824
T H E J O U R N A L OF I A V D C S T R I A L A N D ENGIAVEERI-VG C H E M I S T R Y
ENZYMES OF ASPERGILLUS ORYZAE AND THE APPLICATION OF ITS AMYLOCLASTIC ENZYME TO THE FERMENTATION INDUSTRY By
JOKICHI
TAKAMINE
Received June 8, 1914
HISTORY
A s p e r g i l l u s o r y z a e , a n insignificant species of fungi, belonging t o t h e genus A s p e r g i l l a c e a , plays a n important role i n t h e national economy of J a p a n , on account of t h e particular enzymes it generates during i t s growth. Other species of t h e same genus are also largely employed for production of various dietary articles in almost all countries of t h e Orient besides Japan. Nevertheless, i t s utilization in Occidental countries is singularly lacking. Calmette a n d Bodin’s investigation on amylomyces with a view t o utilizing it in t h e spirit industry is a n isolated instance in Europe, a n d their process, known as t h e amylo-process, has been in operation i n France since 189I. I n 1891,I made a n arrangement with t h e Distilling & Cattle Feeding Co., of Peoria, Ill., a n d carried out on a practical scale t h e application of t h e A s p e r g i l l u s oryzae t o t h e American Distillery. N y experiments, which r a n for a couple of months on a 2 0 0 0 bushel scale a t t h e M a n h a t t a n Distillery, were partially successful, b u t unfortunately t h e process did not a t t a i n general recognition of its merit, because i t still lacked means t o overcome various impediments due t o t r a d e conditions a n d difficulties in adapting t h e process in t h e new field of application. I did not forsake t h e investigation after this first trial, b u t became even more enthusiastic about perfecting t h e method. Improvement after improvement was added a n d I now believe t h a t I can soon demonstrate its usefulness. For m a n y centuries A s p e r g i l l u s oryzae has been employed i n J a p a n for varied purposes: Sake or rice beer, Soy a n d Miso are t h e products which are made b y t h e use of this fungus. T h e fermentation of Sake for t h e fiscal year of 1912contributed t o t h e national treasury t h e goodly sum of $41,974,630revenue. The t a x on t h e production of Soy (Bean sauce) amounted t o $2,048,141. T h e total cost of both a n d other articles produced b y aid of t h e fungus in question is p u t a t a n aggregate of $ ~ o o , o o o , o o o . It will t h u s be seen how important a role t h e fungus is playing in t h e national economy of Japan. If we a d d t o t h e above statement t h e products a n d cost of t h e articles which are p u t out through t h e useful services of this fungus, or other species, rendered in all t h e countries of t h e Orient, t h e grand total is a n enormous amount. Curiously enough this t i n y a n d important hustler has scarcely a t t r a c t e d attention i n t h e Occident, a n d this fact made me determine t o work for i t s introduction t o industrial use in t h e United States. Scientifically, A s p e r g i l l u s oryzae has attracted t h e attention of Occidental investigators as far back as 1873. Prof. Kozai, of Tokyo Imperial University, reviewed’ t h e literature regarding t h e early investigations on t h e subject of A s p e r g i l l u s oryzae a n d its industrial applications, a n d gives credit t o Hoff m a n n a n d Korshelt as t h e first writers upon t h e subject. 1
Centralblalt fur Bakleriologie. 6 (1900), No. 12.
