Selected Writings of Fermentation Chemist S.F. Ashby

This is a very cool and very long selection of works by fermentation chemist S.F. Ashby who came after Charles Allan and Percival Greg. It is not for everyone and probably just for distillers and rum makers interested in getting deeper into fermentation. These works are still cited 100 years later by some of the foremost rum researchers. What I’ve done is made the document more accessible and better indexed so more people will find it in searches.

If you are just a casual rum enthusiast it may still be fun to read or skim. You can see how much they knew and didn’t know about fermentation in the glory days of Jamaican rum. You can also see their methodology for getting a handle on it all and deepening their involvement. Sadly, so much of the rum made these days by the new distillers aren’t that sophisticated. They are still in the “look mom I’m making rum” phase and not exactly sculpting their product yet. As I’ve said before rum is the most malleable of all spirits, but you can’t start to shape it without doing serious homework and pretty involved experiments.

One more notable thing is that towards the very end there is an awesome account of making Jamaican orange wine and orange wine vinegar.

Somewhere in there is also the first mention I’ve ever come across of Marshal Ward’s ginger beer plant which is a type of SCOBY used to ferment the original Jamaican ginger beer.

I spent three weeks in Jamaica a few years ago and hated it. My boss had sent me to supervise a house that was under construction. There was so much that could be done there but no one was doing anything. I scoured the island looking for moonshine and found nothing. So much of the jerk chicken I was coming across was made with Chinese barbecue sauce. I have a dream of returning some day to start a Noma style restaurant with a large beverage focus and revive so many of the great products that used to be there until they consolidated into virtually nothing which was then undermined by cheaper Chinese imports. Give me a few years, I still have a lot more to learn.


It had been established during the three years that my predecessor Mr. Chas. Allan, B. Sc. had worked on the manufacture of Jamaica Rum, that flavour was mainly due to the compound ethers. These bodies were considered as produced by chemical combination of alcohol with various volatile fatty acids during and after fermentation of the wash, and particularly during distillation. The alcohol was the product of the action of yeasts on the sugar in the wash, but the acids were the work of bacteria, being partly preformed in the materials used for setting up the wash, and partly produced in the wash during and after the yeast fermentation. The following acids were found, acetic, propionic, butyric, capryllic, capric, lauric, all of which yielded ethers with alcohol capable of giving varied flavours to Rum. Acetic ether was shown to constitute about 98 per cent, of all ethers in Rum, but contributed little flavour and owing to its volatility was very transient. Butyric ether was found to be more valuable, but the ethers of the higher acids, capryllic, capric, and lauric, were held to be of special importance for giving both body and characteristic flavour.

As the yeasts were considered to be only alcohol producers attention was mainly directed to the study of bacteria producing the valuable acids. One such bacterium was isolated and the conditions under which it works determined (Report 1906, pages 136-137). A microscopical examination of washes showed the presence of two yeast types, distinguished by very different modes of multiplying; to the one type belonged, the oval and sausage shaped forms which multiplied by budding (Saccharomycetes) whereas the other type reproduced by division through the middle of the cell, that is by ‘fission.’ (Schizosaccharomycetes). The oval budding forms were alone seen in cane juice washes, but the fission type was found to be the characteristic fermenting yeast of both common, clean and flavoured rum washes. The latter kind could not be isolated, and indeed no systematic experiments appear to have been made with any of the yeasts.

Mr. Percival H. Greig of Westmoreland was the first to isolate a number of Jamaica Distillery yeasts, and to study their action on washes in a state of pure culture. In molasses and dunder which he took to Jorgensen’s Laboratory in Copenhagen in 1893 the fission type of yeast was discovered and studied for the first time. Greig continued to work with these yeasts in Jamaica till 1896 and published reports of his results in the Bulletin of the Botanical Department (March, August, and September 1895 and January 1896). He observed marked differences in the time required for fermentation, amount of attenuation, and alcohol-yield with different yeast, and drew particular attention to a slow working top fermenting fission form which alone was able to produce an agreeable flavour in washes. He recognised the importance for flavouring of fruit ether in rum, but appeared to think that these bodies in so far as they were not contained in the original juice of the cane, could be produced at will by pitching the wash with a suitable flavour engendering yeast. On these grounds he strongly advocated the employment of pure yeast cultures in Jamaican Distilleries, and insisted that the distiller should strive to suppress the action of bacteria.

As previously indicated Mr. Allan took up the precisely opposite view, pushing the yeasts into a subordinate position and devoted his attention mainly to the search after flavour producing bacteria.

As the yeasts must always be the central factors in fermentations for the production of spirits, it appeared to me natural to devote first attention to them, and to observe in particular whether some are really able to engender flavours of value in Jamaica Rum.


Early in the year I isolated and obtained in pure culture a number of the oval budding yeasts from washes in the Laboratory distillery which were set up from a mixture of fresh cane juice and dunder, and about the same time some fission yeasts were secured from a dead wash sent in from the country. As the result of some preliminary fermentation experiments it was observed that the oval cane juice yeasts worked more rapidly in washes of low acidity, but with an acidity of nearly I per cent, the oval yeasts showed very sluggish fermentation, while the fission type worked as well at the high acidity as at the low.

It seemed desirable to study the effect of three common distillery acids, lactic, acetic, and butyric, on the two types of yeast, and accordingly a number of fermentations were set going in cane juice and dunder washes to which varying quantities of the single acids were added before putting in the yeast. The oval budding yeasts all showed bottom fermentation phenomena, but the fission yeasts all showed strong top fermentation with the production of an abundant fatty head. A vigorous yeast of each kind was selected for the experiment with the acids.

The amounts of the different pure acids added are expressed also as Sulphuric acid by weight per cent, of the wash by volume. The amount of the yeast added was as far as possible the same for both types, except in the butyric acid series, which was carried out at a later date with a larger amount of yeast. The results are set out in Table I. which shows the amounts of sugar fermented at the end of each day to the sixth day. The figures were obtained by daily weighings, multiplying the loss of weight by two and calculating the resulting numbers 011 the total amount of sugar originally present.

With regard to acetic acid the results show that the budding yeast is much more susceptible to it than the fission yeast. In the presence of a half per cent, of this acid the budding yeast showed greatly reduced fermentation during the first three days, whereas the fission yeast was but slightly affected. One per cent, completely prevented the activity of the budding type, but again only slightly reduced the fission yeast fermentation. Both yeasts are very resistant against lactic acid, but even here .7 per cent, showed an injurious influence on the budding yeast, whereas, 1.4 per cent, hardly reduced fermentation by the fission yeast. Butyric acid proved to be very poisonous for both yeasts, but whereas .15 per cent, wholly prevented the budding yeast from fermenting it caused the period of fermentation to be increased by only one day with the fission type. Even .4 per cant, did not completely suppress the latter’s activity, but .5 per cent, prevented all fermentation.

The conclusion to be drawn from these results is that the budding yeasts are suitable only for the fermentation of weakly acid washes, whereas the fission type is at home in washes of high acidity. A notable point which the figures bring out is that where the acidity is low the budding yeasts get to work greatly more rapidly than the fission yeast. This is particularly well shown in the case where no acid was added. Although both yeasts completed the fermentation in five days, the budding yeast multiplied and fermented much stronger in the two first days. The ability of the budding type to multiply and ferment more rapidly from the outset in the weeker acid liquors, like cane juice washes and fresh skimmings, explains why this is the only kind found in such liquors the acidity of which is generally under .5 per cent. In the usual estate washes containing dunder, molasses, acid skimmings, and frequently specially added acid, the budding yeast is largely suppressed, but the more slowly developing and very acid resistent fission type takes possession, and is practically the only form found in washes the acidity of which is 1.0 percent, and over.


In March I collected samples of fermenting washes, dead washes, skimmings, dunder, acid and rum, from several estates in Westmoreland and St. James, and from the washes was able to gain pure culture of many fission yeasts. These cultures were started from a single cell according to the method of Hansen in order to prevent the possibility of any of the growths consisting of mixtures. With ten of these derived from four estates a fermentation series was set going in a wash of the composition :—

The Brix was 17.4, the Acidity .48 per cent, and the total sugar present 14.5 per cent.
The yeasts 3 and 9 although pure fission forms, showed a totally different kind of fermentation to most of the others, the yeast gathering mostly into a coherent mass at the bottom of the vessels, the bubbles breaking on the surface being glassy clear and containing practically no cells. This fermentation was evidently strictly of the bottom kind. Yeast 5 showed mainly bottom fermentation phenomena, but produced also a slight yeasty head. All the other yeasts formed a strong glistening brownish white head at the surface and the bubbles were thickly cloudy, these yeasts were accordingly strongly top fermenting. Under the microscope the two forms could be distinguished easily, the bottom type showing isolated and paired cells, but never more than two together, whereas the top yeasts showed long chains of four or more cells interlaced and apparently branched. Yeast 5 showed no chains but the cells were often united mechanically into flocks.

The bottom yeasts 3 and 9 completed the fermentation in two days less than the top forms, yeast 5 occupying an intermediate position. This character of the bottom yeast to ferment more vigorously than the top kind has preserved itself in all subsequent experiments. The increase of acidity due to the yeast alone, all bacteria having been excluded, amounts to only about .1 per cent. The attenuation was very much the same in all cases, but the highest amounts of proof spirit were obtained from the bottom yeast 9 and the mainly bottom yeast 5. The yield of proof spirit per degree attenuation was good, in four cases exceeding unity. The distillation was effected from glass apparatus with one retort, the liquor being divided into two parts, the first yielding high wines of 20 O.P. and the second portion giving rum of 41 O.P. with the high wines in the retort. The rum could hardly be called by that name, and it showed the same character for all ten yeasts; in no case was any characteristic flavour produced.

