal brewery seeding rates albumoses and peptones but not amino acids came under the head of readily assimilable nitrogen. This supported Bokorny’s view in contradiction of the prevailing belief. At the same time C. H. Field compromised by saying that the nitrogen compounds needed by brewery yeasts were intermediate between peptones and amides. (Ibid 1907, 584-6). Field also considered that too small a proportion of nitrogen led to yeast degeneration, while an excess of non-assimilable nitrogen tended to make the yeast sluggish.

In a number of yeast-growing experiments made by W. Henneberg in 1908, various forms of nitrogenous nutriment were offered. (Ibid, 1908, 108-110). Normal growth was achieved with only two of the compounds examined, peptone and neutralised asparagine. It was noticed that similar conditions of nutrition were frequently productive of similar yeast forms, and that abnormal cell-forms were an indication of the presence of excessively nutritive substances such as the early degradation products of proteins. In the publication of some further work (Ibid, 1908, 399-401) Henneberg explained the significance of neutralised asparagine by stating that in the decomposition of nitrogen compounds in the yeast cell acids are liberated which unless neutralised are harmful. The evidence suggested that the substances most toxic in this way are those which are most diffusible. Hence the presence of acid-destroying salts and bases in the medium is essential, more so with amino acids and amides such as asparagine than with peptone, whoso injurious products are soon nullified. The calcium ion is particularly efficacious in this respect. In the same year V. Stanêk and O. Miŝkovsky found that betaine, which occurs in beer wort in minute amounts, is not available as yeast food. (Ibid, 1908, 190-1.)

The possibility of the fixation of atmospheric nitrogen by yeast attracted the attention of three investigators in 1910, 1911, and 1913. H. Zikes (Ibid, 1910, 203-4) stated that Torula Wiesneri could fix small amounts of atmospheric nitrogen and suggested it was a property possessed by strongly aerobic film-forming yeasts. C. B. Lipman (Ibid, 1911, 703) confirmed Zikes’ statement and extended his observations to S. cerevisiae S. ellipsoideus, S. apiculatus, and a number of mycoderma yeasts and torulae, all of which exhibited the nitrogen fixing property to a small extent, depending on the conditions of culture. A. Kossowicz (Ibid, 1913, 238) noticed a small fixation of atmospheric nitrogen by several yeasts and yeast forms, but on repeating the experiments of Zikes and Lipman two years later (Ibid, 1915, 68) was unable to confirm their observations.

In 1914 H. J. Waterman examined the assimilability by baker’s yeast of a number of nitrogen compounds (Ibid, 1914, 432). Those assimilable were the fatty amines, aromatic amines with the amino group in the side chain, ammonium chloride and ammonium nitrate. Other nitrates or nitrocompounds such as potassium nitrate and nitro-methane were not assimilable, nor were the acid amides except in the case of formamide (always containing ammonia) and the more complex amino-amides such as asparagine. F. Schönfeld (Ibid, 1914, 435-6) placed the assimilable nitrogen in beer wort at 45 to 65 per cent, of the wort nitrogen, a higher figure than that given by H. T. Brown, but agreed with the latter in saving that only 15 to 30 per cent, is assimilated under brewery conditions. He also noted the unexpected fact that high nitrogen worts lost less nitrogen than low nitrogen worts, which he explained by pointing out that with a poor nitrogen supply a yeast “breaks” slowly and is therefore able to continue assimilating and fermenting for a longer time than a yeast which is well fed with nitrogen and breaks rapidly in consequence. Small assimilation of nitrogen is therefore accompanied by low attenuation. There may be some relation between this observation and that of Kusserow and Lange.

