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I S D C S T R I A L A S D EXGINEERING CHEAV1STRY
Vol. 21. No. 7
Molds and Chemical Horace T. Herrick and Orville E. May COLORA N D FARM\I’ASTE DIVISION,BURSAEO F CHEMISTRY A N D SOILS,WASHINGTON, D. C.
ND all the bread of their provision wa. dry and mouldy.” (17)* So far as is known, this quotation from the Bible is the first reference to the action of molds that is found in the literature and is characteristic of many later statements in that it neglects the chemical aspects of the case in favor of the morphological, though it is entirely probable that the Children of Israel made a number of chemical observations when they attempted to put the bread to the use for which it was originally intended. However, these results have unfortunately been unrecorded by the chronicler. I n his ‘(Kovum Organum’’ Sir Francis Bacon touched on almost every phase of the scientific knowledge of his time, so that in a number of places in that great work we find references to molds. “A cut Citron, being left in a closed Room, for three Summer Months, there were grown out of the Pith, Tufts of Hair an inch long, with little black Heads, as if they would have been some Herb.” (12) Sir Francis held that molds arose spontaneously from the reaction of the material in which they were found, and this curious fallacy persisted well down into the nineteenth century, until more modern methods of research, proving that these organisms were really microscopic plants, opened the way to their productive utilization. Popularly speaking, a fermentation is the production of alcohol by microorganisms. Such a n interpretation is not adequate for our studies, however, and it would therefore be well a t this time to give a more general definition of the term as it will be used in the course of this discussion: Fermentation is a chemical decomposition or rearrangement of organic compounds, brought about during the growth or metabolism of living organisms, or by their enzymes. Fermentations in the popular sense have been known almost as long as mankind. Egyptian hieroglyphics, Babylonian cuneiform inscriptions, and the manuscripts of Greek mythology abound in references to the fermented juice of the grape. while the experience of Xoah as outlined in our own Bible is familiar to all. According to Biblical chronology it is a long step, nearly five thousand years, from Koah’s grape juice to the industrial ethanol plants of this country, but the production of alcohol by the action of microorganisms, chiefly yeasts, has gone on continuously since that time. It is not intended, however, to deal with the work of bacteria and yeasts. These organisms have been widely utilized for industrial alcohol (65); for butanol, acetone, and ethanol by the Weizmann process ( I @ , and for vinegar in France and this country (26). The success of these processes should point the way to a similar utilization of the chemical action of the many varieties of molds which have been identified and classified. As a matter of fact, there have been a number of successful attempts along these lines, but in this respect the Orient has been far ahead of the West. The empirical application of molds in the manufacture of sake and soy sauce has been understood for centuries, though it is only recently that the reactions and organisms involved have been placed on a 1 Received February 2, 1929 Contribution 160 from the Color and Farm Waste Division, Bureau of Chemistry and Soils 2 Part of a lecture given before the Institute of Chemistry of the American Chemical Society at Evanston, I11 , July, 1928 * Italic numbers in parenthesis refer to literature clted at end of article.