In another experiment with dunder, molasses, and water, a much larger amount of dunder was used, namely one half the bulk of the wash.

The Brix of the mixture was 18.6, Acidity .7 %.

The Bottom yeast here showed a gain of 2 to 3 days in the fermentation period. The yield of proof spirit was very high. The rum obtained was very light, and gave no difference in flavour with the different yeasts.

In another fermentation series with the yeasts 2 and 9 pure volatile acids were added to the molasses and dunder wash before pitching with the yeast. The Brix was l8.6, the natural acidity of the wash .46. Acetic acid was added equal to .5 acidity, and butyric acid equal to .1 acidity, so that the total acidity before fermentation amounted to 1.06.

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The large amount of volatile acid added had a marked effect in slowing fermentation, the time required as compared with the previous experiments, being 10 days as against 6 days with the bottom form,and 16 days as against 9 with top yeast. The rum showed an improvement in flavour, and with the top yeast contained more than twice as much ether. This was due to the much longer period during which alcohol and volatile acids could react chemically to produce ethers in the wash containing the top yeast.

The conclusion to be drawn from these experiments is that, whereas, none of the fission yeast isolated from the estate washes was able to produce flavour on its own account, the top yeast owing to its slower fermentation admitted a greater amount of chemical ether production in a wash originally high in volatile acids. The latter result is in accordance with distillers’ experience as they consider that a wash showing a strong fatty head due to the top fermenting fission yeast yields the best flavoured rum.


It is well known that the alcohol accumulating during fermentation has, beyond a certain concentration, different with different yeasts, a marked slowing effect on fermentation and finally stops it all together. In order to test the maximum amount of alcohol endured by the Jamaica fission yeasts, it was necessary to set up a wash of very high gravity. In a first experiment with the yeasts 2 and 9, a wash consisting of 4,000 dunder, 1,600 molasses, and 703 water was set up at 30′ Brix. This was practically completely fermented, so that the alcohol formed was below the maximum which the yeasts could endure. In a second series a wash of 30° Brix was set up with molasses and an extract of yeast, and after some days a further quantity of molasses was added. In this case both yeasts stopped fermenting due to the action of the alcohol, while there was still abundant sugar left in the wash. The data and results of the Experiments are given in Table IV.

The first Experiments show a complete fermentation by both yeasts, the bottom form taking 5 days less than the top yeast. The bottom yeast also shows a higher yield of proof spirit. The influence of the accumulating alcohol on fermentation is very marked, for whereas the bottom yeast had produced 16.5 per cent, proof spirit in 7 days, only 7 per cent, more spirit was produced in the following II days.

In the second Experiment the maximum yield of alcohol which prevented all further fermentation was just under 25 per cent, with the bottom yeast and just over 23 per cent, with the top form ; while the bottom yeast yielded 17 per cent, proof spirit in 7 days only 7.7 per cent, more was produced in the following 12 days. A similar effect of the alcohol is shown by the top yeast. The top yeast showed a rather sudden falling off in fermentation with about 18.5 per cent, proof spirit present, but the top yeast gave a more gradual falling off; it appeared, however, to be susceptible at about 16.5 per cent, proof spirit. The mixture of the two yeasts showed throughout intermediate results.

It is evident from these results that the fission yeasts which work the estate washes are capable of yielding very large amounts of alcohol in pure culture with abundant time at their disposal. Fermentation is rapid and uniform for 7-9 days, during which 16-18 per cent, of proof spirit is yielded. This means that a wash containing about 16 per cent. of sugar can be fermented in a reasonable time. Above this amount the loss often becomes serious owing to sluggish fermentation. This fact has been recognised in practical distillery work, so that estate washes are rarely set up with more than 16 per cent, of sugar and usually with less.


This Experiment was devised with a view to observing the effect of varying the amount of sugar in the wash, on time, attenuation and yield of proof spirit. The washes all contained the same proportion of dunder, namely, three-fifths, the gravity being varied by means of the molasses. The results were as follows:

Here as usual the bottom yeast is the most rapid worker, showing 1 gain of three days. The time required is least with the lowest gravity, but there, is a difference of two days between the 25 and 20 settings and of only one day between the 20 and 15 settings. This difference hardly shows itself during the period of the main fermentation. After five days the relative amounts of sugar fermented by the bottom yeast were 35, 51, and 68.

As there was a half more sugar in the 20 setting as in the 15, and twice as much in the 25 setting, these figures indicate that the activity of fermentation was proportional to the amount of sugar present, i.e., in a given time twice as much sugar was fermented in the 25 setting as in the 15 setting, the 20 setting coming half way between. The difference, however, was shown by the time taken by the wash to die off after the main fermentation was over. The 25 setting took 3 days to die, the 28 setting I day, and the 15 setting only a few hours. The yield of proof spirit was as high for the highest gravity as for the lowest, and the bottom yeast gave as usual the best results.

On the other hand there was markedly more sugar left unfermented in the highest setting than in the other two, and the bottom yeast in all three case left more than the top yeast. The dunder employed in this series was a light cane juice product having a Brix of 9 and an acidity of only 1.2. The amount which had to be used (3/5 of the wash) to secure a normal acidity was more than is usual in practical operations, where the dunder has an acidity of over 2 per cent. The result was that the relative amount of sugar in the wash was low, and the attenuation and yield of proof spirit low also.


The yeast produced in some of the fermentations of the last experiment was collected, dried in the air and weighed. The results are shown in pounds for 1,000 gallons of wash.

The top yeast produces a half more yeast substance than the bottom yeast consequently a pound of the bottom yeast is able to ferment a much greater amount of sugar. The amount of yeast produced by the top variety falls away with the reduction in gravity of the wash, so that only one half as much yeast is produced in a 15 Brix setting as in one at 25 Brix. The amount of yeast produced is proportional to the amount of fermentable sugar present for washes from 25 to 15 Brix, but at 30 Brix relatively less yeast is produced, so that the ratio to sugar fermented is wider.

At first sight it seems inconsistent that the top yeast should often attenuate more than the bottom yeast and leave less sugar unfermented, yet give a lower yield of proof spirit. The above results, show however, that it removes no more sugar to build up its substance than the bottom yeast, and owing to its habit of gathering at the surface of the wash in intimate contact with the air, respiration is more active, causing a greater loss of sugar by combustion into water and carbonic acid The bottom yeast is consequently a more economical worker.

Stability of the two Varieties

Distillers often observe that during the advance of the season their fermentations which were at first of the bottom type, tend more and more to top characters, suggesting either a conversion of the bottom yeast into the top or else a gradual displacement of the former by the latter due to some change in the composition of the wash which favours the top yeasts. That top and bottom fermentation may proceed in the same wash, was evident from the fact that both forms were in several cases isolated from the same material.

Some observations have made it seem probable to me that at any rate one of the varieties is not stable. The fission yeast No. 3 when freshly isolated showed wholly bottom-fermentation phenomena, and agreed entirely with the other bottom yeasts. It was allowed to lie for two months under a fermented cane juice wash, and was then freshened up again. I was surprised to find that it no longer showed bottom fermentation, but gave a strongly marked top fermentation. On comparing its behavior with that of yeasts which had always been top fermentation, it was found that it gave quite similar results, i.e., an equally slow fermentation and a lower yield of alcohol than the bottom yeasts. Under the microscope it also was identical with the top form. The view which remained for many years unchallenged in Europe was, that the top and bottom yeasts were distinct types, the one never passing into the other. Quite recently Hansen has shown however, that there is always a tendency to vary, and has actually obtained the one form from the other in the case of a number of brewery and wine budding yeasts. There appears to be a much greater tendency for bottom yeasts to go over into the top form than vice versa. Further observations must show whether the fission yeasts are particularly liable to vary in this way, and whether the change so often seen in distilleries in Jamaica from bottom to top fermentation is due to a variation of the yeast.

Conclusions with regard to the two varieties of Fission Yeast.

I—The bottom yeast is a characteristically more rapid worker than the top yeast giving a gain of 2 to 3 days in the fermentation period.
2—The bottom yeast forms less substance and consequently makes a smaller claim on the amount of food stuff in the wash.
3—The bottom yeast gives a rapid and uniform fermentation during the main period, but the wash dies slowly. The top yeast ferments very uniformly throughout, and shows no sharp transition to the final stage.
4—The yeasts attenuate about equally, but the bottom yeast gives a better yield of alcohol.
5—The top yeast leaves less unfermented sugar in the wash.
6—The bottom yeast gives a higher maximum yield of alcohol, namely 25 per cent., as against 23 per cent., with* the top variety.
7—The bottom yeast shows the injurious effect of alcohol at a higher concentration than the top yeast, viz., II and 16 respectively.
8—Owing to its slower fermentation the top yeast admits of more ethers being produced in the wash than the bottom yeast where volatile acids are present. The rum is consequently better.


Owing to insufficient distillery space or small still capacity, it often happens that molasses have to be stored for weeks during which period they undergo a rather active fermentation. This involves a loss of sugar, so that it seemed desirable to make some experiments with a view to (1) determining the amount of loss arising from the cause. (2) separating and studying the properties of the yeast causing the trouble. (3) finding a remedy for it.

Three yeasts were secured in pure culture from a fermenting molasses, all of which were able to set up fermentation in a liquor of very high gravity.

YEAST (a)—This was a budding form of the pastorianus type which formed spores on the gypsum block at the air temperature in under 18 hours. Transfered to mixtures of molasses and water of increasing gravity it fermented actively at 45Brix, feebly at 60 Brix, and showed no fermentation in molasses alone of 90 Brix. It was therefore not the kind active in the stored material.