The statement that peptones are more assimilable than amino-acids was challenged in 1915 by A. R. Ling (Ibid, 1915, 512-536), who contended that ammonia and amino acid nitrogen was the more assimilable, albumoses and peptones being only slowly assimilated and of more value as producers of palate. He emphasised the dynamic nature of the nitrogen metabolism of yeast; excre-tion of nitrogen was proceeding simultaneously with absorption. This factor had not been overlooked by H. T. Brown in his experiments, but he considered that if observations were restricted to the period of active fermentation the error was negligible (see discussion on “Nitrogen Question in Brewing,” Ibid, 1909, 294-5). Ling, however, considered that most of the interchange of nitrogen took place during attenuation, and suggested that some of the proteins in solution were rendered assimilable in the later stages of fermentation under the action of yeast proteases. In order to test the availability of the nitrogen in “ring” compounds F. Ehrlich attempted to grow mycoderma yeasts on media using as sources of nitrogen such compounds as pyridine, nicotine, quinine, etc. (Ibid, 1917, 82-3.). He found that growth went on for a time and was then arrested, suggesting that the nitrogen in these compounds is available but that poisonous by-products are rapidly formed, (c.f. earlier note on betaine.).

T. Bokorny in 1921 noted the fact that the nitrogen compounds are capable of supplying yeast with its carbon supply in addition to its nitrogen so that the presence of a carbohydrate is not essential (Ibid, 1921, 82-3). In the same year L. H. Lampitt gave an account of his investigations into the life of yeast, in which he dealt at some length with assimilation and excretion of nitrogen (Ibid, 1921, 83-5). His conclusions in this direction were that a yeast of low nitrogen content has a greater assimilative power than one of high nitrogen content; during active fermentation the greater the coefficient of multiplication of the yeast the greater the quantity of nitrogen assimilated by each cell, although during active reproduction the yeast has a lower nitrogen coefficient even with an ample supply of nitrogen (in other words, reproduction outstrips assimilation); and, the final nitrogen coefficient is independent of its initial value and tends to a constant value for set conditions of reproduction, other conditions being equal. From another series of experiments it was deduced that fermentative activity is essential to nitrogen assimilation, although the two are not proportionate and de-amination once started may continue after fermentation has stopped. A rapid fermentation will result in a low nitrogen assimilation (c.f. Schönfeld’s work). Lampitt obtained some important data concerning the excretion of nitrogen, which he observed may reach the high total of 33 per cent. of the total yeast nitrogen. The act of excretion is dependent on the life of the cell and takes place while assimilation is proceeding; the excreted nitrogen under suitable conditions can be reassimilated, and an increase in the amount of sugar available, especially between 1 and 5 per cent., is allied to an increase in excretion. As with assimilation, fermentative activity is necessary for excretion to commence, but the two are not proportionate and cessation of the former does not arrest the latter. Excreted nitrogen does not appear to be in the form of amino acids, about 35 per cent, of it being precipitable by phosphotungstic acid.

A fact of interest discovered by E. I. Fulmer, V. E. Nelson, and F. F. Sherwood (Ibid, 1921, 138-9) was that the optimal concentration of ammonium chloride in yeast-growing media is the same for many ammonium compounds, and coincides with the concentration which causes least swelling of wheat gluten. This concentration varies in both cases with the temperature without disturbance of the relationship. No explanation was suggested by the authors who, working with beer wort at a later date (Ibid, 1924, 1000) confirmed their observation.