scientific basis (28). About the middle of the nineteenth century it was discovered that gallic acid could be manufactured by the action of molds on tannin (,79),and a discussion of this operation can be found in the French literature of the period. The Amylo process for alcohol as operated in France, an outline of which will be given later in the paper, makes use of some of the properties of a variety of mucor. Takadiastase, the enzyme so widely used in textile work, is also produced by growing a variety of Aspergillus o r y i a e on bran (37). The outstanding possibilities of the use of molds a t the present time lie along the utilization of their action on glucose and other sugars or carbohydrates. The earliest work of this type was done in Germany about 1890 (4O),on the production of oxalic and citric acids from sugar solutions by the action of molds grown on the surface of the liquid. Since then experimenters in Europe have worked continuously on various phases of this problem, but until recently the results have been of little value from a strictly chemical viewpoint. Life Cycle of a Mold
Consider for a minute the life cycle of a mold. A solution containing a source of carbon and nutrient salts is inoculated under aseptic conditions from a culture of the mold which it is desired to reproduce. The source of carbon is usually a carbohydrate, though instances are known in which molds have grown and thriven on other organic compounds. The nutrient salts are varied to suit the conditions. though nitrogen, phosphorus, potassium, magnesium, and sulfur must be present in some form or other. Claims are made for the necessity of small amounts of iron and zinc (SO, 5 , SS), but cases are reported in which the cycle of the mold has been completed without the conscious addition of anything more than the elements first enumerated, though it is probable that there may have been traces of iron and zinc in the chemicals and apparatus used (14). About a day or so after the inoculation, the time depending on the variety of mold used, small white spots appear, dotted over the surface of the liquid. In a few days these spots have grown until the surface is entirely covered by this coating which in time thickens and becomes homogeneous. It is in the cells of the coating, or mycelium, as it is known, that most of the work of the fermentation is done. The work may be either direct, in which case the change takes place within the cell, or an enzyme may be discharged into the solution, and the transformation takes place there. The two types of fermentation are known as intra- and extra-cellular fermentations. Contamination and Its Prevention
There is one particular factor in the growing of molds for chemical work that cannot be too strongly emphasized, and that is the necessity for the greatest care in avoiding contamination by other organisms, either bacteria, yeasts, or other varieties of molds. The air is full of microscopic spores; many organic materials contain them, and as soon as the proper conditions of moisture and temperature are reached they germinate and grow. There have been cases in mold fermentations of contamination by an organism which multiplied by division-once every 20 minutes-while the mold
July, 1929
I S D L-STRIAL, A S D E S G I S EE'RISG CHE'3lIZSTRY
with a life cycle of several days was growing from spores. The result was that the mold was literally crowded out and never had a chance t o develop. Even if the stranger is a mold of a different species, growing in the same way but more rapidly, the result is slower but no less sure. There is only one answer-the use of pure cultures, which can be insured only by sterility of apparatus, sterility of material, and protection against the ingress of rival organisms. If the proper precautions are not taken, failure is the result, always. There are two leading methods for guarding against contamination, heating and a proper control of pH. The latter will prevent the growth of many varieties of bacteria, but not of yeasts or kindrcid species of mold. The only sure way is the application of moist heat (steam) under pressure, to all parts of the apparatus and material, for such a time as to insure the destruction of all contaminating spores and organisms. Mold Metabolism
The field of mold metabolism is a debatable ground between botany and chemistry which has been invaded from both sides, by botanists more interested in morphology than in the chemical nature of the materials produced, and by chemists more interested in the by-products than in the exact identity and purity of their cultures. For this reason many of the results reported are not entirely reliable, either chemically or botanically, and in other cases important elements of the investigations have been neglected or overlooked. Even on this basis it is certain that the following acids have been found in the culture medium after the action of molds on various carbohydrates: acetic (34), succinic (34, 35, I S ) , malic (IS, SB), fumaric ( I S , 36, 12, 4I),lactic (13, 36), kojic (%), oxalic ( I O ) , citric (IO), and gluconic (27, 3, 7 ) . A great deal of the work that has been done on sugar has been with sucrose, for it has been important from a n experimental point of view to work with a pure substance, and it is only recently that glucose, in the form of corn sugar of the desired purity, has been available in large quantities. Since the first reaction of molds on sucrose is one of inversion, forming an equimolecular mixture of glucose and letdose, there is no reason to believe that the acids mentioned above could not be made from glucose alone. Use in Chemical Production-Two