YEAST (b)—This was a fruit ether producing yeast forming a dry wrinkled friable skin on ordinary washes. It was a small budding yeast which formed hat shaped spores on the gypsum block in 24 hours. It fermented strongly in molasses and water of 45 Brix, more weakly at 60 Brix and not at all in molasses alone. It was also therefore not the form desired.

YEAST (c)— This was a small spherical or oval budding form characterised by the production of branched chains of cells in weakly acid washes, and a very abundant multiplication. It formed no spores and no skin on cane juice, but merely a yeast ring. It appeared therefore to be no true yeast, but a ‘torula.’ This kind fermented actively in molasses and water of 45 and 60 Brix, and also in pure molasses of 90 Brix. It corresponded to the form most abundantly present in the original material, and was evidently the true agent.

As an alkaline medium acts very unfavourably on yeast fermentation lime suggested itself as the first substance to try as a remedy. In one experiment the molasses were allowed to ferment spontaneously without the addition of lime, and with the additions of 6, 12, and 18 lbs. of dry lime to every 100 gallons of molasses, the lime being added as fresh milk of lime and well stirred in. The same experiment was repeated with sterile molasses into which a pure culture of yeast (c) had been introduced, but here only 3 and 6 lbs. of lime were used. The fresh molasses had a Brix of 90 and contained nearly 70 per cent, of sugars. After six weeks the Brix was determined and found to be as follows :—

The molasses alone fermented strongly with crude and pure yeasts from the outset. With 6 lbs. of lime there was no fermentation for nearly three weeks, when it started, but was much stronger in the pure yeast culture. 3 lbs. of lime in the pure yeast culture did not prevent fermentation from starting within a few days. With 12 lbs. of lime in the crude culture fermentation had only just started between the 5th and 6th week. With 18 lbs. of lime there was no growth of yeast and no fermentation. In the crude there was a maximum loss equal to 15 per cent, of the total sugar, and in the pure culture this loss exceeded 21 per cent. Lime in small amount was therefore capable of checking this fermentation for a time, 6 lbs. to 100 gallons being sufficient to preserve the molasses for nearly three weeks. As the lime gradually losses its alkalimity and goes into the neutral carbonate the fermentation starts afresh. As it is very undesirable to bring an alkaline molasses into a distillery wash as small an amount as possible should be used to check the foaming 6 lbs. of lime to 100 gallons molasses should be used at first, the lime being freshly stirred up into a milk with a few gallons of water, but only enough of the latter to admit of a thorough stirring into the molasses. If after a time foaming shows evidence of beginning again a further smaller amount of lime milk must be stirred in.

The yeast or ‘torula’ (c) ferments very sluggishly in a dilute molasses wash, and hardly at all in cane juice. Judging from the Experiments with the molasses, it is able to produce about 14 per cent, of proof spirit. It cannot invert cane sugar, and hence the feeble fermentation in cane juice, but only attacks the ready formed invert sugar in molasses.


As this yeast in pure culture gave a very marked flavour to washes in which it was fermenting some preliminary experiments were made with it in different media, the Rum distilled off and the Ethers determined therein. It was grown in three washes ;—

(i) Molasses and water Brix 15 Acidity .10
(2) Molasses, half dunder and water Brix 15 Acidity .34
(3) Tempered cane juice and one sixth dunder Brix 15 Acidity 20.

The yeast formed the dry wrinkled surface skin in a couple of days in all the washes, and multiplied abundantly, at the same time the fruity odour was very perceptible. Fermentation was very slow, the time required for the washes to die was;—

In spite of the very high ether content the rum had a pleasant fruity flavour with no trace of ‘pepperiness.’ These result were obtained by a simple distillation without any treatment of lees. The ethers consisted mainly of acetic ether, so that the yeast is able to produce both alcohol and acetic acid. There was no increase of ether production during distillation as a portion of (1) was neutralised before distilling and gave the same amount of ether as the un-neutralised part, namely 18.000.

The increase of acidity during fermentation was inconsiderable, a result which taken from the preceeding one makes it highly probable that ether formation does not occur by a merely chemical reaction in the wash, but takes place in intimate relation with the actively working yeast cell.

Further work is being done on this yeast with a view to its introduction into distillery practice.


Two perfectly different species of Acetic Acid Bacteria were isolated from acid skimmings and dead washes.

I. A form which appears quickly on dead washes both of low and high acidity. At first a delicate blue dry friable film which becomes white when strongly developed, but is always easily broken up. In a glass vessel the film climbs up the sides high above the surface of the liquid. It consists of short rather plump rods which stain yellow or yellowish brown with iodine, but never blue, and forms only short chains. It resembles Bacterium Kutzeanum of Hansen except in its inability to turn blue with iodine.

II. A Bacterium which forms a very tenacious cartilaginous skin in skimmings and dead washes, consisting of long narrow rods. The skin turns blue with iodine and sulphuric acid, and is in all respects similar to Bacterium Xylinum of A. Brown.

In order to observe the highest concentration of alcohol which admits of a development of acetic bacteria a dead wash holding 23 per cent, of proof spirit was exposed to the air. For six weeks there was no sign of an acetic film, and there was no rise in the acidity. Between the sixth and seventh week a film began to form and at this stage the liquor contained 14 per cent, proof spirit, 9 per cent, having evaporated away from the wash.

In another experiment a dead wash containing 24.7 per cent, of proof spirit was diluted with water in varying amounts and seeded with a pure culture of acetic bacterium I. The progress of acidification is shown in the following table, the figures representing the increase of acidity expressed as Sulphuric acid per cent.


More alcohol was added to c, d, and e, after three weeks, and the acid rose to 6.2, 5.8, and 5.3, respectively in another week, but showed no further increase. The greatest amount 01 acid produced was therefore equal to about 7.5 per cent, of pure acetic acid, the largest quantity which the bacterium could endure. The organism could not grow and work in 24.7 per cent of proof spirit, and showed only feeble activity in 16.5 per cent, but in 12.3 per cent, it worked strongly. The evidence shows therefore the amount of alcohol which can undergo vigorous acidification is between 12 and 16 per cent proof spirit, which agrees with the result of the first observation.

The theoretical maximum amount of acetic acid which could be formed from the alcohol in cultures c, d, and e, is 7.3, 5.9, and 4.9 per cent. The actual amounts formed in 20 days were 5.7, 5.2, and 4.6 so that
in c 78 per cent of the possible was formed,
”  d 89
”  e 94

The lower the amount of alcohol in a liquor, the more completely therefore is it oxidised to acetic acid. For practical purposes the highest acidity was reached in a fortnight at about 4 per cent. Bacterium II. proved to be unable to grow and produce acid in a dead wash containing 12 per cent proof spirit, but gave over three per cent acid in a liquor with 8 per cent proof spirit. This bacterium also makes greater claims upon the nitrogenous foodstuff in the liquor than bacterium I. Bacterium I. is therefore the characteristic acetic acid producer in all liquors containing 10 per cent and more of proof spirit, such as ordinary dead washes, while bacterium II. works best in liquors like fermented skimmings and fermented rum cane juice.

The following table shows the amounts of Total and Volatile acid (mostly acetic acid) and the relative amounts of volatile acid to total acid in some distillery liquors. Of special interest are the quantities of volatile acid in such materials as acid skimmings, and flavour, because in these liquors an attempt is made to produce as much volatile acid as possible. The volatile acid shows an average percentage of the total acid of from 22 to 27, or only about one quarter of the acid present is volatile. As the fresh skimmings which comes down from the boiling house are practically neutral the great part of the acid produced in the cisterns is the work of bacteria. Although the skimmings readily undergo fermentation, this is not entirely due to yeast, as the liquor is heavily contaminated by bacteria which produce fixed acids such as lactic from sugar. A number of such bacteria have been separated from the skimmings. They include the well known rice grain bacterium, which can nearly always be found in skimmings. It forms large rounded gelatinous masses when strongly developed consisting of enormous numbers of hand shaped colonies, the rod shaped bacteria being embedded at the ends of finger like processes of the jelly. This bacterium produces lactic acid and forms its jelly at the expense of the sugar present. Another rod shaped organism often develops in fresh cane juice contaminated by dirt from the mill or by soil, at a great rate, and converts the liquor in one day into a thick viscous mass in which yeast can only work very sluggishly. Gas and lactic acid are produced, the viscous substance being formed at the expense of the sugar. The presence of such objectionable organisms account for the poor yield of alcohol in skimmings, and the small amounts of volatile acid. Acetic acid bacteria are wholly dependant upon oxygen for their work of converting alcohol to acetic acid, and require therefore that the liquor in which they are working should expose as great a surface as possible to the air. This is only being imperfectly attained in distilleries even in the trash cisterns. It is proposed therefore to Experiment on a practical scale with a view to the more rapid and more abundant production of acetic acid from alcoholic liquors.


By S. F. ASHBY, B.Sc, Fermentation Chemist.