I701. 6 , SO.I O
Korshelt’ made a n important contribution t o t h e knowledge of t h e fungus in Europe. His report was upon Sake fermentation with special reference t o a n amyloclastic enzyme which occurs in t h e culture of the fungus on rice a n d which he named Eurotius. I n his “Chemistry of Sake Brewing,” Atkinson discusses t h e function of t h e enzyme just named. T h e fungus was t h e n known as E w o t i u m o r y z a e , being first identified by Ahlburg in 1876 b u t Cohn later investigation led t o renaming i t A s p e r g i l l u s oryzae.2 I t was re-examined by Bosgen, Schroter a n d later Wehmer who gave full morphological descriptions of it. 0. Kellner a n d his pupils’ investigations on invertase amylase a n d maltase are worthy of note. Controversies t h e n existed with regard t o t h e function of A s p e r g i l l u s oryzae i n Sake fermentation. It was thought, on the one hand, t h a t t h e conidia (spores of t h e fungus) may transform, under certain favorable conditions, t o peculiar yeast cells whose action induces t h e saccharified mash of rice t o Sake, containing 1-15 per cent alcohol. Korshelt propounded this theory b u t was opposed by Atkinson. I , myself, confess t h a t I shared t h e same view with Korshelt, b u t later was led t o abandon it. Juhler, confirmed b y Jorgenson3 a n d H a n ~ e n ,also ~ gave opinions agreeing with t h e transformation theory. Kozai a n d Yabej showed t h a t Aspergillus a n d Saccharomycetis have nothing in common. Klocker, Schioning, Seiter a n d Sorrel finally established t h e error of t h e transformation theory. Acclimatizatioiz oj’ A s p e r g i l l u s Oryzae-For a few years I have been working on acclimatizing A s p e r g i l l u s oryzae t o various kinds of antiseptics. The a r t of acclimatizing fungi t o antiseptics is not new, e. g . , t h e growth of yeast in a medium containing fluoric acid or other antiseptics has been tried a n d also p u t into practice. Effront’s a t t e m p t t o increase fermentation products in t h e distillery by means of his process is well known. I t consists of acclimatizing yeast t o hydrofluoric o r some other inorganic acid a n d then employing it in a mash containing t h e acids so t h a t yeast can multiply without being disturbed b y t h e various bacteria which gradually infect t h e mash. So far as I know, nobody has yet tried t o acclimatize A s p e r g i l l u s oryzae for practical purposes. This is because t h e utilization of this interesting fungus is very little known, or is ignored in t h e U . S. a n d Europe, while t h e use of antiseptics is rather unnecessary in t h e fermentation industry of t h e Orient, since it prefers mixed culture t o t h e pure, on account of t h e flavor or bouquet of products still thought to be imparted from a mixed culture. Since m y object in producing t h e culture of A s p e r gillus oryzae is chiefly concerned with t h e utilization of its amyloclastic property developed during i t s growth a n d multiplication, i t matters little whether t h e culture medium employed contains antiseptics 1 Mitlheilungen deulschen Gesellschajt f u r Natur und 1.oikerkunde Ostasiens BY T o k y o , H e f t 16.
2
Bd.
“Wakreobericht der Schleisiochen Gessellsrhaft fur Vaterl.,” Kulter..
LXI (18831, p. 226. 3
4 5
Centralblall f u r Bakteriologie, 11, Bd I, pp. 16, 326. LOG.cil., p . 65. Centrolblaft f u r Bakleriologie, I1 A b t . , Bd. I , p. 619.
Oct., 1914
T H E JOCR,VAL O F I ; V D C S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
or n o t , provided I can get t h e culture which is possessed of maximum enzymic function. T o t h e growth of A s p e r g i l l u s o r y z a e on wheat bran as a culture medium I gave t h e convenient name of Taka-Koji a n d have employed i t for a number of years t o distinguish i t from t h a t known in J a p a n as Koji which is a culture on steamed rice. Taka-Koji is designed for a substitute for malt as a n amyloclastic agent in varied fermentation a n d other allied industries. I t s proposed use is encouraged b y t h e fact t h a t t h e cost of malt is subject t o fluctuations according t o t h e crop conditions of barley while bran is exempt from similar market conditions. Besides, t h e transformation of bran into Taka-Koji can be accomplished i n 48 hours, while malting needs three or four times as long for completion of t h e process. T h e making of Taka-Koji, as i t was formerly practiced, was described b y myself' some years ago, before t h e New York Section of t h e Society of Chemical Industry.' Later, several improvements were made a n d quite recently a radical