INDUSTRIAL AND ENGINEERING CHEiMIXTRY
1172
the laboratory and some mechanical difficulties were encountered. A change in construction and operation routine has now made it possible to operate the plant smoothly and the yield has equaled or bettered those obtained in the laboratory. The present estimate is that from 5 to 10 cubic feet of gas can be obtained per pound of cornstalks, and that the rate of production will be from to 1 cubic foot of gas per day per cubic foot of tank volume. Taking the lower figure, a ton of cornstalks would furnish gas for 400 people for one day, allowing 25 cubic feet per capita per day. From the data given by Webber (12) for yields from regions where 30 per cent of the land is planted to corn, an area with an 8-mile radius will produce enough cornstalks to supply a city of 80,000 inhabitants with gas continuously. I n other words, the cornstalks from one acre will produce the gas for one person for a year. Saturally, the bacteria require some nitrogen, and this may be supplied from domestic wastes. I n the experiments discussed above the digestion was not complete. The pith and finer fibers are digested first, leaving behind that portion of the cornstalks which is most valuable for paper-making. According to Sutermeister (11) the removal of the pith is a serious handicap in the manufacture of cornstalk paper. If the pith is removed by digestion, with the production of methane, the process should be more profitable. The volume per pound is decreased by 25 to 30 per cent as the result of removing the pith. This is considered an advantage in paper-making. As intimated above, the more fibrous and resistant portions of the stem are too slowly attacked to be allowed to remain in the digestion tank. This residue is the most desirable portion of the stalk for the manufacture of wallboard and paper. I n fact, the first step in the production of either wallboard or paper is to remove the pith. It is probable that the anaerobic fermentation may serve instead of the usual cooking process to prepare the fibers for subsequent use in manufacture.
1‘01.
22, No. 11
As the percentage of carbon dioxide in the gases from cellulose digestion is much higher than that in the usual commercial sources of carbon dioxide (7, 8) its recovery would seem feasible. Conclusion
It is believed that the completion of some development work now in progress will make it possible for farms and ranches to install digestion tanks in which various crop residues may be converted in considerable amounts to a gaseous fuel of high heat value. The undigested residue could be composted and returned to the soil. The operation can be combined wit,h other routine farm work in such a way that the cost of the gas should compare favorably with city gas prices. It is also probable that small towns located in the corn belt could be supplied with gas in t’he same way. I n this case the undigested residue would be baled and shipped to a nearby wallboard or paper mill. As our coal, oil, and gas supplies become exhausted the installation of pipe lines fed by fermentation plants located along them a t short distances would seem the most probable line of development. Literature Cited (1) Boruff, “Anaerobic Fermentation of Cellulose and Cellulosic Materials,” University of Illinois, Ph.D. Thesis, 1930. (2) Boruff with Buswell, IND. ENG. CHEM.,21, 1181 (1929). (3) Boruff with Buswell, Ibid., 22, 9 (1930). (4) Buswell, Ibid., 21, 322 (1929). (5) Elder with Buswell, Ibid., 21, 560 (1929). (6) Fowler, J . I n d i a n Inst. Sci., 3, 39 (1920). (7) Killeffer, I N D .E N G . CHEM.,19, 192 (1927). (8) Martin, Refrigerating Eng., 16, 33 (1928). (9) Neave and Buswell, J . A m . Chem. Soc., 62, 3308 (1930). (10) Sen, Pal, and Gosh, J . I n d i a n Chem. Soc., 6, 673 (1929); C. A . , 24,. 642 (1930). (11) Sutermeister, “Chemistry of Pulp in Paper Making,” Wiley, 1929. (12) Webber, IND.E N G .CHEX, 21, 270 (1929).
Some Minor Industrial Fermentationslr2 0. E. May and H. T. Herrick COLORA N D FARM WASTE DIVISION,BUREAUOF CHEMISTRY A N D SOILS, WASHIXGTON, D. C.