Viewpoints
There are two viewpoints on the use of molds in chemical production which exercise a marked effect on the methods of investigation. One is that any given mold will produce what may be desired in the way of chemical output-granting, of course, the limitations in chenlical composition of the material on which it is allowed t o act-the governing factors being variations in nutrient salts, temperature conditions, time of reaction, concentrations, and pH. The other is that there is somewhere a particular mold for any given purpose, the problem being t o find it and then work out the factors governing its maximum output of the substance desired. It is believed that the second is the more reasonable of the two viewpoints, since it is only natural t o assume that anv given organism will do best the Tvork that it is peculiarly fitted to do, and that there will be less difficulty in limiting its activities in other directions. Many organisms in their normal state produce two or more acids, and t o attempt to incarease their activity along this line would seem t o introduce unnecessary complications into a task already sufficiently difficult. The Aspergilli and Citric Acid Production
Three groups of molds have been sufficiently studied to give us an accumulation of information comprehensive enough t o be worth while. These are the Aspergilli, the Penicillia, and the llucors. At present the Aspergilli are by far the
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most important, not alone because of actual ability t o do useful mark, but because their mycological characteristics make them easier to handle. Chief among the fermentations produced by Aspergilli, and also chief among all mold fermentations, is the production of citric acid, which is of especial importance a t this time because of the export tax placed by the Italian government on citrate of lime. The chief commercial fermentation by molds in this country is founded on work done by J. S . Currie, formerly of the Department of Agriculture ( I O ) , who was exceptionally fortunate in haring access to the collection of molds built up in the Bureau of Chemistry bacteriological laboratory as a result of years of work by Doctor Thom and Doctor Church. H e was also able by their assistance to avoid the pitfalls that lie before the unwary chemist who strays into microbiology. Men may exist 1%-hoare competent enough in the two fields to go forward fearlessly, but the chemist who has the good fortune to enlist the aid of an expert mycologist in such work carries far better insurance for success. Currie substituted a strain of Aspergillus nzger for the varieties of the appropriately named citroniyces that had previously been used in this work, and was successful in his attempts t o work out the optimum conditions of production. Probably the most important feature of his work mas the adjustment of nutrients so as to limit the formation of oxalic acid, which is found with citric acid in most solutions of sugars which have been fermented with Aspergillus nzger. This process is now being operated on a large scale. Essentially it consists in the inoculation with spores of a special strain of Aspergtllus niger of the contents of large shallow pans, filled with sterile sucrose solution containing the necessary nutrient salts. I n from 2 to 4 days a continuous felt of mycelium forms over the entire surface of the solution, and citric acid formation begins. The fermentation has usually run its course by the end of the tenth day after inoculation. The solution is then drained off, the mycelium is pressed to remove any acid present in the tissues, and the acid is recovered in yields of approximately 50 per cent by weight of the sugar taken. Many difficulties were encountered in going from a laboratory t o a plant scale, but the nature of these difficulties and the means employed in solving them have been kept as carefully guarded plant secrets. The manufacture of citric acid is of especial interest to chemists concerned with fermentations because it offers an outstanding example of actual chemical synthesis by a mold. Most microbiological reactions, such as the production of the alcohols, acetic acid, lactic acid, and oxalic acid, may be described as examples of catathesis, rather than synthesis, since they involve the passage from larger to smaller molecules, and according to the usual conception synthesis involves a n increase in the complexity of the molecule. But the production of citric acid from a six-carbon sugar is a true synthesis, which starts with a six-carbon straight-chain compound and ends nith a branched-chain compound. Of equal interest is the fact that citric acid has been synthesized by molds from glycerose, a three-carbon sugar, and xylose, a five-carbon sugar (1). The Penicillia and Gluconic Acid
Sext in importance are the Penicillia. Their appearance is undoubtedly familiar t o all. Though markedly different morphologically, t o the untrained eye these molds at the beginning of their growth look much the same as the Aspergilli. Upon development, however, the more common species have a grayish green color, instead of the black so characteristic of Aspergillus niger. X fair example ir Peniczllietm ropuefortii, the mold of roquefort cheese. The Penicillia have been studied and classified, but most experimenters have turned for chemical studies to more familiar fields.