departure was made in t h e mode of effecting t h e culture. I will, however, describe briefly t h e process as practiced some years ago, so as t o facilitate m y exposition of t h e subject m a t t e r . T h e process consists of first moistening a n d t h e n steaming wheat bran, so as t o sterilize t h e material a n d a t t h e same time t o gelatinize t h e starch. After cooling t h e steamed mass down t o 40' C. a small q u a n t i t y of t h e spores of fungus are intimately mixed with it. It is now carried i n t o a room where t h e floor '2 is cemented, a n d spread in a t h i n layer of about inches thick. I t is still better if p u t into a number of t r a y s with wood or metal frames, a n d provided with a false b o t t o m of wire netting, fine enough t o hold t h e particles of bran. T h e layer can be made in this case a little thicker since air supply can be obtained from both t o p a n d bottom. Such t r a y s are placed upon a specially constructed rack t o hold t h e t r a y s one above t h e other a n d a b o u t 2 inches a p a r t . T h e temperature of t h e room is kept a t a b o u t 3 0 " C. a t t h e beginning b y means of opening steam jets direct into t h e space. This also keeps t h e room moist a n d warm. Within 1 6 t o 18 hours t h e fungus commences t o multiply, which action is easily followed b y t h e gradual rise in temperature in t h e room. F r o m this time on steam is t u r n e d off gradually a n d a t last entirely s h u t off. After 2 0 t o 24 hours from t h e t i m e of inoculation t h e growth a n d multiplication of t h e fungus becomes so vigorous t h a t i t is necessary t o cool t h e room by t h e introduction of fresh, cool air. Carbon dioxide resulting from t h e vigorous growth of t h e fungus becomes very conspicuous a n d t h e room needs t h e renovation of air which is effected b y t h e air draft introduced for cooling purposes. Care should be t a k e n t h a t t h e air is supersaturated with moisture, so t h a t t h e culture medium can always be kept from drying. T h e t e m perature of t h e room often goes u p as high as 4-42 O C. A s long as t h e growth of fungus is comparatively pure a n d t h e mass is kept moderately moist t o furnish t h e necessary a m o u n t of water for t h e culture, such a high temperature does not interfere with t h e genera-
' Journal of ..lpplicd
Socivly of Chemrcal I n d u s l r y , London, Feb. 28, 1898
82j
tion of amyloclastic enzyme i n fungus cells a n d its secretion into t h e medium. T h e optimum temperat u r e of t h e fungus growth lies, however, between 303 j" C. It is, of course, desirable t o keep t h e temperat u r e as nearly a s possible between these limits. F o r 8 or I O hours t h e multiplication of fungus is most vigorous a n d there high temperature prevails. T h e n a gradual decrease in temperature is noticed until t h e culture medium is replete with t h e mycelia of fungus within 48 hours from t h e time of inoculation. At this point vigorous a n d numerous conidiophores are already t o be seen with yellow or greenish yellow conidia (spores). T h e diastatic strength of Taka-Koji has now reached i t s maximum a n d t h e Taka-Koji is ready t o be t a k e n out for use. When t h e vigorous growth of fungus has ceased t h e medium is liable t o be easily infected with injurious bacteria which m a y destroy t h e diastase already generated. I t is, therefore, necessary t o conduct d r y air into t h e room where Taka-Koji is manufactured, so t h a t i t shall be dried a n d made exempt from destructive infection. When t h e moisture of Taka-Koji is reduced t o IC-I j per cent, it is immune from infection of bacteria a n d can be k e p t for several months. By use of antiseptics such as formaldehyde, benzoic or salicylic acid bacterial infection of Taka-Koji can be easily avoided. While mould fungi in general can tolerate quite a q u a n t i t y of antiseptics, nevertheless, if t h e y are acclimated t o same such toleration will a t t a i n t o a conspicuous degree. One p a r t of formaldehyde t o 2 5 0 0 p a r t s of a culture medium does not considerably hinder t h e development of fungus. As t o benzoic or salicylic acid, one p a r t t o 300 parts of a medium is almost immune t o fungi. I h a d .Aspergillus o r y z a e acclimated a n d t h e result of using i t enabled me t o obtain Taka-Koji I O O per cent stronger i n amyloclastic power t h a n t h e product obtained b y t h e old process. D R Us1 E X P E R I M E N T S