U S G I are utilized ind u s t r i a l l y to bring
F
A review is given of the production of fermentation of citric, gluconic, and gallic acids, and glycerol. Raw materials, methods of fermentation, and yields are given, together with theories concerning the mechanisms of the reactions concerned in the processes.
about t w o g e n e r a l types of chemical reactions, oxidation and h v d r o l v s i s . The first type is -exem;lified by the citric and gluconic acid fermentations, in which the fungus mat rests on the surface of a liquid substrate and brings about the oxidation of the sugar through the agency of intracellular enzymes. The manufacture of gallic acid from tannin is a good example of the second type of reaction. Here the organism secretes the enzyme tannase, which hydrolyzes the tannin to gallic acid. Citric Acid
Wehmer, around 1890, was the first to recognize the microbiological formation of citric acid from sugars (25). He isolated pure cultures of a species of fungus which showed unusual activity in the production of citric acid from sucrose. Since the organism did not fit exactly into the morphological Received October 4, 1930. 184th Contribution from the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S.Department of Agriculture. 1 2
classifications for the genera Aspergillus, Penicillium, or Mucor, Wehmer gave it the a p p r o p r i a t e name “Citromvces.” Considerable work was d o n e b y Wehmer and others on the citric acid fermentation, and a summary of this early work may be found in Lafar’s “Technischen Mykologie” (15).
Largely as a result of the attempts to classify fungi on the basis of biochemical behavior, it was assumed for several years that the vigorous and wide-spread strains of the black and brown Aspergilli were exclusively oxalic acid-forming organisms. Nevertheless, Zahorski in 1913 obtained a patent on a biochemical process for the production of citric acid from sugars, utilizing a strain of Sterigmatocystis nigra, a name often used in the literature synonymously with A . niger ( 2 7 ) . Within the next few years Thom and Currie, in a series of experiments, showed conclusively that the black Aspergilli were capable of producing appreciable quantities of citric acid under definite conditions of culture ( 2 3 ) . Shortly thereafter Currie undertook what i s coming to be considered a classic investigation of the factors controlling the production
Kovember, 1930
INDUSTRIAL A-VD ENGINEERING CHEMISTRY
of citric acid by a srlected strain of A . niger (8). By using the proper concentration of sucrose (15 per cent), supplying the nutrient nitrogen in the form of low concentrations of ammonium nitrate, and adjusting the initial p H to 3.5 by the addition of hydrochloric acid, Currie was able to suppress the forination of oxalic acid and obtained a rapid fermentation with no lois of citric acid as long as any sugar remained in the culture -olntion. The more important of the recent publications concerning tlie formation of citric acid by fungi deal mainly with factors influencing the fermentation and with attempts to gain some knowledge of the mechanism of the reaction. The fact that a branched-chain compound is formed from sugar- in which no such hranclied structure exists is of great theoretical interest and has occasioned a good deal of speculation. Thus far no agreement has been reached concerning the inechanism of the reactions. There are t x o general Tiens-one being that the citric acid results from a chain of reactions represented by the change- hexose + gluconic acid + saccharic acid + 37-diketo adipic acid --+ citric acid, the other that it is synthesized from reactive substances of lower niolecular weight rcmlting from the fragmentation of the sugar molecule. There is a considerable body of evidence to support each hypothesis, but more work is needed before any positil-e statement can be niade concerning the mechanisin of the fermentation. One interesting fact has been apparently established, hov ever, by several investigators n-orking independently. I t is that higher yields of citric acid are obtained froin fructose or from disaccharides capable of yielding a fructose inolecule on hydrolysis. Sucroqe has been found consistently to give higher yields of citric acid than most of the other carbohydrates, and Bernhauer has suggested that the peculiar structure of fructose in the sucrose molecule may have wine significance. d bibliography of most of this work has been compiled by Fulmer and Werknian ( I S ) . It is known that citric acid is being manufactured on a large scale in this country by t h r mold fermentation of sucro5r. The details of operation have not been niade public, but the process undoubtedly consiits of a shallow-pan fermentation of