A. Tait and L. Fletcher in commencing their work on the development and nutrition of yeast (Ibid, 1922, 597-621) turned their attention to the disparity between Stern’s observations with asparagine and the value of this compound under brewery conditions. They found that malic acid was produced from the asparagine, and that this acid in the absence of sufficient neutralising substances exerted a toxic action on the yeast. This is a confirmation of Hcnneberg’s statement. Therefore, although asparagines added to malt wort was found to increase the nitrogen assimilation, it was considered an unsuitable food for S. cerevisiae. Ammonium salts were also considered to be of little value to brewery yeast. The authors suggested that an undue proportion of amino acids in wort might be the cause of a thin tasting, unstable beer; they also gave as their opinion that growing yeast does not excrete nitrogen, and that albumoses and peptones are superior to amino acids as yeast nutrients. In the same year F. K. Swoboda tested a number of amides and amino acids as yeast nutrients. (Ibid, 1922, 632-3) Asparagine, succinamide and succinimide were found to increase the growth of yeast, especially in the presence of ammonium sulphate. In such compounds as asparagines which contain two differently combined amino groups, it proved to be only the acid nitrogen that was available. This part of Swoboda’s work appears in marked contrast to Waterman’s experiences with baker’s yeast. Continuing, Swoboda found the nutrient value of edestin was improved by boiling it with acid, but diminished again if the boiling was prolonged. Cystine, histidine, glucosamine, and a mixture of cystine and tyrosine retarded growth, while tyrosine alone, tryptophane, lysine and arginine augmented it. In the case of the last amino acid increase in the concentration beyond a certain point had a deleterious action on the yeast. Experiments to determine the effects of various amines on the fermentative activity of yeast were made in the following year by J. Orient (Ibid, 1923, 72), with these results: methylamine and creatine in dilute solution were unfavourable but in stronger solution became favourable; di and tri-methylamines were favourable at all concentrations; choline guanidine, muscarine, and betaine were favourable in very weak solutions, but unfavourable at higher concentrations—reversing the behaviour of methylamine and creatino; and aldehyde ammonia was strongly activatory in 1·6 per cent, solution but inhibitory in concentrations exceeding 3·2 per cent. In 1924 E. I. Fulmer demonstrated anew that S. cerevisiae will grow using the atmosphere as its sole source of nitrogen, and hazarded the opinion that aeration of worts is beneficial on account of the nitrogen as well as the oxygen that is dissolved.

Ever since the time of Pasteur’s experiments it has been known that yeast can utilise ammonium salts as its only source of nitrogen; that ammonia is not only absorbed but excreted was shown by A. Fernbach and D. Triandafil (Ibid, 1924, 523-4) who inferred that the ammonia excreted is a constant product of metabolism and that its accumulation in a wine was in direct proportion to the time the yeast was in contact with the liquid. An historical summary of the work of the assimilation and excretion of nitrogen made by Triandafil (Ibid, 1924, 627-9) would suggest that investigators into the nitrogen requirements of yeast cannot afford to overlook the interfering factor of excretion. Triandafil, as a result of his own experiments (Ibid, 1924, 629-30) came to the conclusion that amino acids are not completely assimilable even when present only in small amounts, and that yeast excretes ammonia but not amino acids. In this he confirms the views of Lampitt.

P. Petit and J. Raux, dealing with bottom fermentation yeasts, stated that in a normal wort the nitrogen assimilated is from 22 to 24 mgrms. per 100 c.c, and at maximal attenuation 26 mgrms. (ibid 1924, 524). A fresh argument against the presence of excessive nitrogen in wort was provided by their statement that the yeast liberates basic substances, probably ammonia, from the excess nitrogenous matter, thereby reducing the wort acidity to an unstable point.

That potassium nitrate, although useless as a source of nitrogen, has potentialities as an accelerator of fermentation, was shown by A. Fernbach and S. Nicolau (ibid 1924, 787-8). The presence of this salt only in a medium enabled complete attenuation to be obtained, but when other inorganic salts were present attenuation was not complete. The absence of any nutrient value in potassium nitrate was recognised by the authors, who noticed that it weakened the yeast and checked multiplication. Baetslé found that vitamins and amino acids not only stimulate yeast growth, but fermentation also (ibid, 1924, 932-3), especially with aeration. The maximum yield of yeast is obtainable with a mixture of vitamins and amino acids, but the former are not indispensable. R. Moenart found that assimilation of amino acid nitrogen from a wort varied inversely as its original gravity (ibid 1925, 472), thereby recalling Schönfeld’s statement that high nitrogen worts lose the least nitrogen.