1. Useful Information’ Regarding Estate Distillery Materials.

Skimmings or Scummings—A mixture of liquor and solid matters skimmed from the surface of juice in clarifiers and coppers (if used) together with wash water from coppers, etc. The solid matter a mixture of pulverised cane fibre (trash), phosphate of lime, pectic and waxy matters, and coagulated albumen. According to the amount of solid matter and of dilution the gravity may vary when quite fresh from that of the juice (15-20 Brix) to under 10 Brix. The reaction to litmus is either neutral, faintly acid or faint alkaline.
Dunder—The liquor left in the still after distillation is completed. A yeast extract. The gravity varies according to materials fermented from under 10 Brix to over 25 Brix, and the same applies to the acidity which varies from about 1 per cent, to over 3 per cent. It is never free from sugar which varies from 0.2 per cent, to over 1 per cent. Sugars other than hexoses (pentoses) and allied bodies may be present which reduce Fehling’s solution but are not fermentable by yeast. On an average about 1 percent, of glycerine has been found in Dunder. It is never free from volatile acid.
Its high density is due to cane and yeast gum and caramel (especially if still is direct fired.)
Molasses—The sweet viscous syrup separated from the crystalized sugar by the centrifugals. It is markedly acid (about 0.5 per cent.) has a specific gravity of about 1.45, contains about 40 to (50 per cent, cane sugar, and 10 to over 20 per cent, glucose. One gallon (imperial) contains 8-10 pounds of fermentable sugar.
Acid—Skimmings, normal cane juice, or rum cane juice, allowed to sour. The production of acetic acid is the object sought. The volatile acidity rarely exceeds 40 per cent, of the total and is usually under one third the total.
The souring is carried out either with trash cisterns or without the addition of trash. The liquor ferments (yeast) and sours simultaneously.
Lees—The liquor left in the retorts after distillation is completed. It contains a high proportion of volatile acid.
Wash—The liquor (prepared from the mixed materials) which is actually fermented and distilled for rum. The mixing of the materials is called “setting up.” When fermenting it is “live” wash, and when fermentation has ceased it is “dead” wash.
Flavour and “Muck Hole”—(See description in first Sugar Experiment Report.)
Rum—The early portion of the alcoholic distillate; (the preliminary runnings if cloudy are rejected) its strength varies from 36 to over 40 proof as determined by the “bead.” It is water clear (white Rum). Before leaving the estate “Rum store” it is coloured by caramel boiled by the distiller. Each estate has its own standard of colour.
High Wines—The running from the still which follows the rum; collected to a strength of about 20 over proof.
Low Wines—The subsequent runnings collected till all alcohol has distilled over. The strength varies from 40 to 60 under proof.
Retorts—Copper vessels inserted between the still and the coil. The vapours from the still must pass through them. Most estates have one retort which contains the high wines of a preceding distillation. Some estates have both ”high wines” and “low wines” retorts, the latter next to the still. The retorts have a capacity of about 1-10 that of the still.
Low Wines Rum—Some estates with one retort (high wines) add the low wines to the wash in the still; other estates, however, distill the low wines independently (they run about one low wines still to 5 or 6 ordinary wash stills) and obtain “low wines rum” a product of inferior quality and price.

Types Of Rum.

The two main kinds of Rum are “Common Clean” and “Flavoured or German.” The individual estates confine themselves to the manufacture of one of these kinds. Nearly all the “Flavoured” Rum is made in the parish of Trelawny.

Common Clean Rum—may be divided into two kinds depending on the materials used.
1. From washes set up with a mixture of skimmings, dunder, molasses and water. The materials are not allowed to sour. Several estates with up-to-date boiling house plant (vaccuum pans, etc.) and a consequent large out put of skimmings and molasses employ this method. The materials must be used rapidly, and fermentation rendered of as short duration as possible. The wash is set up with 1/3 skimmings, 1/3 dunder, and molasses and water to give an initial gravity of about 10 Brix. The wash attenuates in about 4 days to 3 or 4 Brix. The initial sugar content is about 11-13 per cent, and the attenuation from 11-13 degrees. The rum is light in body and of low ether content, and is mainly consumed locally.
One or two estates which do not make sugar boil their juice and ferment it with dunder. (Appleton).
2. From washes set up from the same materials and also with “acid” prepared either from skimmings, rum cane juice or normal cane juice. The composition of the wash varies:—

The gravity of the setting depends largely on that of the dunder which varies from 10 to 20 Brix. As a rule the setting is not lower than 18 Brix. and may be as high as 24 Brix. The initial sugar content varies from 10 to 14 per cent, and the attenuation corresponds to that. The fermentation period depends on both the acidity of the dunder and on the quantity and acidity (especially the volatile) of the “acid.” The wash ferments from 5 to 9 days and is often allowed to lie for a couple of days when dead.

The only acid produced is evidently “acetic” and some of these rums may contain over 1,000 ethers (Swanswick, Long Pond) where much “acid” is used in the wash.

The yield of proof spirit is from 0.85 to 1.0 per cent, on the sugar fermented and on the attenuation 0.8 to 0.9 per degree. From 5 to 10 per cent, is lost in distillation.

The yield of rum 40 o.p. varies from 60 to 90 gallons per 1,000 gallons wash in still.

The fermenting cisterns (sunk in floor of distillery built of wood and backed by puddled clay) and vats are usually of 1,200 gallons capacity and the still will receive the contents of one cistern. Two stills are usually run per day (daylight). The stills are heated by steam coil or by direct fire. The rums made with ‘common clean’ materials vary in ether content from under 100 parts to over 1,000. Acetic ether is practically the only one present, and its amount depends entirely on the quantity of acid used in the washes and on the length of time the wash ferments and lies when “dead.”

Flavoured or German Rum.—These rums are made on estates having old fashioned boiling house plant where the manufacture of sugar is of secondary importance. The usual common clean materials are employed and in addition “flavoured.”

“Acid” is prepared from cane juice or skimmings in the usual way in a succession of trash cisterns. A “muck hole” outside the distillery is the receptacle for the thick matter deposited from the dunder, and the wash (dead wash bottom) to which is added cane trash and lees. The matter consists to a large extent of dead yeast and is therefore highly nitrogenous. It undergoes slow fermentation and putrefaction and its acidity is kept low by the addition of marl. When ripe it contains large amounts of butyric and higher fatty acids, both free and combined with lime. It is added to a series of acid cisterns outside the distillery where the butyric and other acids are set free. This complex acid material is the “flavour.” The flavour enters the wash after fermentation has begun owing to the presence of acids in it which are injurious to yeast, the fermentation is prolonged and the sugar is never very completely fermented out. Fermentation lasts 9 to 10 days and the dead wash lies for several days longer. An example of the kind of wash follows:—

This means a yield of 48 galls, rum per 1,000 galls, wash whereas the attenuation would indicate a yield of about 78 gallons. Only a portion of the high strength distillate is therefore collected as rum of first quality.

These rums show an ether content as a rule from 1,000 to 2,000. While over 95 per cent, of the total ethers is “acetic” there is always present several per cent, of butyric ether and still smaller amounts of esters of higher fatty acids (capryllic, caproic and lauric). Most of these rums find their way to Germany for blending and particularly for “stretching” potato or molasses spirits.


Yeasts.—Practically three yeasts perform all the conversion of sugar into alcohol in the Jamaica Distillery.
1. Bottom fermenting oval budding yeast.
2. Top fermenting chained fission yeast.
3. Bottom fermenting unchained fission yeast.

Oral budding yeast.—A typical bottom fermenting yeast the cells of which do not form chains. It is oval in shape and often rather pointed at one end. The average dimensions are 7.5-9 m long by 6-7 m. broad. It does not form a film on dead wash but at most a yeast ring. It forms spores on the gypsum block (as a rule four in a cell) in 24 hours at air temperature. It readily inverts and ferments cane sugar. This yeast is present on the rind of the cane and is always found in freshly milled juice. Spontaneous fermentation of juice is therefore always brought about by this yeast. In fresh juice it multiplies quickly and sets up a rapid fermentation. It displaces all other native yeasts in a favourable liquor like juice. The optimum temperature for its multiplication lies above 30 C. but it appears to ferment best at that initial temperature. It will work practically all the sugars out of an undiluted juice if not interfered with by acid-producing bacteria. The fermented liquor has an agreeable odour. In the experimental work at the Sugar Station Distillery where either cane juice or cane juice, molasses, and dunder are usually worked with, this yeast alone sets up and carries through normal fermentation.

On estates where the first type of common clean rum is made (i.e. without “acid “) this yeast possesses the wash owing to its properties of quick multiplication and rapid and intense fermentation. Such washes heat up quickly and temperatures as high as 108 F. have been observed. These high temperatures mean injury to the yeast, imperfect attenuation, and marked loss of alcohol by evaporation. Like most bottom fermenting kinds this yeast is markedly susceptible to unfavourable conditions such as poor food supply, excessive temperature and especially high acidity. Volatile acidity injuries it very readily (see experiments in second S.E.S. Report.)

It is injuriously affected by the fixed acids of dunder and works best where the initial acidity of the wash does not exceed 0.3 per cent. In washes with an initial acidity of 1 per cent and more it gradually gives place to more acid-resistent yeasts. On estates using acid the wash contains both this and fission yeasts, the relative proportion depending on the amount of acid employed. In common clean washes with an acidity exceeding 1.5 per cent, and a volatile acidity of 0.5 per cent, the writer found it entirely displaced by fission yeasts even quite early in the season.

Top Fermentiny Fission Yeast.—A typical top fermenting chained yeast. On washes of high acidity which are not working very intensely this yeast throws up a characteristic light or dark golden yellow thick moist creamy or fatty head which may completely cover the surface of the liquor. The bubbles of gas escaping through the head are cloudy. The head consists mainly of short, rectangular cells in chains of four or more, often in clumps and showing a kind of false branching. When shaken up in a wash the yeast forms into loose flocks which rapidly deposit. There is considerable variation in the size and shape of the cells: the size varies from 6-12 m by 4.5 to 5.5 m. and the chain cells are usually small viz., 6-7 m. long by 4.5 m. broad.

Spores are freely formed in the wash during fermentation. There are four oval spores in a cell and their walls stain blue with iodine (in iodide). The spores are very frequently found in bridge shaped sporangia formed by the reunion after division of two cells or by the union of two neighbouring cells. This yeast has a high optimum for multiplication and fermentation between 34 to 37 C. It endures high acidity (over 3 per cent. total) and is greatly more resistent to volatile acid than the budding yeast. At ordinary temperatures 24 to 27 C. the fermentation is slow but the sugar is efficiently worked out. In pure cultures the attenuation and the yield are as good as from the oval yeast.