sucrose by a strain of A . niger, the reaction being completed in probably less than 9 days. The patent literature concerning this ferinentation, whik revealing little of plant operation, is iiitcresting. I t dates back to 1894, TT hen Kehiiier, recognizing the industrial possibilitiei of his discover\- of the citric acid fermentatinn, secured broad patents covering the procew ( 2 6 ) . The industrial application of Weliner's patent lvas un5uccessful. X a n y difficulties were encountered. one of the most troulilesoiiie being that the organism attacked the citric acid as soon as it reached a concentration of around 8 per cent. I n order to overcome this, calcium carbonate had t o be added to the fermenting solution. However, it was then found that the resulting neutral solution not only retarded the rate of fermentation, but n as very wsceptible to infection by yeasts and bacteria. The coinhination of the length of time required for the production of paying quantitie.: of tlie acid and trouble from infection- by other organisms proved too coitly, and the process was abandoned. In 1913 Zahorski was granted a patent for the production of citric acid from sugars by the fungus Sterignzafocysfis nigra. This organism was claimed to have a good resistance to high concentration. of citric ac4d and to give higher yields of acid tliaii had previously been obtained ( 2 7 ) . Falck has patented a process for the production of acids based on the use of a solid starchy substrate and organisms of the Aspergillus, Pcnicilliuni, or Citroniyces groups ( I O ) . The production of citric, malic, tartaric, and succinic acids is claimed, but 110 details as to methods of recovery or individual yields of the acids are given. Bleyer has included the idea, developed by Currie, of a preliminary acidification in the specifications of
1173
a patent granted to him ( 3 ) . The acidity was brought to p H 3.3 and sterilization then effected by heat. The desirability of introducing an abundant supply of sterile air to the fermentation pans for the purpose of holding the temperature constant and eliniinating carbon dioxide is also mentioned. Szilcs, in a patent granted in 1928, claims the production of citric acid from molasses by fungi of the Citromyces, Mucor, Aspergillus, or Penicillium groups ( 2 2 ) . With a strain of Aspergillus it Tvas clainied that temperature? around 20" C. suppressed oxalic acid formation, inhibited infections, and gave high yields of citric acid. Ferribacli and Tuill have patented a process in Tvhich the dark Aspergilli are used a3 the fermenting organisms. The novelty of this process lies chiefly in the elimination of sterilization by heat through the addition of sufficient hydrochloric or sulfuric acid to the sugar solutions to give a pH of around 1.8 (12). According to the specifications, this treatment inhibits the growth of foreign yeasts, bacteria, and molds nithout affecting tlie activities of the citric acid organism. Recently Cahn, according to the patent specifications, has succeeded in producing citric acid in good yields in a very short fermentation period by the mold fermentation of sections of sugar-containing plants, such as sugar beets and artichokes ( 5 ) . Good yields of acid were also obtained by inoculating plant residues, from which the sugar had beenextracted, impregnatedvith sugar solutions. I n a recent paper Amelung has reported some experiments which may have a decided bearing on the industrial utilization of the citric acid fermentation ( 1 ) . He investigated the production of the acid in deep layers of sugar solution by the submerged growth of A . niger. Air in finely divided bubbles \vas forced through the solution, and weight yields of citric acid as high as 19 per cent were obtained in 40 days. The rate of fermentation is, of course. too slow to make the process one of immediate industrial interest, but the fact that appreciable quantities of citric acid were formed by a yeastlike growth of a characteristic surface-growing organism is quite significant. The writers attempted to produce gluconic acid by such a process in 1926, and have repeated the experiment frequently siiice that time under varying conditions with discouraging results, little acid being produced. I n view of the large quantity of information concerning the citric acid fermentation available in the journal and pateiit literature, it might be wondered why tlie industrial production of the acid has not assumed larger proportions. The chief reason is the difficulty in transforming the laboratory fermentation into a working plant process. The problems involved in the operation of such a process are those connected with the organism and the engineering difficulties inlierent in a shallowpan fernleiitation