Encouraged b y this result, I thought of growing t h e acclimated fungus on t h e culture medium placed i n a cylinder similar t o a pneumatic malt d r u m . This is not a new idea. I carried o u t a series of experiments nearly t w e n t y years ago, b u t t h e n without t h e use of t h e acclimatized variety of .ispergillus o r y z a e . h l y experiments were t h e n a total failure, which made me t h i n k t h e fungus could not grow on t h e material: in motion. If we consider t h e case of sprouting barley in a pneumatic d r u m , i t is, of course, seen t h a t t h e development comes from. within, where t h e vitality of t h e seed resides; hence t h e slight impediment, due t o frictions between t h e moving mass, is not sufficiently severe t o totally inhibit t h e sprouting of t h e grains. T h e case is different when t h e fungus is grown on t h e culture medium in motion, since i t must develop a n d multiply where frictions must always be injuring delicate mycelial cells of fungus. I n m y early experiments, i t was noticed t h a t t h e fungus shows a n initial growth indicated b y t h e rise of temperature a n d also observed under t h e microscope, b u t i t gradually retrogrades. Meanwhile, bacterial infection sets in a n d totally destroys t h e fungus growth.
826
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
T h e subject once almost forgotten, returned t o my mind as I said above, a n d I decided t o repeat experiments again. A small experimental cylinder revolving a few times per minute was made out of a Mason jar a n d clock mechanism. About 3 0 grams of steamed bran were p u t in i t a n d mixed with t h e new variety of acclimated fungus a n d t h e whole was kept in a n enclosure maintained a t 30' C. Unexpectedly a fair result was obtained in which I noticed a very interesting f a c t , namely, one side of t h e bran particle is covered with t h e fungus. I t is t h e inner side of t h e bran. This side is coarse in structure a n d is rich in starch a n d protein matter. If t h e bran is moistened a n d steamed, this particular side shows a tendency t o curl inward a n d assumes t h e shape of pericarp previous t o its severing from t h e kernel. When each particle of t h e bran has curled, t h e inner side is naturally protected from t h e friction between particles when t h e y are p u t in cylinders a n d motion is imparted t o t h e mass by revolution. M y success a n d observations encouraged me t o construct larger apparatus for I O O grams, I kilo, j kilos, 20 kilos a n d finally 7 0 kilos capacity. I was very glad t o confirm in every experiment my belief t h a t t h e process of making Taka-Koji in a pneumatic d r u m is feasible. Therefore, I ordered a d r u m of about 4800 pounds capacity for Taka-Koji manufacture, which is t h e size for a n 8-ton malt drum. I n this d r u m many new devices were introduced, b u t t o my grief, such elaborate devices became in fact stumbling blocks and t h e experiments were far from successful. T h e new devices were taken out one after another until a t last t h e d r u m remained as simple as t h a t of my own laboratory make. At length, after repetitions of more t h a n fifty experiments, I was enabled t o obtain a fairly good outcome a n d was able t o show t h a t the process could be accomplished successfully on a large scale. T h e first d r u m became so dilapidated from t h e alterations a n d changes t h a t I had t o order t h e second apparatus. I t is now ready a n d I a m soon t o t r y it out. I t s success is hardly t o be doubted. T h e drum consists of a huge, plain iron cylinder provided with inlet on one side a n d outlet on t h e other, through which air can be passed by means of a suction fan located on t h e outlet side. The apparatus is furnished with mechanism t o t u r n t h e cylinder a t t h e rate of once per minute. An iron pipe runs through t h e center of t h e cylinder independent from i t . I t branches out in several places along t h e whole length, each branch ending in a spray nozzle, through which water or steam can be turned upon t h e material. T h e Koji manufacture in this d r u m is carried out as follows: T h e wheat bran is dumped in, t h e n t h e necessary quantity of water a n d finally t h e steam is added, t h e mass being p u t in motion by revolution of t h e drum. As soon as t h e mass is properly steamed, it is cooled t o 45' C. cool moist air. I n t o this mass a n aqueous solution of antiseptics a n d t h e n spores of t h e fungus suspended in water are sprayed. The temperature of t h e mass is generally a t 38-40' C. The d r u m is now brought t o a standstill a n d left fo;:about I 2 hours, when t h e gradually decreased temperature
Vol. 6 , No.