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INDCSTRIAL A N D EhTGINEERING CHEMISTRY
When corn sugar came to the fore recently as a substitute for sucrose, attention was called to the fact that it could be produced cheaply, in large quantities, and in a high state of purity. These qualities brought it forward as a desirable raw material for fermentation, and the Color and Farm Waste Division of the Bureau of Chemistry and Soils initiated an investigation of the effects of the growth of molds on varying concentrations of this sugar (24). The objective of the research was tartaric acid, which has never yet, so far as has been recorded, been produced in appreciable quantities by mold action. A number of molds were studied, and those producing citric or oxalic acid, of which there were many, were discarded. There were found, however, a number of specimens, chiefly of the Penicillium group, which gave in large quantities an acid that did not respond to the tests for either of these acids. After some examination the product was identified as gluconic acid. Heretofore gluconic acid has been known chiefly as the product of a chemical reaction characterized by many objectionable features, with a list price of more than a hundred dollars a pound. Further study of the molds and conditions (15) surrounding their production of this acid has resulted in the development of a process which can be transferred to a larger scale and utilized for commercial production. At present the chief interest in gluconic acid is centered in its calcium salt, which promises wide application as a source of calcium, both for human and animal needs (31, 23). Preliminary experiments have indicated that calcium gluconate can be injected into the tissues without causing necrosis, and that it has unusual effects in increasing the thickness of eggshells of hens previously suffering from a calcium deficiency. Gluconic acid is closely related in structure t o glucose, the body's natural food, and its part in body metabolism might possibly be explained by this observation. The Penicillium that produces this acid is unusual in that it apparently has no other action on the sugar, nor does it oxidize the gluconic acid t o form other products. The Mucors for Production of Alcohol
A third class of molds which finds industrial application is the Mucors, a few groups of which are used in France in a symbiotic fermentation with yeast to produce industrial alcohol. I n this operation, which is known as the Amylo process ( 6 ) ,the strains of hlucors used have been found to possess an amylolytic power far superior to that of either diastase or acid. A solution or emulsion of starch coming directly from the grain is treated for 48 hours with these molds with agitation to keep them submerged. At the end of this time the splitting of the starch to glucose has so far progressed that yeast can then be added to bring the fermentation to completion. A significant feature of this particular fermentation, brought about by a mold which when submerged develops yeast-like forms, functioning practically as yeasts, is that it has all the advantages of deep-tank fermentation, as contrasted with the shallow layer operation outlined in the manufacture of citric acid. The reduction in overhead and simplification of operation of deep-tank fermentations are obvious. The only trouble is that many organisms won't work t h a t way-but the possibilities are there. Characteristics of Industrial Fermentations
The three industrial fermentations produced by molds as outlined above have various individual and general features. I n the firsteplace, the three operations have widely different purposes: the use of a mold t o produce a substance that had hitherto been found only in natural products; the use of a mold to dispense with complicated and costly chemical reactions; and the use of a living mold as a chemical reagent.
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The chemistry involved is equally varied, comprising an oxidation and rearrangement, a simple oxidation, and an hydrolysis. All three fermentations have the advantages of an even and smooth-running process, little supervision, no costly reagents, good yields, few impurities, and low labor costs. Opposed t o these are the facts that all the organisms are sensitive to any but carefully adjusted conditions, and they are extremely liable to contamination or infection. A process that has been carefully studied has all the advantages, but a n immature or premature plant development of a laboratory fermentation is guaranteed to cause more varieties of trouble than the average plant operator has ever known. The Future of Industrial Fermentation