by a surface-growing organism. The chief requisites for a satisfactory organism are good vegetative and biochemical vigor, ability to withstand sudden variations in environment, and ability to maintain liiochemical characteristics in artificial culture. Tht. engineering difficulties center around the probleni of sterilization and handling of large numbers of comparatively small pans and other accessory equipment. The capital involved in the production of citric acid in large quantitiei by fermentation must be considerable, which, coupled with the uncertainty of biological processes, makes the prospects for tlita conservative investor somewhat unattractive. That the problem is no small one is attested by the fact that apparently x-ery few industrial concerns have succeeded in producing citric acid by fermentation in quantities large enough to be noticeable in the inarket-. Gluconic Acid
The discovery of the microbiological production of gluconic acid is credited to Boutroux, who in 1878 isolated an acid from the culture solution of Mycoderma acefi on dextrose which he
1174
INDUSTRIAL A N D ENGINEERlNG CHEMISTRY
concluded was lactic but which two years later he recognized as gluconic acid (4). It has since been frequently found in culture solutions of bacteria on dextrose (6). I n 1922 Molliard isolated the acid, along with citric and oxalic acids, from cultures of A . niger on sucrose, and since then it has been found by several investigators. During a survey of the action of fungi on solutions of commercial dextrose, the authors observed the formation of gluconic acid in good yields by a strain of Penicillium. A study of some of the factors governing the fermentation resulted in obtaining consistent yields of around 60 per cent of theory when carried out on a laboratory scale. These yields, together with the fact that no citric or oxalic acids were formed during the fermentation, resulted in an investigation of the possibilities of placing the fermentation on a semi-plant basis. The main difficulty was in finding fermentation pans of suitable material. The chief requirements for such a material were resistance to corrosion, non-toxicity to the organism, low cost, and durability. Preliminary tests indicated that nickel, lead, copper, and monel metal, while acid-resistant and durable, were markedly toxic to the organism. Zinc, iron, and to some extent the ordinary grade of aluminum were corroded by the acid. High-grade acid-resistant enamel was quite satisfactory except that pans coated with this material were heavy and unwieldly and also expensive. If the enamel were chipped or cracked through careless handling, the pans would be useless and would have little scrap value. Finally, pans made of a high-grade aluminum were obtained. This metal contained 99.45 per cent aluminum and less than 0.1 per cent copper and manganese. With pans 43 by 43 by 2 inches made of this material in a battery of seven, several successful fermentations were carried out with yields averaging around 57 per cent of theory (15). Inasmuch as only one unit of seven pans was used, there still remain the difficulties involved in the handling and sterilization of hundreds of units if the process were put into actual plant operation. The process, however, has industrial possibilities, even though, unlike citric acid, it must compete with acid obtained by the chemical oxidation of dextrose. The formation of gluconic acid does not involve the complex series of reactions occurring in the fermentation of sugars to citric acid. Muller has isolated from Aspergilli and Penicillia an enzyme capable of transforming dextrose to gluconic acid, which he has termed “glucose-oxidase” (17). Bernhauer (2) also obtained experimental results from which he concluded that the formation of gluconic acid by fungi was a strictly enzymic process induced by an oxidase which he termed “gluc-oxidase.” He noted that toxic materials added to the fermenting solutions in concentrations sufficient to inhibit vegetative growth did not interfere with gluconic acid formation by a mycelium already developed but completely suppressed citric acid formation, showing that fundamentally there is a wide difference between the mechanisms of the reactions leading to the formation of the two acids. It is also worth noting that, in addition to gluconic acid, oxalic acid was produced by the enzymes obtained by triturating the mycelia of A . niger with calcium carbonate. Citric acid was, however, never found in these experiments. Gallic Acid