Referring once more to the question of nitrogen excretion by yeast, H. von Euler and V. Sandberg clarified the question by pointing out that in yeast metabolism synthesis predominates, and that the extent to which degradation and excretion occur is dependent on the conditions (ibid 1926, 42). Von Euler, in collaboration with H. Fink dealt with this matter experimentally at a later date (ibid 1927, 174) and showed that ingestion and excretion of nitrogen proceed simultaneously, the observed change in the medium representing the difference between the two processes. E. I. Fulmer and L. M. Christensen returned to the subject of fixation of atmospheric nitrogen (ibid 1926, 87), and showed that it was a function of the pH of the medium. At 30° C, optima were found at pH 6·0 and Ph 7·9, but any actual gain in nitrogen content of the yeast was only obtainable after incubation for 6 to 8 weeks. In the opinion of P. Petit (ibid 1926, 231-2) assimilation of nitrogen is affected by the state of the yeast cell wall, in the same way as it has been shown to affect fermentation (see C. Ranken, “The Surface of Yeast as a Factor in Fermentation,” ibid 1927, 76-84).

An addition to our knowledge of the influence of certain amines and amino acids on fermentation, due to Orient, was given by H. Zeller (ibid 1927, 303-4). Glycocoll and histidine hydrochloride do not affect fermentation, aspartic acid and alanyl alanine increase its rate by 33 per cent., alanine and tryptophane by 50 per cent., and asparagine by 100 per cent. G. Seliber and R. Katznelson found that the nature of the peptone supplied to a yeast has an influence on the cell weight (ibid 1927, 551). W. Windisch, P. Kolbach, and E. Hennecke contributed to the difference of opinion concerning the relative merits of amino nitrogen and peptone nitrogen by stating, in 1928, that amino nitrogen is more readily assimilable than any form of nitrogen, and that it forms about half the total assimilable nitrogen in beer wort (ibid 1928, 575-7). The complex proteins are not appreciably assimilable. Since the action of the malt proteases is to increase both the total assimilable nitrogen and the amino nitrogen, the increase in the former not being wholly accounted for by the increase in the latter, it is concluded that bodies intermediate between amino acids and peptones or proteins are also assimilable.

In reviewing the diversity of data and opinions it is difficult to dissociate the quantitative and qualitative aspects of assimilation. The quantity assimilated is dependent on so many factors beyond the kind and amount of nitrogen offered ; on the amount of reproduction (and therefore degree of aeration of the wort and the other diverse influences that reproduction is subject to) and on the nitrogen content of the yeast if the seeding rate is high, according to Lampitt; on the concentration of the carbohydrates, according to Thomas; on the rapidity with which the yeast “breaks,” according to Schönfeld; and on the state of the cell wall, according to Petit. The question of the actual amount of assimilable nitrogen necessary to produce the best results has received two answers; Stern, experimenting with asparagine, says 0·025 per cent, (as nitrogen), and Petit and Raux find that a maximum of 0·026 per cent, of nitrogen disappears from the wort under brewery conditions. These values are in remarkable agreement when one considers the differences in the conditions of observation.

In turning next to consider the subject qualitatively, to find what state of combination of the nitrogen is most propitious, there are three standards of judgment—yield of yeast, fermentation, and type of yeast produced. A substance that may conform to one standard may not conform to the others; for example leucine, asparagine, and ammonium sulphate have all been quoted as compounds that yeast can readily grow on, but Pringsheim has shown that their presence is detrimental to fermentation. It is not enough that a compound should promote yeast growth, the yeast grown must be normal and productive of normal progeny. Stern used asparagine as a yeast food with every satisfaction, and ammonium compounds can supply yeast with all the nitrogen it requires, yet Tait and Fletcher have condemned both these sources as food for the S. cerevisiae of the brewer. It is in part to the existence of these three standards that we owe the seeming confusion of data, and a further complication is the disturbing effect, as pointed out by Thomas, of one nitrogen compound on another. A knowledge of the nutrient properties of a number of individual compounds will not necessarily enable us to forecast the corresponding value of their mixture.