IO
again commences t o go up. This means initial growth of t h e fungus. The d r u m is now p u t in motion a n d a slight air current is passed over t h e mass, care being taken t h a t t h e temperature should not be lower t h a n 30' C. At t h e eighteenth hour t h e temperature shows a tendency t o rise quickly a n d a t t h e twenty-fourth hour it reaches its maximum. The force of t h e air current is increased accordingly t o keep t h e temperat u r e a t about 38' C. A suction p u m p of 1600 t o 1800 cubic feet capacity per minute was p u t in full action yet t h e mass of 2400 pounds was barely within t h e limit of 38' C. According t o my small experiment, a higher temperature of 42' C. did not impair t h e diastatic activity formed by t h e growth of t h e fungus; b u t if i t is kept within 40' C. it is so much better for t h e process, t h a t t h e air is previously cooled and saturated with moisture by drawing through a freely sprayed coke tower. I n 48 hours the process is concluded. T h e Taka-Koji manufactured in the drum is more compact a n d glossy t h a n t h a t grown otherwise. The inward side of t h e bran is covered with a felt-like growth of mycelia which do not branch out many conidiophores bearing spores; hence t h e color is whitish. The labor needed for this process is reduced t o onesixth of t h a t necessary in t h e old process. The saving of space is also considerable a n d t h e quality of TakaKoji is excellent. T h u s t h e process promises well for furnishing a substitute for malt in alcoholic fermentation a n d other industries, where amyloclastic enzyme is required. Dr. Niels Ortved, Chief Chemist of Hiram Walker & Sons, of Walkerville, Ontario, Canada, kindly carried out a series of experiments in his distillery a n d published his results in his paper contributed t o t h e last Congress of Applied Chemistry, held here in New York. T o quote briefly, he said: ' I On account of t h e numerous great variations in t h e price of barley malt (in t w o consecutive years t h e price varied I O O per c e n t ) , i t would be of great value t o t h e distilling industry if a converting medium of moderate a n d more uniform price could be employed instead of barley malt. Eliminating, therefore, t h e different grains a s a source of converting medium, I turned t o the diastase produced by a microorganism, t h e Aspergillus o r y z a e . Takamine was t h e first t o introduce t h e Koji process in America. As far back as 1889 he advocated t h e use of Koji in t h e distilling industry. Instead of growing the fungus on rice, Takamine employed a material far cheaper for this country, namely, wheat bran. An extract of the wheat bran, on which t h e A s p e r g i l l u s o r y z a e had been allowed t o germinate, contained t h e diastase, produced by t h e Aspergillus, a n d this extract was mixed with t h e mashed grain, bringing about t h e conversion of t h e starchy materials. Lately, I understand, he has succeeded in adapting a modification of t h e GallandHenning malt d r u m system t o his process. This should be a great improvement over t h e old floor system, in so far as i t makes it possible t o work under absolutely sterile conditions. For my experiments I decided t o use t h e Taka-Koji itself instead of t h e
Oct., 1914
n
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
diastatic extraction of same a n d a d d i t t o t h e mash in t h e same y a y a s malt. Before beginning t h e practical experiments in t h e distillery, laboratory experiments were conducted on a small scale t o ascertain the a m o u n t of Taka-Koji which was necessary t o convert a certain amount of starch into sugar, a n d also t h e optimum temperature a t which t o conduct t h e conversion. It was found t h a t 4 g. of Taka-Koji was sufficient t o give a complete conversion in a mash made from 96 g. of corn a n d rye, t h e corn containing 1 j . o per cent of moisture a n d t h e rye 14.0 per cent. Three experiments were made in t h e distillery. For t h e first experiment only a 14 gallon can was used a n d a portion of our ordinary mash from t h e mashtub was employed, t h e mash being t a k e n from t h e main mash just before malt was going t o be added for conversion. T h e second experiment was performed on a somewhat larger scale. Instead of using mash material from the mashtub, t h e mash was made separately. It consisted of 500 kg. altogether, of which 20 kg. were Taka-Koji. T h e third experiment was performed on a good-sized working scale. Two mashes, each consisting of 3,401.94 kg. (of which 1 3 1 . j kg. were Taka-Koji), were prepared. T h e two mashes were filled in T u r n N o . 