Unlike the production of citric acid and gluconic acid by fermentation, the preparation of gallic acid from tannin is an instance of the hydrolytic action of certain molds. It is, moreover, distinguished from these two fermentations by the fact that the mold ferment will act as efficiently in the absence of the living organism as when it is present. The parent substance, tannin, is found in a number of different plants, prominent among which are varieties of oak and sumac, but its
Vol. 22, No. 11
most important source lies in the gallnuts which are produced on sumac and oak as a result of the stings of insects. Apparently there are three broad general classes of tannin, each one of which produces a different material upon hydrolysis, but the largest group and the one with which we are most concerned includes the gallo-tannins, which produce gallic acid upon suitable treatment. The structure of these tannins has been the occasion for considerable dispute, and the matter is still open to discussion. Apparently, however, gallotannin is a glucoside of gallic acid in some form or other, the number of molecules of gallic acid varying according to the theory of the investigator. Gallic acid itself was first discovered by Scheele some time prior to 1787 (20). He noticed that a cold-water infusion of gallnuts had deposited a sediment which, although it gave a black precipitate with iron sulfate, was not identical with the original tannin because it had a sour, rather than an astringent taste. Wishing to study this product further, he pulverized a pound of gallnuts and extracted the powder with water. After allowing it to stand for a brief period, he filtered off the residue, placed it in a flask, and allowed it to stand exposed to the air for several weeks. At the expiration of this period he noticed that a thick mold pellicle covered the surface and that, while there was no sediment, the solution itself did not taste so astringent as formerly. A second examination, following the expiration of a further period of several weeks, gave evidence that the pellicle had increased in thickness and that there was now a deposit on the bottom of the flask, while the astringent taste had entirely disappeared. Scheele filtered off the sediment and examined it very thoroughly by the methods which were available at that time. A study of his results gives abundant evidence of the fact that the material, which he named “gallnut acid,” was the substance which we now know as gallic acid. Without attempting to review thi! literature on this subject thoroughly, we will take up the next important milestone, which is a paper by Van Tieghem (24). This investigator made a thorough study of the matter and reached a number of conclusions, some of which were correct and some erroneous. Apparently there had been some discussion as to whether the formation of gallic acid from a solution of tannin was the result, of an air oxidation or that of a soluble ferment already present in the tannin or whether there was another agent responsible for the transformation. He succeeded in proving that a solution of tannin prepared from gallnuts remained unchanged in the absence of air and also that, when the solution was thoroughly sterilized and only air admitted, no change resulted. He then proceeded to demonstrate that there were two groups of molds, Aspergillus niger and Penicillium glaucum, which would produce gallic acid by their growth on a tannin liquor. He also showed as a corollary that air was necessary for the growth of both organisms. I n this investigation, however, he reached the mistaken conclusion that the enzyme produced by the organism would not operate except in the presence of the living plant and that the reaction was therefore presumably intracellular. Several years later two other investigators, Fernbach (11) and Pottevin (19), took up the matter independently, but about the same time, and reported that the enzyme tannase could be produced by the growth of Aspergillus niger on solutions containing tannin and suitable nutrients and could then be separated and used to hydrolyze solutions of pure tannin to gallic acid, The results of these two investigations prove conclusively that the hydrolysis of tannin to gallic acid is an extracellular reaction and therefore of a very different type from those hitherto mentioned. A further interesting step in the develbpment of t h e production of gallic acid from tannin is evidenced in the patent obtained by Calmette (6). I n this patent Calmette reviews
November, 1930
.
INDUSTRIAL AND ENGINEERING CHEMISTRY