From an examination of the published work it appears that the standard of assimilability has been most often applied, the standard of effect on fermentation much less so, and the third standard of the brewing qualities of the yeast produced almost ignored. It must be pointed out that the majority of those who have contributed information have not been concerned with this last standard, and that only investigators with the requirements of the brewer directing their work would feel the necessity of taking cognisance of it. This limits the value that the brewer can attach to their observations.

Summarising the results obtained with compounds of known chemical constitution, there are only three that pass both standards one and two (in the order just given); urea, tryptophane and asparagine. About the latter there is much divergence of opinion. The compounds that pass standard one but fail standard two are leucine and ammonium sulphate, while those which have only been tested by standard one are:

(a) pass:—Guanine, allantoin, uric acid, ammonium tartrate, ammonium carbonate, neutralized asparagine, ammonium chloride, ammonium nitrate, succinamido, succinimide, tyrosine, lysine, and arginine (weak);

(b) fail:—Creatine, creatinine, betaine, potassium nitrate, arginine (strong), cystine, histidine and glucosamine. Those which have only been tested by standard two are

(a) pass:—Creatine (strong), methylamine (strong), di- and tri-methylamine, choline (weak), guanidine (weak), muscarine (weak), aldehyde ammonia (below 3·2 per cent.), aspartic acid, alanine, and alanyl alanine;

(b) fail:—Methylamine (weak), choline (strong), guanidine (strong), muscarine (strong), and aldehyde ammonia (above 3·2 per cent.).

The compound which fails both standards one and two is creatine (weak). Of these compounds only two have been examined by standard three, asparagine and the ammonium compounds, and neither has passed. Concerning elemental nitrogen there appears to be some doubt, but even if assimilable the conditions are such as to make it a source of no practical value.

Dealing next with less particularized evidence, substances in which the nitrogen is not combined either as amino nitrogen or ammonium nitrogen, such as the alkaloids, pyridine, etc., are of no value. Proteins, nitrates (other than ammonium) and, according to one authority, acid amides, excepting formamide and those of a complex nature, are non-assimilable. Peptones, fatty amines, aromatic amines with side-chain nitrogen, vitamins, diastase, and amino acids in general have been stated to be assimilable.

It is remembered that H. T. Brown sub-classified total assimilable nitrogen into readily-assimilable and assimilable; it is this distinction which is considered to account for the failure of a yeast under brewery conditions to assimilate all the total assimilable nitrogen of the wort. It appears that what is absorbed by the yeast is the whole of the readily-assimilable and only a variable fraction of the assimilable. It therefore becomes important to see that there is an adequate supply of, not merely assimilable, but readily-assimilable nitrogen in the wort. Opinions have been evenly divided on the question as to whether the intermediate protein degradation products such as peptones and polypeptidcs, or the ultimate degradation products, the amino acids, form the readily-assimilable portion of the total assimilable nitrogen in malt wort.

In a subject so fraught with difficulties it is easy to criticise but less easy to make constructive suggestions. Perhaps the most promising step towards unravelling the tangle of circumstances is to make those circumstances as invariable as possible. The nitrogen compounds under test should be the only variables; all other conditions, since they are proved to have such interfering influences, should be rigidly standardised and, if the work is primarily in the interest of the brewer, adapted as far as possible to resemble brewery conditions. For the same reasons the yeast used should be brewers’ yeast of known type, and the test should be threefold: effect on the growth of the yeast, on the attenuation, and on the racial characteristics of the new yeast. In connection with the 6rst part of the test the distinction of readily-assimilable from total assimilable nitrogen would appear to merit attention. Finally, in the investigations into quantitative assimilation which would follow a successful issue of the qualitative examination, the same standardization of conditions is essential, and it is probable that allowance must be made for the excretion of nitrogen by the yeast.