2 5 of F r i d a y , M a y 26, 1911. T u r n N o . 2 5 was distilled separately a n d t h e yield was 36 liters of I O O per cent alcohol per I O O kg. of mash material, just a trifle higher t h a n t h e yield of t h e other mashes which were made t h e same d a y . I n judging t h e adaptability of T a k a - K o j i for use in distilleries several questions must be asked a n d answered: “ I s Taka-Koji capable of giving a complete conversion of t h e starchy materials in t h e mash? “Yes, 4 per cent of t h e air-dried Taka-Koji will in I j t o 20 minutes give a complete conversion of well prepared mash material. “ I s t h e fermentation a satisfactory one? “While it is accompanied by a strong odor, which is prevalent in t h e fermenting room, t h e fermentation, however, is very rapid a n d complete, a n d on this account should give rise t o t h e least a m o u n t of infection. “ I s t h e yield of spirit satisfactory? “Yes, t h e yield obtained was a little higher t h a n t h e yield gotten from t h e barley malt mashes, although t h e t o t a l fermentable extract available in t h e mash material was less. T h e yield of 36 liters of I O O per cent alcohol per I O O k g . of mash material is of course only a comparative yield. I n distilleries which employ cookers a n d boil t h e corn under pressure, a higher yield would naturally result. “Therefore, I should say a s a final conclusion t h a t in distilleries which make commercial or potable neutral spirit, t h e Taka-Koji process could be introduced t o advantage. Aside from a probable higher yield in spirit, t h e saving in malt bill would be worth while in years with normal malt prices a n d very considerable in years when t h e malt prices become abnormal.” TAKA-DIASTASE
An aqueous extract from t h e Taka-Koji can be easily made b y percolation and a n enzyme can be precipitated b y adding alcohol t o such extent as t o contain 7 0 per cent by volume of same in t h e mixture.
827
T h e precipitate is dehydrated by means of strong alcohol, dried a n d powdered. It is a white or yellowish white powder of hygroscopic nature. I t is marketed in this form for medical use under t h e name T a k a Diastase. Though known a s a n amyloclastic agent, it contains various enzymes; nevertheless, amyloclastic a n d proteolytic enzymes predominate. 0. Kellner a n d his pupils early reported’ t h e presence of invertase maltase beside amylase. Newcornbe* established t h e presence of cytase, while Aso reports, in a Bulletin of t h e Agricultural College of J a p a n , t h e presence of orydase in Koji extract. According t o Brunstein, Koji contains a n emulsin-like substance t h a t decomposes helicin i n t o salicylic aldehyde a n d glucose which former is later oxidized into salicylic acid; amygdalin is decomposed i n t o cyanhydrin a n d glucose, which former is later oxidized t o mandelic acid. Investigations on peptase a n d ereptase carried out by S. H. Vines3 are replete with interesting observations. He found t h a t Taka-Diastase contains t h e above enzymes a n d effected t h e separation of one from t h e other. By treating Taka-Diastase with 50 per cent ethyl alcohol a n d leaving it over 48 hours ereptase went i n t o solution while peptase remained behind. Equally valuable a n d interesting are researches of J . W ~ h l g e m u t h . ~He states t h a t T a k a Diastase contained a n enormous amyloclastic function; t h e valuation of same according t o his method showed = 6 2 , joo (“ D ” Diastatic power digested t o be D;: a t 38’ C. for 24 hours). Testing t r y p t i c action of Taka-Diastase he found t h a t one gram corresponds t o nearly I O O cc. of t h e pancreatic juice of man a s well as dog; hence he recommended t h e use of Taka-Diastase for therapeutics in cases of general debility. Wohlgem u t h also proved t h e presence of maltase, chimosin, ereptase, a n d lipase in Taka-Diastase. Quite recently, Kitas reports t h e discovery of a specific enzyme in Taka-Diastase or in a Koji extract, whose function is t o transform starch direct into glucose. He doubts very much t h e opinions entertained by preceding investigators t h a t Taka-Diastase first transforms starch t o maltose a n d t h a t t h e l a t t e r