hIicrobiologica1 chemistry is the chemistry of the future. hZost of Nature's growth processes are catalytic, by the action of enzymes. When the chemist or engineer attempts to duplicate them, he takes acres of ground, tons of machinery, the productive labor of hundreds of men to imitate what Sature has done in the stem of the plant or the leaf of the tree; and too frequently he makes a bad job of it. When Nature wishes to synthesize a product she takes a few elements from the soil, calls on the sun and air for aid, and the work is done. Tartaric acid is formed in the grape from the same materials from which the dextrose also found there is produced, and tartaric acid can also be manufactured from dextrose by a biochemical reaction involving the use of peroxidase from the rye germ (11). There is undoubtedly a mold somewhere that will do the same work-but the task is to find it and put it to work in the conditions under which it will work most happily. For molds are temperamental, but so is human labor, and molds have their advantages. They do not sleep on the job, they work 24-hour shifts, there is no strike, no turnover. All they need is infinitesimal quantities of the proper food, a comfortable home in a temperate climate, and protection from their enemies, and they will work for you uncomplainingly until their work is done. The chemical action of molds has been found to be more broad and diversified than is generally known. These organisms will produce phenols from saturated cyclic compounds, (8),and cyclic compounds from carbohydrates (58). Coloring matters (19, @, sugars (go), starch (4), fats (29), urea (16),and alcohols (%I), have been found among the products of mold metabolism, and in its essentials the production of citric acid from dextrose is as difficult a task chemically as can be found (9, 22). Since molds have these possibilities, why are they not being investigated more extensively? The literature of Europe is full of references to the work that is being done there on microorganisms. Are we going to be found asleep again, as we have been in the past, or are we going to keep in step? Let industry and our universities furnish the answer. The field is crammed with problems of practical and theoretical interest, but someone must take them up. Of course there is money in it eventually, but remember this-the dollar rolls more willingly along the road constructed and made smooth by the hands of scientists. Literature Cited Amelung, Z . physiol. Chem., 166, 161 (1927). Barber, J . Soc. Chem. I n d . , 46, 200T (1927). Bernhauer, Biochem. Z., 163, 517 (1924). Boas, Boten. Cenfr., 36, Abt. I, 136 (1919). Bortels, Biochem. Z., 182, 301 (1927). Boullanger, "Distillerie Agricole et Industrielle (Paris)," Vol. I , p. 394. Butkewitsch, Biochem. Z., 164, 177 (1924). Butkewitsch, I b t d . , 169, 395 (1925). Challenger, Walker, and Subramaniam, J. SOL. Chem. Ind., 46, 1047 (1927). Currie, J . Bid. Chem., 31, 15 (1917). Diamalt Akt.-Ges., German Patent 426,864 (1926). Ehrlich, Ber., 44, 3737 (1911).
INDUSTRIAL, A S D E;\’GIXEERISG CHEMISTRY
July, 1929
Ehrlich, Ber., 52, 63 (1919). Frouin, Compl. rend. roc. biol., 89, 986 (1923). Herrick and M a y , J . Biol.Chem., 77,185 (1928). Ivanov, Z.physiol. C m m . , 170, 274 (1927). Joshua, 9 : s . Killeffer, IND.EKG.C!HEM.,19, 46 (1927). Klocker, Chem. Zentv , 87, 11, 672 (1916). Kostychev, Z . physiol. Chem., 111, 236 (1920). Rostychev a n d Afanassjeva, Jahrb. miss. Bolan., 60, 638 (1921). Rostychev and Teschesnokov, Compt. rend. acad. uniol; repub. Soviet social., 13, 195 (1927). Kottman, Schmeiz. d l e d . Wochen., 57, 409 (1927). h l a y , Herrick, Thorn, and Church, J . Biol. Chem., 75, 417 (1927). McIntosh, “Industrial Alcohol,’’ Greenwood & Son, London, 1923. hlitchell, “Vinegar-Its Manufacture and Examination,” London, 1926. Molliard, Compt. vend., 178, 4 1 (1924).
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Oshima and Church, IND. END.CHEM.,16, 67 (1923); Church, U. S. Dept. Agr., Bull. 1152 (1923). Pearson and Raper, Biochem. J . , 21, 875 (1927). Roberg, Cenlr. Baht., Parasitenk., I1 Bbt., 74, 333 (1920). Rothin, Schu’eiz. Med. Wochen., 67, 388 (1927). Shaw, “Philosophical Works of Bacon,” Vol. 111,p. 284, London, l i 3 3 . Steinberg, Botan. Gaz., 70, 465 (1920). Takahashi and Asai, Proc. I m p . Acad. Japan, 3, 86 (1927). Takahashi a n d Sakaguchi, Bull. Agr. Chem. Soc. Japan, 3, 35 (1927). Takahashi and Sakaguchi, Ibid., 3, 59 (1927). Takamine, U. S.Patent 525,823 (September 11, 1894). Tamiya, d c t a Phytochim. ( J a p a n ) , 3, 51 (1927). Van Tieghem, Compl. rend., 65, 1091 (1865). TVehmer, “Beitrage zur Kenntnis einheinscher Pilze,” Hanover, 1893, No. 1. Wehmer, Ber., 61, 1663 (1918).