briefly the previous industrial processes for the manufacture of gallic acid from tannin by fermentation. This transformation up to that time had been carried on by allowing a moistened mass of fresh gallnuts to ferment in the air without taking any particular precaution for the sterility of the fermentation or the identity of the organism. Calmette claims that the yield by this method is not particularly satisfactory and the fermentation period much too long, and outlines a very different procedure. According to him a specific organism, which he names “Aspergillus gallomyces,” is found in small quantities in all gallnuts. By suitable methods he prepares a pure culture of this organism, claiming that it will do the work more efficiently than any other mold previously employed. He then gives a method of operation which is radically different from any employed previous to that time. He places the clear tannin extract, which may be obtained apparently from any one of a number of raw materials, in a sterile tank equipped with an agitator and with a means for introducing air a t the bottom, and sterilizes the liquid by the introduction of steam. After this liquid is cooled to a temperature between 35” and 42” C., he inoculates it with a culture of his Aspergillus gallomyces and allows the fermentation to proceed. An essential and very interesting part of his patent is the fact that the organism is kept submerged during the fermentation, both by means of a mechanical agitator and by the introduction of large quantities of sterile air. Samples of the mixture are taken from time to time until analysis shows that the tannin has entirely disappeared. The fermentation is then terminated and the gallic acid recovered in the usual manner. The industrial production of gallic acid in this country is no novelty, as it has been placed on the market by several firms for a number of years. It is well known, however, that the usual method for the production of this substance is a chemical one, involving an acid or alkaline hydrolysis of a tanninbearing material and the subsequent recovery of the gallic acid by purely chemical methods. Though no definite data are obtainable on the subject, it is entirely probable that the gallic acid produced in Europe was made by a fermentation process, since many references to such a process are found in the literature. It is understood that a t least one concern in this country has been producing gallic acid by fermentation during the last few years. The details of operation are carefully guarded secrets, and very little can be said about them at this time. It is entirely improbable that the crude process mentioned in the European literature is employed, and it is much more likely that the gallic acid is produced in shallow pans, as is citric and gluconic acid, or by some variation of the Calmette patent. This patent gives rise to some exceedingly interesting speculations. The difficulties inherent in a shallow-pan process are obvious. High labor cost, small production per unit of operations, and expensive apparatus are the first objections that come to mind. On the other hand, a fermentation process based on principles similar to those outlined in the Calmette process offers advantages which are obvious to anyone giving the matter the slightest consideration. As a matter of fact Amelung’s ( 1 ) work along these lines has already been mentioned. Gallic acid finds a number of uses in industry. During the war, when the supply of German indigo and German vat dyes was cut off and before the development of such products in this country, gallic acid was used in the production of gallocyanine, with which the sailors’ uniforms were dyed. At present it is the basis for making a very fast dyestuff known as alizarine brown, which is used for dyeing woolens and for printing on cotton. When condensed in sulfuric acid it also yields hexahydroxyanthraquinone, giving a synthetic entrance into the anthraquinone series not generally appreciated. With ferrous sulfate it forms the permanent black constituent of writing
1175
inks, is used as a remedy in skin disorders as the subgallate of bismuth, and as the starting point for pyrogallol, which is a very important photographic developer. Glycerol
The industrial production of glycerol by the yeast fermentation of carbohydrates may be said t o be a by-product of fundamental biochemical research of a strictly non-utilitarian nature. I n the middle of the nineteenth century Pasteur, in the course of his classic investigation of the alcoholic fermentation, recovered glycerol in comparatively small quantities from the fermentation products, but its occurrence could not be explained satisfactorily owing to the lack of knowledge of the chemical processes involved in the transformation of sugar to alcohol. As the result of the work, begun in 1910, of Neubauer and Fromherz, Xeuberg, Fernbach, and Schoen, Muller-Thurgau and Osterwalder, it was definitely established that pyruvic acid, CH,CO. COOH, is an important intermediate in the alcoholic fermentation, that it is decomposed by the enzyme carboxylase into carbon dioxide and acetaldehyde and that the latter