is converted t o glucose by t h e action of t h e maltase present. His experimental d a t a have not yet been confirmed b y a n y other experimenter. Such a n enzyme as K i t a reports probably exists b u t confirmation by more exhaustive proofs t h a n he has furnished must be had. The resistance of amyloclastic enzyme of T a k a Diastase toward acid is reported by many investigators. This fact becomes very significant when applied t o t h e practical conversion of grain mash. I n a grain mash where I O t o ~j per cent sugar is t o be finally produced a n addition of mineral acid, say sulfuric acid, t o a n amount oi I part t o 2000 p a r t s of mash accelerates t h e diastatic action a t least I O t o ~j per cent. An equivalent of hydrochloric acid gives a Zeitschyijt f u r fihysiologische Chemie, 14, P a r t 111. “Annals of Botany,” 13 (1889). ICo. 49. 3 I b i d . , 24 (1910). N o . 93. ‘Biochemische Zeitschrift, 39, Parts 3 a n d 4. 1 2
5
THIS JOURNAL, 6
(1913). 220.
8 28
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
similar result. hlalt diastase is entirely inactive with such a high percentage of acid in mash. Taka-Diastase possesses a n important property as a medical agent; i. e., i t is more stable t h a n t h e diastase of malt. T h e l a t t e r loses i t s activity gradually a n d within several months its activity dwindles, while Taka-Diastase remains almost unchanged for several years. How this stability is imparted t o i t is a subject full of interest for investigation. Taka-Diastase contains generally I O t o I j per cent of a s h ; this can be reduced t o 4 t o j per cent b y reprecipitations b u t t h e activity of t h e enzyme does not increase even b y this apparent purifying process b u t on t h e contrary a loss of activity is occasioned in most cases. T h e a u t h o r extends his t h a n k s t o h l r . Wooyenaka for his untiring a n d valuable assistance a n d t o Parke, Davis & Co., for affording every facility for carrying o u t t h e " D r u m Experiments." 552 WEST 1 7 3 ST., ~ ~ N E W YORK
ALCOHOL I N THE MANUFACTURE OF P H O S P H O R I C ACID AND P H O S P H A T E S By PAULJ.
Fox'
Received J u n e I , 1914
T h e increasing tendency i n t h e fertilizer t r a d e i n t h i s country t o produce a concentrated complete fertilizer makes t h e question of t h e production of cheap phosphoric acid of special interest. Whatever process of separation is used, it is clear t h a t t h e original source c a n be only t h e n a t u r a l , tri-phosphates of lime-phosphorites, apatite, a n d t h e rock a n d pebble phosphate of t h e sedimentary beds? F o r t h e separation of t h e phosphoric acid, a large number of methods have been patented. I t is not t h e writer's intention. t o review or even enumerate these methods. He desires merely t o make a suggestion. It is difficult t o see, when t h e energy relations are considered, how anything cheaper can be found t h a n sulfuric acid as t h e prime separator of phosphoric acid from calcium phosphate. When we consider t h a t sulfuric acid is in p a r t formed b y t h e combustion of a substance very common i n n a t u r e a n d very cheaply obtained, a n d moreover is a product which must be manufactured i n some cases t o avoid damage t o vegetation, i t is clear t h a t with t h e progress of chemical technology, we m a y expect t h e present price of a b o u t four dollars a n d a half per t o n t o go down rather t h a n up. I n view of these considerations, i t appears t h a t t h e cheapening of t h e production of relatively pure phosphoric acid is rather a question of extraction t h a n of some other method of setting free t h e phosphoric acid from phosphate rock. T h e writer suggests t h e use of denatured alcohol for this purpose. T h e plan would be t o t r e a t t h e phosphate rock with t h e theoretical a m o u n t of sulfuric acid of a b o u t 50' BaumC, taking care t o bring a b o u t thorough incorporation Scientist in Physical and Chemical Investigations, Bureau of Soils. It is n o t meant t o imply t h a t there is a n y such definite chemical compound as calcium tri-phosphate. T h e material of approximately this composition is probably a solid solution. See Cameron and Seidell, Jour Am. Cltem. SOC.,27 ( 1 9 0 5 ) . 1503; a n d Cameron a n d Bell, Zbid., p. 1512. Apatite always contains chlorine or fluorine. See Cameron and McCaughey, Jour. Phys. Chem., [ 5 ] 16 (1911). 463. 1 2
1-01. 6 , No.