In all washes of high total acidity (over 1 per cent.) and especially of high volatile acidity this yeast is generally present and often carries out the entire fermentation. It is the typical yeast of the “Flavoured Rum” washes.

Bottom Fermenting Fission Yeast.—This yeast produces no head in washes, the escaping bubbles being glassy clear. The cells are found single and in pairs, and when the wash is stirred the cells distribute themselves in a fine clay-like suspension, which clears slowly. The cells are variable in shape and size averaging 6-14 m. long by 4-5.5 broad. Spores are formed with the top yeast.

This yeast has a somewhat lower optimum temperature than the top yeast, and like most bottom yeast yields markedly less substance and is more susceptible to external factors than the top yeast. It is often found in acid washes together with the top yeast. It increases more rapidly and ferments more strongly than the top yeast at ordinary temperatures. The attenuation and yield of alcohol in pure cultures are the same as for the top yeast; it appears to leave more unfermented sugar in the dead wash. In a sample of soured cane juice having a total acidity of 2.1 per cent., and a volatile acidity of 0.90 per cent, this yeast alone was found. In the wash set up with this “acid” having an acidity of 1.6 per cent, and over 0.50 volatile, the top yeast was the characteristic worker. It would seem that in highly acid washes the more resistant top yeast gradually increases with the advance of the season. Comparative experiments with these two fission yeasts in Laboratory washes at air temperature indicate that the bottom yeast ferments the wash in one or two days less time.

A bottom yeast with slightly top phenomena has also been isolated. It forms no chains but the cells agglutinate more than the typical bottom yeast. In its properties it comes between the two extremes.

The undermentioned yeasts isolated from distillery materials play no evident part in the actual fermentation of washes.

Fruit Ether Yeast.—Isolated from ”foaming” molasses. Forms a dry white friable wrinkled film on material containing sugar. A small oval budding yeast with cells very variable in size. Forms spores on the gypsum block in 18-24 hours at air temperature. The spores are “hat shaped.” The yeast is therefore an “anomalus” variety (Willia anomala). It inverts and ferments cane sugar and will ferment diluted molasses over 50 Brix.

Produces a very high amount of acetic ether, the distilled wash containing from 12,000 to 40,000 ethers. The fermentation is slow occupying two weeks or more to attenuate 12. In ordinary washes it is easily displaced by more active yeasts. (See second S.E.S. Report, and experimental data in Laboratory records).

Pastorianus Yeast.—Isolated from “foaming” molasses. A top fermenting yeast which forms spores abundantly on the gypsum block in 18 hours. Will ferment diluted molasses of 50 Brix. Inverts and ferments cane sugar but is easily surpassed by the oval budding yeast and fission yeast in appropriate washes. It yields a fermented product of good aroma and has been used successfully in the preparation of orange wine.

Torula from Molasses.—Does not form spores and cannot invert and ferment cane sugar. A small chained oval or spherical yeast which ferments the glucose in molasses of the highest gravity. The cause of “foaming” (see second S.E.S. Report)

Large celled Oval Yeast.—A spore-forming top fermenting yeast which works badly in estates’ washes.

Ludwig’s Yeast—Found occasionally in small amount in fermenting cane juice and also present in “acid” (Long Pond and Swanswick). Is probably present on the cane. Corresponds in size, division, and spore formation to Saccharomyces ludwigii.

Mycoderma Species.—Commonly present as grey and white wrinkled films on dunder, sour skimmings, and dead washes which are allowed to lie two or more days. Produce no fermentation, but oxidize alcohol to carbonic acid and water. No spores formed.

Culture Media.—The medium which had answered well for the cultivation of all yeasts is a cane juice peptone broth. Prepare as follows:— Fresh cane juice is tempered with milk of lime, heated to the boiling point and filtered. Care must be taken to avoid excess of lime or the liquor will darken excessively. This liquor may be transferred to a Carlsberg Can and boiled for half an hour on two successive days to sterilize it.
The medium has the composition:—
tempered cane juice 100
peptone 0.5
potassium phosphate 0. 05-0.1

The cane juice should first be diluted to 13-14 Brix. The broth is heated in the steamer and filtered and then rendered distinctly acid with Hydrochloric acid or Lactic acid. The acidity should be between 0.05 and 0.1 per cent. If not clear the medium is allowed to stand a day and filtered again. It is distributed into Frendenreich flasks (a few o.c. in each) and sterilized by heating ½ hour in the steamer (Koch’s) on three successive days.

To prepare a solid medium 1.5 per cent, agar is dissolved in the broth. The medium should have a more or less pale sherry tint and should not be reddish brown.

Media containing dunder are very dark and difficult to clear. The acid of dunder affects the solidifying power of agar. 10 to 15 per cent of gelatin may be used instead of agar but cultures must then be kept in the cool incubator at 20 to 22 C. The yeasts grow much better in the cane juice medium than in a purely artificial one. The oval budding yeast retains its vitality well in the cane juice broth for over a year. The fission yeasts die out more easily but living cells are present in fair numbers after twelve months.

Fermentation experiments are carried out in flasks containing 1 litre of wash and plugged with cotton wool. Washes with an acidity exceeding .7 per cent, are generally sterile after ½ hour steaming. The progress of fermentation is determined by daily weighing, the loss being taken as carbonic acid.

The following factors are determined in all experiments (Laboratory or distillery).

The wash should be quite dead and the yeast well settled. If yeast is suspended the spindle gives a reading 0.15 to 0.4 too high. After 48 hours the reading will be correct. The weight of sugar fermented is very closely double the loss of weight.

On estates the Arnaboldi or Jamaica Saccharometer is still frequently employed. It is corrected for 80 F. and gives a reading roughly half as high again as the Brix spindle.

Sending Yeasts To Estates.

At the start of crop the distiller gets a spontaneous fermentation in whatever liquor he can get. This is either skimmings, cane juice of low gravity and purity (rum cane juice) or, if he is fortunate, fresh juice from the first mill. Dunder left from the preceding season is often used to mix with the juice. This dunder often contains matters which inhibit or interfere with the growth of the yeast. It is improved by vigorous boiling with or without the addition of lime. As soon as he has molasses and fresh dunder he can set up a normal wash. If he has to start on skimmings they often contain very little or very feeble yeast, and easily get spoilt by bacteria which render them ropy or viscous. Much difficulty is therefore often experienced in getting a good start to fermentation. In any case the yeast which develops is the oval budding kind. If “acid ” is made and used from the outset in quantity the oval yeast frequently works badly and gives place gradually to the more suitable fission yeasts. In the meantime there may be loss by bad attenuation and accumulation of materials.

In sending yeasts to estates at the start of each crop the object of the Laboratory had been to get in suitable yeasts from the outset and curtail the period of uncertainty. For estates not making “acid” the oval budding yeast, and for estates making acid—one or both of the fission yeasts.

Yeasts were sent to a few estates in December 1907, and January 1908, to still more in December 1908 and January 1909, and to over twenty estates in December 1909 and January and February 1910.

The following estates got yeast this crop:—

Yeasts are required from the middle of December to the middle of February. The estates taking them later had really started weeks earlier. The number of Common Clean Estates willing to test the yeasts could doubtless be doubled for the crop 1910-1911. They would need to be circularized in November.

Preparation Of Yeasts For Estates.

The yeast is first grown in the Frendenreich flasks in the cane juice broth. Two or three transfers should be made when fermentation has almost ceased; ½ c.c. suffices after shaking up the liquor. This is done with sterile pipettes in the glass chamber after washing down the latter with 2 per 1,000 mercuric chloride solution,; 5 c.c. are then added to 60 c.c. sterile wash in small Pasteur flasks. After fermenting in these flasks for 3 to 4 days they are shaken up and the whole liquor poured into flasks plugged with cotton wool containing 1 litre sterile wash. When fermentation has nearly ceased these flasks are shaken and the liquor poured into large flasks containing 10 to 12 litres sterile wash. The wash is allowed to die completely and the yeast permitted to settle out (24 hours after wash is dead). The covering liquor is then poured carefully away and only sufficient left to give a thick muddy suspension when the yeast is is shaken up with it. The mixture is poured on to moistened filter paper in Buchner funnels and the moisture drawn out as effectively as possible by means of the Geryk air pump. The yeast and the filter paper are lifted out, wrapped in dry filter paper with a covering of glazed paper, packed in a small tin with cotton wool and mailed by Letter Post to the Post Office nearest the estate without delay. The estate must be advised to get the yeast working on the day of arrival.

The washes in the Pasteur, litre, and large flasks should, for preference, be set up from a mixture of molasses, dunder and water, using 1/3 to 1/2 dunder (according to its acidity and gravity). The gravity of the wash should be such that it will attenuate 12 if allowed completely to die. The Pasteur and litre should have added to them 0.2 per cent, asparagin, and the wash in the large flasks 0.1 to 0.2 per cent, ammonium citrate or ammonium sulphate. In the absence of molasses muscovado sugar or concentrated cane juice may be used. If neither dunder or molasses are available the wash may be set up with muscovado sugar and citric acid (1 per cent, of a gravity of 12 Brix). To this should be added .2 per cent, asparagin for the Pasteur and litre washes, and .2 per cent, ammonium citrate for the large flasks, .05 per cent, potassium phosphate should also be added.

Before adding the yeast the wash should be warmed to 30 C. The litre and large flasks should be packed round with saw dust or better fibre packing to keep up the temperature and make the fermentation more uniform. The flasks containing the litre washes should be weighed daily to judge if fermentation is vigorous and normal. If a yeast is to go to several estates within a short period a little may be kept back in the large flasks after decanting off the dead liquor and this will serve to start another large flask (or more than one). Before sending away, a little of the yeast should be examined under the microscope to observe if it is true to its type and free from living bacteria.