product is the immediate forerunner of alcohol (21). It was also observed that when the acetaldehyde was fixed by the addition of sodium sulfite to the fermenting solution increased quantities of glycerol appeared. This discovery was utilized on an industrial scale in Germany and Austria-Hungary during the Great War because of the acute shortage of fat stocks in those countries. The details of the process were made public by Connstein and Ludecke ( 7 ) and by Zerner (28) after the cessation of hostilities. The process as used in Germany was based on the following procedure: One kilogram of sucrose, 50 grams of ammonium nitrate, 7.5 grams of dipotassium phosphate, and 400 grams of sodium sulfite were dissolved in 10 liters of water, 100 grams of fresh yeast were added, and the fermentation was allowed to proceed for from 48 to 60 hours a t 30” C. The fermented mixture was distilled to remove the easily volatile constituents, the residue treated with lime and calcium chloride to remove the sulfite, and the excess calcium removed with sodium carbonate. The liquor, consisting chiefly of a solution of glycerol and sodium chloride, was distilled under reduced pressure to give a technically pure glycerol. I n some cases the finished product was contaminated with small quantities of trimethylene glycol, the presence of which was ascribed to the action on the glycerol of bacteria in the mash. Fermentation of 1 kg. of sucrose produced from 200 to 250 grams of glycerol, 300 grams of alcohol, and 50 grams of acetaldehyde. KO difficulty was experienced in carrying out the process on a large scale, and during the war a technical grade of glycerol was produced a t the rate of more than 1000 tons per month. According to Henneberg one plant used 60 tons of sugar and 6 tans of yeast daily, the fermentation being carried out in iron vats each of about 125,000 gallons capacity (l/t). Shortly after the entry of the United States into the World War, the problem of the production of glycerol by fermentation was assigned to federal chemists for investigation. Eoff, Linder, and Beyer succeeded in fermenting blackstrap molasses in the presence of sodium carbonate to give promising yields of glycerol (9). They found it advisable to use a special strain of yeast. Molasses solutions containing from 17 to 20 grams of sugar per 100 cc., to which a small quantity of ammonium chloride was added, were used. Sodium carbonate was added a t intervals during the fermentation, the final concentration not exceeding 5 per cent. Twenty to twenty-five per cent of the sugar originally present mas transformed into glycerol, and no difficulty was experienced in recovering a satisfactory grade of technical product. A mechanism expressing the reaction leading to the forma-
IAVDUSTRIAL A,VD EXGINEERIAVG CHEMISTRY
1176;
tion of glycerol in yeast fermentation which does not conflict with experimental facts, has been put forward by Neuberg (18). He conceived the yeast fermentation of glucose as being capable of taking place in three different ways, depending on the conditions of culture. All three of these forms involve the breakdown of the hexose into two molecules of hydrated methylglyoxal, which is then transformed to pyruvic acid, setting free active hydrogen according to the equation: C6H1206 +2CHaCO CH(0H)z +2CH3CO COOH
+ 2H2
The pyruvic acid is then attacked by the enzyme carboxylase, breaking it down into acetaldehyde and carbon dioxide as folloTT’s: CH3 CO.COOH
+CH3 CHO + COz
I n a normal alcoholic fermentation the acetaldehyde is then reduced b y the active hydrogen, giving ethyl alcohol, the complete process being represented by the following equations : C6H12O6 + 2CH3CO. CH(0H)z + 2CH3, CO . COOH 2CH3, CO. COOH +2CH3. CHO 2C02 2CH3, CHO 2Hz +2C2Hs. OH
+
+
C6H12Oe
--+ 2ClH,OH
+ 2Hz
+ 2C02
I n the presence of sodium bisulfite, for example, the aldehyde is fixed and removed almost completely from reaction with the hydrogen, which then attacks the methylglyoxal, giving rise to glycerol, according t o the equations:
__
CnHI>Or+2CH3 CO CHiOH), +CH,” CO COOH 4- H7 CH3”CO COOH CH3 CHO-+ C 0 2 CH3 CHO NaHS03 +CH3 CHO HS03Sa CH3 CO CH(0H)z HA+CH3Hj(OH)3
+
+
I n the presence of alkalies a third series of reactions occurs in which two molecules of the aldehyde, following CanniZaro’s reaction, undergo mutual oxidation and reduction, giving rise t o a molecule each of et’hylalcohol and acetic acid, thus permitting t’lie active hydrogen to react Tvith methylglyoxal to form glycerol, according to the equations: 2CBH1206 +4CH3. CO. CH(0H)z +2CH3. CO. COOH+2H> 2CH3, CO , COOH +2CH3. CHO f 2COa
Vol. 22, No. 11
+ CH3. COOH
2CH3. CHO +CzHsOH 2CH3. CO. CH(OHj2 2H2
+
2C6Hiz06
+2C3Ha(OH)3
+2CsHj(OH)3 + CzHbOH
+ CHJCOOH + 2C02