IO
of t h e acid a n d consequent decomposition of t h e rock, b y mechanical mixing. T h e mixed mass of phosphate rock a n d sulfuric acid is t h e n leached with denatured alcohol, t h e residue being filter-pressed. This yields a pure alcoholic solution of phosphoric acid, t h e sulfates, undecomposed phosphates, etc., being left behind.' The most favorable strength of alcohol, whether 95 per cent or more dilute, t o be used in t h e process, would have t o be determined under factory conditions, a n d would depend on t h e subsequent t r e a t m e n t adopted. T h e same remark applies t o t h e sulfuric acid. T h e method involves nothing new in theory, b u t so far a s t h e writer knows has no application on a n industrial scale. Besides t h e obvious advantage of yielding a phosphoric acid substantially free from calcium, t h e plan offers t h e following further features: If i t is desired t o evaporate t h e extract t o produce pure phosphoric acid, t h e specific heat of alcohol is only 0.j452 t o t h a t of water as unity. T h e boiling point of alcohol is 78.4' as against 100' for water. T h e l a t e n t heat of vaporization of alcohol is 206.4' (at 78' C.,Schall) as against 536 ( a t 100' C.) for water. T h e figures for specific heats a n d l a t e n t heat of vaporization refer, of course, t o unit weight. When we consider unit volume, we see t h a t t h e thermal capacity of a given volume of alcohol is (taking density of alcohol at 20' = 0.789) only 0.430 t o water as unity, a n d t h e l a t e n t heat of vaporization similarly computed is 0.304 t o water as unity. Another phase of t h e question is of interest. I n case i t is desired not t o separate t h e phosphoric acid, b u t t o prepare immediately a concentrated fertilizer of saline character, m a n y double decompositions a n d precipitations can t a k e place i n alcoholic solution which are impossible in aqueous solution except a t a great concentration. For example, various potassium a n d ammonium phosphates m a y be more easily precipit a t e d in alcoholic solution t h a n in aqueous. I n particular various phosphates can be precipit a t e d b y merely adding aqueous ammonia, i n slight excess, t o a n alcoholic solution of potassium chloride a n d phosphoric acid, or better b y conducting in gaseous ammonia. The double decomposition depends on t h e solubility of potassium chloride i n alcohol of various concentrations a n d t h e insolubility of potassium a n d other phosphates i n t h a t medium. A4gain, by merely digesting solid potassium chloride with alcoholic phosphoric acid, potassium phosphate results, though t h e progress of t h e double decomposition is limited b y t h e hydrochloric acid set free. T h e progress of t h e double decomposition m a y be followed i n a roughly quantitative manner with a microscope provided with nicol prisms, as t h e potassium chloride is isotropic a n d t h e potassium phosphate anisotropic. If t h e hydrochloric acid t h u s set free, however, is neutralized b y aqueous or gaseous ammonia, ammonium phosphate a n d potassium phosphate are precipitated, certainly as a mixture of t w o or more salts a n d possibly in p a r t as 1 Dr. Cameron has suggested t h a t a plan might be worked out whereby t h e phosphate rock would be treated in one process with a mixture of alcohol and sulfuric acid.