Directions For Working The Yeasts On The Estates.

Set up ten gallons of fresh wash in a clean keg; the wash to consist of dunder 1/3 molasses and water, and to be of such a gravity as to give an attenuation of 12-13 Brix (18-19 Arnaboldi) if the wash were allowed to die completely. The temperature should be 86-88 F. Stir in the yeast, cover the keg and allow to stand in a warm place. When this wash has lost 9-10 Brix (14-15 Arnaboldi) by attenuation, stir up properly and pour the entire liquor into 50 gallons fresh wash. When this has attenuated to a like extent stir up and pour the whole into 500 gallons freshly set wash. When this is working well (after 24-36 hours) make up to 1,000 – 1,200 gallons. A freshly set 1,000 gallon wash can be started again from that by adding to it 50- 75 gallons when the attenuation has fallen 9-10 Brix and after thoroughly stirring up. The yeast can be got through the distillery more rapidly by keeping back 10 gallons of the fermenting 50 gallon wash and using it to start a fresh 50 or 100 gallon wash in the same puncheon (with the head knocked out) which may be poured into 1,000 gallons when it has attenuated 9-10 Brix. Skimmings should not be used in setting up wash except in the last 500 gallons.

When circularising the estates they should be asked if they propose to use “acid” in the coming crop. Content, Kent, Cinnamon Hill. Running Gut, Ironshore, Gale’s Valley, and Swanswick have already employed the top fission yeast with success. Catherine Hall, Albion and Parnassus would be best suited with the oval budding yeast. Sevens, Spring, Appleton, Denbigh, Bog and Llandovery, Green Park and probably the Belleisle Estate Co. might get both oval and bottom fission yeasts. Orange Valley should get both fission yeasts. If two yeasts are sent together the distiller should be advised to grow- them separately in 10 and 50 gallons and then pour the two 50 gallon washes together into 1,000 gallons of ordinary estate wash. The yeast better adapted to the conditions would then get the upper hand.


Acetic Bacteria.—Forming a film on liquors containing alcohol oxidizing it to acetic acid. They can be isolated from dead washes, “acid,” etc., by means of cane juice peptone agar to which 2 per cent, of alcohol has been added. The commonest forms are B. kutzingianum (or an allied species), B. xylinum and B. xylinoides. The first named does not give the blue stain with iodine. It forms a blue to white delicate very friable ascending film and clouds the liquor strongly. (For experiments with this species see second S.E.S. Report and Laboratory records). B. xylinum develops the characteristic tough thick white skin on any nonfermenting liquor containing cane sugar and not less than 10 per cent, proof spirit.

B. xylinoides forms a very similar skin on “acid” and liquor containing over 10 per cent, proof spirit. One or more of these species are always present in fermenting cane juice or estate washes and cause a rise of acidity by forming gluconic acid from sugar. When the wash is dying and especially when it is dead they produce acetic acid.

Saccharobacillus pastorianus has also been found in fermenting washes and especially in soured skimmings and cane juice. It is present as long narrow rods often covered with small particles of matter deposited on them from the liquor. The liquor is strongly clouded and the bacteria cause the appearance of silky waves. This organism grows freely in the presence of alcohol and therefore increases with the yeast during fermentation. In cane juice broth it gave rise to 0.8 per cent, total acidity of which 30-35 percent, was volatile (acetic). The fixed acid is lactic acid.

“Acid. “—This is prepared either from skimmings or cane juice by allowing them to ferment and sour in special cisterns. As a rule trash is added to the liquor which on some estates is pumped into a succession of cisterns in each of which an increase of acidity occurs. The acidity of the ripe acid rarely exceeds 2.5 per cent, and of this as a rule less than 1/3 is volatile. The highest volatile acidity hitherto observed was 0.9 per cent, out of a total acidity of 2.1 or about 43 per cent. The liquor is fermented by yeast (oval or bottom fission) and the acid increases rapidly at the same time. This increase frequently stops attenuation when several per cent, of sugar is still present. Hence “acid ” shows a most variable gravity according to the relative activities of the yeast and bacteria. The amount of acid formed is the same whether attenuation has been good or bad. (See data in Laboratory records on Swanswick “acid.”) The trash not only infects the liquor with bacteria but increases aeration. It also seems to carry on a strong infection when fresh juice is added. No marked film forms on the acid so that the acetic bacteria do not have a chance to unfold their full oxidizing activities. B. xylinoides, B. xylinum and Saccharobacillus pastorianus have been isolated from ripe acid.

The acetic bacteria form gluconic and acetic acids. The Saccharobacillus form lactic and acetic acids. The fixed acidity preponderates. (See first and second S.E.S. Reports.)

Jelly and Slime forming Bacteria.— Certain species readily form jelly and slime in weakly acid liquors containing cane sugar. Skimmings and cane juice often undergo a viscous fermentation with the production of gas and the skimmings may frequently be drawn out into long threads (ropy skimmings). This condition interferes with the yeast fermentation which is prolonged and incomplete.

The liquor shows the presence of small cocci single, paired and less frequently in chains. The viscous or ropy condition of the liquor is due to the very diffluent cell walls of the bacteria. The acidity produced in cane juice does not exceed 0.3 per cent, a trace of which is volatile. The growth on cane juice agar is very moist and slimy but no slime is produced on ordinary glucose agar. In glucose broth the chains are very marked so that the organism is a true Streptococcus.

Growth is very rapid in cane juice at 27-40 C. A nonslimy variety has also been isolated. The condition is most marked at the start of crop in both cane juice and skimmings and is due to dirt from the cane and the mill. Owing to its slimy capsule the Streptococcus is not destroyed during tempering in the clarifiers. Thorough cleaning of gutters and skimmings boxes and diluting the hot skimmings with cold water have successfully checked this condition.

Rice Grain.—This occurs not infrequently in washes on estates where no acid is employed. The wash becomes almost filled with gelatinous spherical grains about 1 mm. to 2 m.m. in diameter. The fermentation by the yeast is prolonged and often incomplete. It is caused by a rather thick rodshaped bacterium 1.5-3m by 1 m. Three varieties of this organism have been isolated (see Laboratory records). It strongly resembles the Bacterium vermiforme of Marshall Ward (ginger beer plant). In cane juice the acidity does not exceed .2 per cent. It grows rapidly at 30 C. and just as well in cane juice with 6 per cent, of alcohol by volume as in cane juice without alcohol.

It produces no change in litmus milk and grows out into long more or less cocoid chains without gelatinous sheath in glucose broth. It probably enters the wash from the skimmings.

This organism evidently does not injure the yeast by means of its chemical products. Its interference is physical as the gelatinous masses attach themselves to the yeast cells and grow over them excluding the yeast from contact with the liquor and preventing the cells from rising and whirling in the wash, a condition necessary for active fermentation. A similar kind of interference must be attributed to the streptococcus of viscous ropy skimmings or juice.

Termo bacteria, B. subtilis and B. mesentericus vulgatus have been found in cane juice. These motile forms have not been closely investigated.

The ethers varied from 30,653 to 46,030, and consisted almost wholly of acetic ether. As the ether was formed at the expense of alcohol the yields must be regarded as satisfactory particularly that for the molasses wash to which ammonium sulphate was added. The acid of dunder appears to have an injurious effect on this yeast. Chemical esterification in such liquors could not account for the enormous amount of ether produced. It is evident that both alcohol and acetic acid are formed in the yeast cells by the enzymes, zymase, and oxydase (the latter probably the same enzyme as the oxydase of the acetic bacteria) and are at once brought into union by another enzyme, the whole process occurring within the cell. Certain acetic bacteria are known to yield a vinegar containing a marked amount of acetic ether while other species are quite unable to do so. A yeast is also known which oxydises alcohol to acetic acid and some nonfermenting mycodermas are capable of producing acetic ether in alcoholic liquors. The cells of some species contain, therefore, only the oxydase, others both oxydase and ether producing ferment (esterase). Some of the mycodermas and acetic bacteria which form films on dead washes in Jamaica distilleries may esterify in the way indicated although such forms have not as yet, been isolated in the Laboratory.

Experiments With Fission Yeasts In Dunder And Concentrated Cane Juice Wash.

The juice was boiled down to the consistency of thick syrup without any tempering. The dunder was derived from cane juice and dunder washes from which all alcohol had not been distilled out. This dunder, therefore, had undergone some souring, and was rather high in volatile acidity.—

The yeasts were first grown in a mixture of the cane juice and dunder without added water in sterilized flasks containing 1 litre.

Bottom and top fission yeasts were employed. The wash died in 6 days with bottom yeasts; the wash died in 7 days with top yeasts.

The yeast sediment was then added to 10 litres in large flasks: —

The bottom yeast washes were again dead in 6 days, and the top yeast washes in 7 days.

In this experiment both bottom and top yeasts gave identical attenuations and yields. In spite of the high volatile acidity (45 per cent, of the total) the washes were rapidly (6 and 7 days) worked down with little residual sugar and with excellent results on attenuation and sugar fermented. The rums were high in ethers. Even when distilled as soon as the wash was dead, the rum contained 971 ethers and when the dead wash was allowed to lie six days they were increased to 1404. In washes high in volatile acidity considerable esterification occurs during actual fermentation, and is again greatly increased when the dead wash is allowed to lie.

Grown in pure culture in sterile wash, the top yeast does not yield a rum of higher ether content than the bottom yeast.

Experiment With Poor Dunder.

Washes in litre flasks were set up with dunder water and muscovado sugar.

Oval and fission yeasts were used; the same amount of yeast was added to the sterile wash (in 1 litre flasks) in cash each. To some flasks 10 c.c. of 10% asparagin solution was added at the outset. The loss in weight day by day was as follows:—

The fermentation was normal active with both oval and  fission yeasts in the presence of asparagin. In the absence of asparagin the yeasts scarcely multiplied and the fermentation was very feeble. When, however, asparagin was added on the third day to the latter cultures multiplication set in, and 48 hours later they were fermenting strongly.