These mechanisms express quite well what actually takes place in yeast’ fermentations under varying conditions. It should be st’ated, however, that the existence of methylglyoxal in any of its many labile forms in the alcoholic fermentations is largely hypothetical, as it has never been definitely isolated in quantity in the course of fermentation studies. The extent to which the fermentation process for the production of glycerol is being used today is not known definitely, but it is certainly much less than a decade ago, owing to the comparatively plentiful supply of fat products and to the increased use of synthetic glycols. Nevertheless, the process is available and m u l d undoubtedly’prove very valusble in time of emergency or if the world were faced with a depleted supply of natural fats. L i t e r a t u r e Cited (1) (2) (3) (4)
Xmelung, Chem.-Zlg., 6 4 , 118 (1930). Bernhauer, Z . phjsiol. Cizem., 177, 86, 270 (1928). Bleyer, German Patent 434,729 (1936). Boutroux, Compl. r e n d . , 86, 60.5 (1878); 91, 236 (1853). ( 5 ) Cahn, French Patents 675,236, 675,237 (1929). (6) Calmette, Gerrnm Patent 129,161 (March 1, 1902). (7) Connstein and L.idecke, Bev., 62, 1383 (1919). (8) Currie. J . B i d Chem.. 31, 15 (1917). (9) Eoff, Linder, and Ueyer, J . 1x0. Esc. CHEI., 11, 8 4 2 (1919). (10) Falck. G e r m i n Patent 426,926 (19261. (11) Fernbich, Compt. r e n d , 131, 1214 (1933). (12) Fernbich and Yuill, U. S. Patents 1,631,965, 1,691,966 (1938). (13) Fulmer and W e r k m m , “Chemical Action of hlicro5rgnnisms,” p . 6, 129, Charles C. Tnomas, Springfield, I l l , 1930. (14) Henneberg, “Handbuch der Garungsbacteriologie,” Val. 11, p . 385, Berlin, 1926. (15) Lafar, “Handbuch der Technischen hlykologie,” Vol. I\’, p . 242, Jena, 1905-7. (16) XIiy, Herrick, Moyer, and Hellbach, IND. Exc. CHBM.,21,’1198 (1929). 117) Muller. Biochem. Z.. 199., 136 (1928): . . 205.. 11 (1929). . (181 Seuberg, I b i d . , 96, 175 (1919); 98, 141 (1919); 106, 281 (1920). (19) pottevin, compt. r e n d . , 131, 1215 (1900). (20) Scheele, Creil’s chem. .Ann., I, 3 (1787). (21) Schweizer, Chimie et industrie, 6, 149 (1921).
iii; 2:i t2i)
(231 126) (27) (28)
Van, Compl,
(1916),
6 6 , 1091 (1867),
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[ E N DOF SYMPOSIUM]
Uses of Radium The principal use of radium, for the treatment of cancer, is quite well known, b u t there are various other uses for this rare and costly metal which are not so often referred to, according to the United States Bureau of Mines. Substantial amounts of radium, estimated a t as much as 10 per cent of the production in some years, are employed for the manufacture of luminous paints used on watch and clock dials, electric switch buttons, keyholes, and like products. War-time radium luminous material eliminated lights t h a t would have betrayed the presence of troops to the enemy. Maurice Curie has described experiments that indicate the profitable use of radioactive elements as fertilizers and for agricultural purposes. Tailings from Cornish ores were formerly shipped to France as fertilizers, but opinion in this country is that no good effect is obtained from radioactive substances as fertilizers. Food-preservative receptacles have been made from mixtures of radiferous (carnotite) ore with white Portland cement, the idea beitg t o prevent bacterial action through radioactivity. A few tons of carnotite are used annually in the manufacture of vessels designed to produce radioactive drinking water. Radium emanation has been used in testing the minute leak-
age of air through rubberized fabric for gas masks, and there are numerous scientific and technical problems in which radioactivity may be employed as a convenient indicator of conditions that the most refined mechanical measurements fail t o reveal. Radium is used to eliminate fire hazards in a large Russian rubber factory by preventing sparks of static electricity. I n the laboratory radium has wrought a revolution in many of the preconceived notions of scientists. The existence of radium and of its decomposition products proves the transmutation of metals as an accomplished fact; and though the process of decomposition is far too slow t o satisfy the commerical need for prompt returns, the fact that an atom, formerly considered an immutable and indivisible unit of matter, is actually a complex structure that may be altered t o other atoms less complex goes far toward realizing the dream of alchemists. The study of the spontaneous disintegration of radioactive elements has given the scientific world an insight into the actual composition of matter; and radium rays, especially the alpha particles, have given physicists a new instrument with which t o investigate the fundamental properties of material substance.