Physico-Chemical Studies on the Mechanism of the Drying of Linseed Oil’ I-Changes
in Density of Films
G . L. Clark and H. L. Tschentke DEPARTMENT OF CHEMISTRY.UKIVERSITY O F ILLINOIS. C‘RBASA, ILL
Linseed oil films, raw or containing driers and produced under regulated conditions, were subjected to the effects of exposure to ozonization, to ultraviolet rays, and to direct sunlight and weathering. Using the flotation method, the density of the film was determined before exposure and then at stated intervals after exposure. The conditions under which each film was formed, DUSTRIAL AXD EXGIXEERINGthe density before exposure, and the densities a t succeeding periods of exposure are recorded and some CHEMISTRY dealing with varisignificant data are plotted. From the numerical ous phases of linseed oil redata certain definite generalizations are made which search.* However, there is bear on the facts and mechanism of oxidation and much disagreement as to the drying. findings in the work under-
URING the last few
D
years many investigators have studied the properties of linseed oil and much information has been obtained. I n the last four years no less than twenty papers have appeared in Ix-
taken and there has been little correlation in the results. The object of this investigation was to carry on some precision work on the physico-chemical properties of linseed oil, especially density, which in turn leads to a study of volume changes. This was undertaken in the hope that, a better control method for the study of the aging of paint and oil films might be developed and that a further insight into the drying mechanism might be obtained. An increase in density after a film is dry, with perhaps only negligible increase in weight means a contraction in volume. If, however, a paint or patent leather film is firmly attached to a base, then a strain is set up which may result in cracking and failure of the film. The practical significance of accurately determined and comparable densities is a t once apparent. Comprehensive x-ray diffraction studies form the second series of related investigations. These will be reported in a forthcoming paper. Materials
RAWCOMMERCIAL LIWEEDOm-The purest obtainable oil was secured from a source assuring constant and representative properties. I t s constants were: density, 0.9285 at 2.jo/25O C.; iodine number, 172.2; refractive index (25’ C . ) , 1.4835. D R I E R S - P ~ ~ blue, ~ S ~prepared U~ in laboratory from potassium ferrocyanide. 1
Received January 21, 1929.
* See list of papers at end of article.
Ferric oxide, c. P. and aIso japanner’s brown. Cobalt oxide, prepared by ignition of cobalt carbonate t o constant weight. White japan drier, commercial, obtained from paint factory.
Operations of Film Making
SPINK1NG-L4 h o r i z o n t a 1 spinning table was turned by a belt driven by a small motor. To the top of the spinning table n.as clamped a 23-cm. disk of thin amalgamated tin. Linseed oil was then dropped on the table which-was rotated a t slow speed until completely covered and then more rapidly until a thin film remained. This film was then placed on a level surface and baked in a drying oven kept a t i o ” C. FLOW-A 23-cm. disk of thin amalgamated tin was glued to the plane surface of a dished spinning table top and allowed to cement under pressure. The oil was then poured in and flowed back and forth until completely covered. Then the whole was placed on a n accurately leveled table inside of a drying oven and baked a t 70” C. BAKINGON hfERCuRY-After numerous difficulties with various amalgamated metals, the following apparatus was set up. A sheet of platinum foil 0.05 mm. thick was cut into strips 1 cm. wide. These were spot-welded end to end into a long strip, which was then bent into the shape of a rectangle, approximately 20 X 6.7 cm., to serve as a box without top or bottom. Then the platinum was carefully amalgamated; into a shallow glass tray was poured thrice-distilled mercury; and then the platinum rectangle was dropped through the surface so that about half the height was above the mercury surface. If the platinum was uniformly amalgamated, a slightly concave meniscus was formed between mercury and platinum and a perfectly clear surface, free from amalgamation scum, was obtained. The tray was then placed in the drying oven, and a calculated and weighed amount of linseed oil poured onto the surface and spread out carefully.