These results show clearly that washes set up with a poor dunder are often deficient in nitrogenous food for the yeast with the result that attenuation is feeble and incomplete. In practice such washes are quickly overrun by bacteria, show rapid increase in acidity and become still more unsuited for a vigorous yeast fermentation. In the experimental distillery washes set up with similar dunder worked and attenuated very feebly; 24 hours after addition of 0.15 per cent, ammonium sulphate (equal to 15 lbs. per 1,000 gallons wash) they began to work vigorously and showed a normal attenuation.

Distillery Experiment.

The following experiment selected from a number of such carried out in the Experimental Distillery in 1908, will show what the Jamaica fission yeast are capable of yielding under well regulated conditions.
The fission yeast was of the bottom fermenting type.

It was first developed in a molasses and dunder sterile wash in a flask containing 12 litres (nearly 3 gallons). The yeast was then added to 10 gallons of similar wash in a keg after sterilizing the latter with superheated steam and cooling it to 86 F. When fermentation was almost completed in the keg the contents were stirred up and the liquor added to 50 gallons fresh wash (not sterilized) in a puncheon. When fermentation has started a further 50 gallons wash was added and fermentation allowed to proceed till the wash was quite dead.

The was dead in from 4 to 5 days.

The wash was divided into two portions; the first was distilled for high and low wines (the retorts also receiving charges of wash). The high and low wines were used in toto to charge the retorts for the second distillation 42 gallons wash being introduced into the still.

Taking 6 gallons of rum for every 1° attenuation per 1000 gallons wash as an ordinary average yield 80.4 gallons rum would have been expected on that basis; the actual yield was however 7 per cent, in excess of that amount and must therefore be regarded as highly satisfactory. The above yields are expressed in imperial gallons. In terms of wine gallons the figures are 1-5th. higher, viz., ordinary average yield 96.5 wine gallons. Actual yield 103.2 wine gallons.

Observations Of Estate Materials.

The following determinations made on materials at the estate and on samples at the Laboratory illustrate the composition of dead washes, dunder, and “acids” produced in some “Common Clean” distilleries where rums are made containing 900—1200 parts of ethers per 100,000 alcohol. On such estates no “flavour” is employed so that the ethers in the rums consist wholly or almost wholly of acetic ether.

The acidity of the ripe “acid” varied from 1.9-2.4 per cent, and the gravity from 3.5-10 Brix.

The “Rice Grain” Bacterium.

This organism with remarkably gelatinized cell walls has been already referred to as causing trouble in “common clean” washes particularly in distilleries where only fresh materials are fermented and no “acid” is made.

A sample of dead wash containing the organism was after appropriate dilution plated out in cane juice peptone agar. After some days a variety of colonies including those of yeasts appeared in the medium. Three different types of more or less gelatinous colonies of bacteria could be distinguished.

1. Of no particular shape, raised into a mass and breaking through the agar by rupturing it. These colonies at the surface were smooth, milky, dull and pasty and easily rubbed into a milky homogeneous suspension in water. Such a suspension showed under the microscope small flat, or irregular spherical gelatinous grams about 18-20 microns in diameter and free from any tendency to coalesce with each other. The flat grains showed a more or less circular outline with alternating deep and shallow depressions as indicated in the figure. Embedded in the jelly near the ends of the arms formed by the main depressions and transverse to the surface were two more highly refracting rods separated by the secondary depressions. The almost spherical grains had a convoluted appearance with a fundamentally similar structure, the rods being also transverse to the surface at the ends of the involved arms. This condition was evidently the final state of development of the grain. The resultant appearance was due to the division of rods with gelatinous cell walls, having the property of gelatinous thickening on one side particularly.

The rods are coloured yellow by aqueous iodine in iodide with darker staining granules; the jelly is hardly tinged. The aniline stains colour the rods intensely but do not affect the jelly. The individual rods vary much in length especially in the spheres where they are elongated into threads exceeding 10 micron. The minimum length is 1.5 microns and the breadth about 1 micron. In cane juice peptone broth the organism increases to a finely granular loose deposit in three days at air temperature but multiplies markedly more rapidly at blood heat. The deposit is easily brought into suspension by snaking whereby the minute flocks (grains) are rendered just visible to the naked eye. The liquor overlying the deposit is practically clear. The liquid culture shows a similar appearance under the microscope as the agar material.

Perfectly free rods are not to be found, hence the clearness of the overlying liquor. The organism grows as rapidly and abundantly in cane juice broth containing 6 per cent, alcohol by volume as in broth free from alcohol. The increase of acidity in the broth (equal to 0.1 per cent, at the beginning) does not exceed 0.25 per cent, and only a trace of this is volatile. The acid formed is probably lactic. Gas production is absent or doubtful. In litmus milk the organism produces no change in fourteen days. In nutrient broth and in glucose broth (containing 0.5 per cent glucose) growth is slow with formation of a flocky white deposit. Under the microscope long chains of short rods (almost coccoid) are visible without gelatinous envelopes. The chains grow out from the rods embedded in the grains of the inoculation material, the individual cells being 1.2-1 .5 microns in diameter.

2. Spheres with rough (facetted) surface, translucent, shining, and gelatinous like the agar. The spheres break through the agar and split it into radiating rents. The masses are at least 1 Millimetre in diameter and as a rule from 1-2 m.m. The whole sphere can be lifted away on the loop of the platinum needle. In water it cannot be reduced to a homogeneous suspension but breaks into fragments of jelly. Under the microscope the fragments of jelly are very irregular. The structure is however, very similar (though greatly more irregular) to that of the grains of No. 1. The rods are either transverse at the ends of gelatinous arms or they may be equally gelatinized on both sides. By the rupture of the jelly, the rods often project freely. The length of the rods is very variable, long threads up to 50 microns being frequent. The diameter like No. 1 is almost 1 micron.

In cane juice peptone broth this organism increases by the formation of large irregular masses of jelly or by a gelatinous deposit difficult to raise and then breaking into lumps of jelly. The liquor is distinctly cloudy which is due to the presence of large numbers of free cells with or without gelatinous capsules. The cells are often paired and also form chains of three to ten or more cells. This form also grows equally freely in juice containing 6 percent, alcohol and behaves quite similarly to No. 1 in litmus milk, nutrient broth and glucose broth. The increase of acidity in cane juice broth is also under .25 per cent, and growth at blood heat likewise very rapid.

Grown in conjunction with a bottom fission yeast, both organisms increase freely and the fermentation is 1-2 days more prolonged than in pure yeast culture. The physical interference of the organism with the yeast has been already referred to.

3. Colonies on the surface, transparent, convex, watery (mucoid, not ropy), entire, round and shining. Where colony has broken to the surface a central gelatinous mass, showing under the microscope similar but less marked gelatinous fragments as No. 2 with rods similarly embedded.

The watery part of the colony under the microscope shows rods, single paired and chained with or without an indistinct gelatinous capsule equally developed all round the cells.

In cane juice broth the organism forms an abundant translucent deposit and the liquor is still more cloudy than with No. 2. When shaken up the liquor becomes opaque due to an abundant homogeneous suspension. The dimensions of the rods are the same as for 1 and 2. Its behaviour in litmus milk, nutrient broth and glucose broth is also quite similar. In cane juice broth with 6 per cent, of alcohol the growth was less rapid than in the broth alone. The increase of acidity in cane juice was also under .25 per cent.

The appearance of the colonies applies also to the cane juice agar slants. On this medium each of the three types shows its characteristic growth. It is evident that the three forms are varieties of the same organism. In No. 1 the development of the grain is much more limited than in No. 2. In No. 3 the jelly is less robust and more diffluent, and may be compared with agar which has lost its property of solidifying by heating in a strongly acid liquor. Reference has already been made to the strong resemblance particularly of variety No. 2 to Bacterium Vermiforme of Marshall Ward.

Orange Wine.

Enquiries from several sources came to the Laboratory in the autumn of 1907 and again in the spring of 1908, as to the best way of making orange wine by direct fermentation of the sweetened juice. No experiments had at that time been carried out in connection with the orange wine making and there appeared to be no literature on the subject. The so-called orange wine on the market appeared to consist of diluted rum flavoured with orange essence (or the essential oil from the rind), and highly sweetened. This was more in the nature of a cordial or liqueur and could not be regarded as in any way a true wine.

A preliminary experiment was therefore started in March and April 1908.

A bottom fermenting fission yeast was selected to carry out the fermentation owing to the known resistant properties of fission yeast in general. In order to acclimatize the yeast to a liquor containing a high proportion of citric acid it was first grown in a mixture of molasses, water, and citric acid; the composition was—

The yeast attenuated this wash in four days to 2 Brix, and while it was still working 100 c.c. was used to start a fresh wash prepared from orange juice.

The orange juice was obtained by squeezing the juice of ripe oranges with the rind entirely removed, through a linen cloth.

Gravity of juice—13.8 Brix.
Acidity of juice—1.08 per cent.

To 1,500 c.c. of this unsterilized must, in which cane sugar had been dissolved was added (as stated above) 100 c.c. of the fermenting wash containing the fission yeast. The gravity fell in 7 days from 23.5 to 0.5 Brix, and the final acidity was 1.18 per cent.

After allowing the greater part of the yeast to settle out, the still very cloudy wine was decanted off and bottled. The bottles were filled almost to the corks which were sealed with paraffin. The bottles stood at air temperature for 8 months during which the wine had become perfectly clear and of a dark sherry colour.

The wine had a pleasant aroma of orange and an agreeable though rather marked acid taste. The palatability of the dry wine was improved by the addition of 10 per cent, pure white cane sugar. After this addition the wine was readily appreciated when drunk alone and was also found to be very refreshing beverage when consumed with the addition of two parts of Soda Water. It was pointed out, however, that this wine was not so strongly flavoured as that made by orange growers, and this was attributed to the fact that the oranges had not been squeezed with the rinds still on. The usual practice was to cut the entire oranges into quarters and squeeze out the juice in a wooden press operated by hand. In this way a part of the essential oil contained in the outer rind was set free and entered the juice. To this juice it was customary to add a small proportion of lemon juice (a sample showed a gravity of 10.4 Brix and an acidity of 3. 25 per cent.) to improve the flavour and increase the acidity. White albion sugar was then dissolved in the juice until the gravity was raised to 22-24 Brix. To get this must fermenting a “starter” was then added.

This was prepared by mixing muscovado sugar with warm water to a gravity of about 15 Brix and allowing this to set up a spontaneous fermentation occasioned by the cells and spores of yeasts contained in the sugar. As soon as this was working strongly it was poured into the orange must. If a successful fermentation was set up in the must the latter worked for one to two weeks and finally stopped before all the sugar was worked out, or was intentionally stopped by decanting off the liquor from the yeast deposit. It was pointed out that this method of fermenting the must had some disadvantages namely:—

1. The “starter” spoiled the natural flavour of the wine owing to the characteristic taste of the sugar used.
2. Fermentation often failed in the must after the addition of the “starter” or the fermentation rapid at first fall away quickly and left a product containing insufficient alcohol and too much sugar. This cleared badly and often turned sour (vinegar).

A pure culture yeast acclimatized to orange juice at the Laboratory appeared therefore to offer the most promising solution to the problem. The fission yeasts, well suited to acid distillery washes do not give a pleasant aroma to fermented must. On the other hand a pastorianus yeast isolated from molasses was found to yield a product of very agreeable aroma. It has therefore been employed in the experiments detailed below. Some preliminary work with this yeast indicated that one or more substances contained in the rind of the orange exercised an injurious effect on it when present in the juice from the outset. Juice was accordingly prepared from the fruit after removing the rind. When fermentation was active the liquor obtained by squeezing the rinds separately was added in order to increase the flavour of the finished product.

The yeast was first grown in a wash of molasses, citric acid and ammonium phosphate, then in a mixture of that wash with increasing amounts of orange juice and finally added to the orange juice must. The juice as squeezed from the fruit showed—
Gravity—11. 85 Brix.
Acidity—1. 12 per cent.

The gravity of this juice was increased to 20 Brix by the addition of white crystal sugar, and 0.1 per cent, ammonium citrate added. To start this must one-tenth its volume of a fermenting juice was added which had been attenuated by the pastorianus yeast from 19 Brix to 8.3 Brix. Two days after fermentation began one-sixth of its volume of liquor squeezed from the rinds alone was added. The gravity fell from 20 Brix to 0.3 Brix in 11 days and the must was then dead. After the bulk of the yeast had settled the wine was bottled and kept at air temperature for 15 months. An examination of the perfectly clear dark sherry coloured wine after that period yielded the following figures:—
Acidity—0.64 per cent.
Alcohol as proof spirit—21.43 per cent.

The wine had a fine sherry like aroma and was very palatable after the addition of 10 per cent, cane sugar.

Another must fermented by the same yeast a week later was set up from a juice of—
Gravity—12.05 Brix.
Acidity—1. 18 per cent.

Sugar was added to raise the gravity to 20.8 Brix. This must underwent a more prolonged fermentation and ceased to work with an appreciable amount of sugar unfermented. The must attenuated from 20.8 Brix to 2.8 Brix in 24 days. It was then bottled and cleared very slowly. Fifteen months later the perfectly clear wine showed:—
Total acidity—1.10 per cent.
Volatile acidity—0.15 per cent.
Alcohol as P.S.—18.57 per cent.
In aroma and taste it scarcely differed from the other wine.

When fresh juice is allowed to ferment spontaneously it works slowly and finally dies before all the sugar is fermented. A white dry film usually forms on the surface consisting of mycoderma or a species of Monilia while Apiculatus yeast is often abundant in the deposit. The Apiculatus yeast can only ferment the invert sugar and leaves the cane sugar untouched. As the juice contains about half the total sugar as cane sugar the attenuation stops half way.

A juice worked for 6 days and attenuated from 11.85 to 6.45 and went no further. In another portion of the juice a little added fission yeast reduced the gravity from 11.70 to 1.75 Brix owing to the power of inverting the cane sugar.

When used in larger bulks (10-15 gallons) of sweetened orange juice prepared by pressing the oranges with the rind on, the pastorianus yeast has several times failed to yield a satisfactory fermentation, results which raised the question as to whether this yeast is really well adapted for working in sweetened juice as usually set up. Fermentation certainly sets in more rapidly and vigorously if the sugar is previously melted in hot water to a consistency of syrup and then raised to the boiling point after the addition of 5 per cent. citric acid. This causes the inversion of the bulk of the sugar. The first experiment indicates that the fission yeasts work readily in sweetened juice and it will probably be safer to employ such yeasts in spite of the fact that they do not yield such a good flavoured wine.

The data set out in this paper must be regarded as of the pioneering order and should prove useful as a start in the solution of the difficulties connected with the making of genuine orange wine.

Orange Vinegar.

This is a product with which the wine maker has often hail involuntary acquaintance. About 2½ gallons of an excellent vinegar have been made at the Laboratory in the following way:—
Juice was extracted from the fruit freed from rind.
The gravity was:—10.6 Brix.
Acidity—0.80 per cent.

To this was added sugar inverted by boiling with 2% citric acid. The gravity of the sweetened must was 16.5 Brix. It was pitched with the pastorianus yeast. After 9 days the liquor was dead and showed a gravity of 0.5 Brix. It was allowed to stand in a large flask with a loosely fitting cotton wool plug. After a few weeks an acetic film developed on the liquor and after a further month this had broken up and the liquor was fairly clear.

The total acidity was—5.35 per cent.
Volatile acidity—4.0 per cent.
equal to nearly 5 per cent, of acetic acid. The vinegar was rendered practically clear by filtration through cellulose (filter or blotting paper pulped in water).

Yeast Cultures In Cane Juice Peptone Broth.

Inoculated 20 May, 1910. Frendenreioh flasks.

1. Beer yeast from Jorgensen’s Laboratory, Copenhagen maintained in cane juice broth at Hope. Sets up a speedy fermentation after 12 months in the broth. Used in top fermenting breweries in Denmark. Has been employed successfully in Kingston in a wort of brown sugar, hops and water.
2. American whisky yeast—same source—dextrin fermenting power not tested.
3. Sacchs. thermantitonum—same source—an oval building yeast, with an alleged high optimum temperature for both growth and fermentation. This strain shows nothing striking in those respects.
4. Bottom fermenting oval buckling yeast—the typical yeast of cane juice and washes of relative low acidity. Has been sent to estates in 1908, 09 and 10.
5. Top fission yeast—has been three years in culture. Isolated from a Bluecastle wash. This culture has been used for supplying estates in 1908, 09 and 10.
6. Bottom fission yeast—three years in culture; originally from Mesopotamia wash. A typical bottom yeast.
7. Bottom fission yeast with slight top phenomena from Friendship wash—three years old. Has preserved its power of vigorous fermentation better than No. 6. Has been sent to estates as “bottom yeast” in 1909 and 1910.
8. Bottom fission yeasts—isolated in spring of 1910 from a sample of Swanswick “acid.” Its fermenting power not yet tested in 1 litre portions of wash.
9. Bottom fission yeast—from Long Pond “acid” in Spring 1910. Not yet tested.
10. Top fission yeast—from Long Pond wash, 1910. Not tested.
11. Top fission yeast—from Swanswick wash 1910. Not tested.
12. Sacchs. ludwigii—from Long Pond “acid” 1910 apparently top fermenting.
13. Same as 12—from a different plating.
14. Chained budding yeast—fron Swanswick “acid”. Not investigated.
15. Narrow oval budding yeast—from Long Pond “acid.” Not investigated—may be a variety of No. 4.
16. Pastorianus yeast—from molasses—three years in culture. Used in “orange wine” experiments.
17. Budding yeast—from Parnassus and Moneymusk discoloured crystal sugars. Very abundant in sugars. May be a Torula identical with the torula causing foaming of stored molasses. Does not ferment cane sugar.
18. Willia anomalous (“Anomalus” fruit ether yeast) three years in culture from molasses. This culture was used in experiments with the fruit ether yeast.
19. Mycoderma sp.—from Long Pond dead wash.
20. Mycoderma sp. mixed with B xylinoides—from Swanswick “acid” (See remarks on “ester formation”).
21. B. xylinoides—from Long Pond ”acid” About 1% alcohol was added to the broth.
22. B. xylinoides—from Swanswick “acid.” Alcohol added to broth.
23. Oval yeast and Rice grain variety 2.
24. Large oval yeast—three years in culture.

Literature Of “rum” And “fermentation.”

Rum.—Literature very scanty.
In Library:—
Uber Brauntwein .. Eugene Sell.
Articles by P. Greg (Mesopotamia Estate) in Bulletin Botanical
Department, Jamaica.
—Vol. 2. pts 3, 8, 0.
—Vol. 3. pt. 1.
Sugar Experiment Station Reports 1 and 2.
Bulletin Dept. Agr. (new series) Vol. 1. Xo. 1.
Bulletin Dept. Agr. (new series) Vol. 1. No. 3.
Regarding “artificial rum” see
—Rum Arrack etc., by Gaber.
—Report Whisky Commission 1908.
Fermentation —

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