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INDUSTRIAL FERMENTATION Papers presented at the General Meeting under the auspices of the Divisions of Industrial and Engineering Chemistry, Organic Chemistry, and Cellulose Chemistry at the 80th Meeting of the American Chemical Society, Cincinnati, Ohio, September 8 to 12, 1930
Introduction ANY of the symposia presented at the meetings of the AMERICAN CHEMICAL SOCIETYowe a part of their interest at least to the novelty of the reactions or processes described. While there is obviously much that is new in the field of fermentation chemistry, many of the industrial applications are based on arts which have been practiced since before the dawn of history. Alcohol, vinegar, leavened bread, the souring of milk, and numerous other food fermentations are examples of the empirical employment of microorganisms. It is for this reason that a historical, as well as chemical, aspect is apparent in many of the papers of the symposium. While the fermentation industry is second to few in the importance and variety of its products, it may well take first place in the antiquity of the processes which are the basis of its prosperity. Although fermentation chemistry has its roots buried deeply in the past, the importance of its practical applications and the fact that on their present scale they are largely developments of the last thirty years are sufficient evidence of its right to a place in the sun of our modern industrial civilization. The future is also worthy of careful consideration. Such a development as the production of millions of pounds of citric acid annually by the action of molds on cane sugar ranks in importance as an achievement with any other of the industrial novelties which are attracting the attention of chemists a t the present time. It is particularly such syntheses
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as that of citric acid which turn our eyes toward the future of this branch of chemistry. Many fermentation reactions have been duplicated by other chemical means, but there are some so complex that the very nature of the mechanism is in question and ordinary methods of synthesis have proved inadequate. Although starting with the simplest of elementary constituents and working within limited ranges of temperature, the chemistry of nature is extremely complicated. It is not unreasonable, therefore, to expect the reproduction of her reactions for industrial use by means of microorganisms rat,lier than by the more common methods of research laboratory and factory. I n any event, while it may be possible for economic reasons to substitute other types of chemical synthesis for some of the simpler fermentation applications, there are a number of the latter now in use for which such replacement is not to be anticipated, and a shift in economic factors in the former might easily alter the relative status of the two methods of production. Moreover, when we consider the number of microorganisms of the different varieties whose chemical action has not been studied, we will readily see that the surface has hardly been scratched. To paraphrase Sir Isaac Newton, we have only been picking up sea shells on the shores of the great ocean of fermentation chemistry.
HORACE T. HERRICK, Secretary
The Chemical Approach to Problems of Fermentation’ Ellis I. Fulmer CHEMISTRY DEPARTMENT, IOWA STATE COLLEGE, AMES,IOWA
Fermentation Terminology
EFORE proceeding to the main topic under consideration it will be of interest to trace the evolution of the word [‘fermentation” and to point out the confusion caused by its use in several m e a n i n g s . The word comes from the Latin, fervere, “to be boiling hot.” It was used by Pasteur to designate microbiological action, especially upon saccharine materials, in which such quantities of gas were evolved as to produce an appearance of boiling or effervescence. The agencies bringing about this type of change were called [‘ferments.” Later it was learned that dead cells as well as extracellular products were capable of bringing about fermentations in the original s e n s e t h a t is,.produced gas during their action. Hence the terms “organized” and “unorganized” ferments. The latter name then became
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Received September 15, 1930.
synonymous mith e n z y m e s and the term “fermentation” came to include any enzymatic action whether or not gas is evolved. It is seen, then, that the phenomenon of fermentation was originally not synonymous with utilization. That i s , t h e statement that an organism would not ferment a certain substrate simply m e a n t t h a t g a s was not evolved, not that the material failed to serve as a metabolite. Later the statement became more inclusive and carried the connotation that the organism could not utilize the substratethat is, was not capable of bringing about chemical transformations, leading to its utilization in the living processes of the cell. Hence the statements in the older and newer literature in regard to the ability or inability of an organism to ferment a given substrate may not have the same meaning. I n discussion involved with industrial fermentations the term “fermentation” refers to the microbiological trans-
The complexity of the chemical changes brought about by the action of microorganisms on carbohydrates and the profound effect of physical and chemical environment on the nature of these changes are pointed out. The importance of synergistic phenomena and their connection with industrial processes is discussed. The need for adequate quantitative methods of analysis is stressed, since the whole field of the biochemistry of fermentations is dependent on and limited by such methods. A chart is included which gives the interrelationships between substrates and products of fermentation.
November, 1930
INDUSTRIAL AND EiVGINEERIiVG CHEMISTRY CHAR T f. THE
SUESTRATES
47 Trimethylene
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FERMENTATWE /NTERRELATlONSHlPS OF THE ~ 1 C R O B l O L O G l C A L DISSIM~LATIONPRODUCTS O F THf CAQBCWYDRATES
Svmmuritfd from f u h n e r and Werhman (1930) and 5u=:h?nun and Fu/mer (1930).
g/uco/
48 V o / e r i c a o d
formations, predominately of the carbohydrates and their dissimilation products. The carbohydrates are emphasized, since these materials furnish the important sources of energy for the organisms and for the zymotechnical production of chemicals on a large scale. From a chemical viewpoint zymology (fermentation) deals with catalysis (or rather autocatalysis) in heterogeneous system. Industrial catalyses and zymotechnical syntheses differ in that in the former case the catalyst, usually a simple
Pnoouc T s
Trimethy/ene glycol
47
V a / e r i c ac/d
48
type of chemical, is manufactured outside the reacting mixture and then added to the reactant or reactants under controlled conditions. I n the latter processes the catalysts, the enzymes, are manufactured during the course of the reaction. This involves a knowledge of the nutrition and characteristics of the organism used and the conditions under which it will produce in the highest degree the particular catalysts desired. Different organisms bring about different chemical changes in the same medium and the chemism
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of a given organism varies in the same substrate if nutrients and physical chemical environment are altered. For example, molds commonly produce citric acid from carbohydrates, while bacteria do not. One set of products may predominate under anaerobic conditions, while others are in excess under aerobic conditions. The butanol and acetone fermentation industry depends for maximum yields upon the use of the appropriate organism under anaerobic conditions. Yeast produces only small quantities of glycerol relative to ethanol in the normal or slightly acid medium, while in an alkaline medium or in the presence of sodium sulfite the relative yields of these compounds are reversed. These facts are basal to the commercial production of glycerol by fermentation. The chemical yields are likewise influenced by the temperature aside from the effect upon reaction rates. Moreover, there are many instances known in which two different organisms growing together bring about chemical actions which neither alone could effect. This phenomenon is known as “synergism.” Synergistic action is very complex in detail and a real understanding of the phenomena involved awaits a great deal of research and the development of more adequate chemical technics. Evidently pure-culture industrial fermentations are not always the most desirable, and it is likely that some of the most striking advances in zymotechnology lie in the utilization of synergistic phenomena. Zymotechnology differs in approach from industrial syntheses in that in the latter the catalyst brings about a practically complete final reaction without a complexity of intermediate products-that is, the action is drastic. I n fermentation we are dealing with a series of graded products or stages in oxidation and reduction. The formation of the fermentation products is the process whereby the organism secures the energy for growth and maintenance. Evidently the organisms do not usually secure the maximum energy yield by a complete oxidation of the carbohydrates to carbon dioxide and water. It is owing to this fact that there is such a thing as the fermentation industry. The various theories of the mechanism of fermentation attempting to rationalize the complex oxidation-reduction relationships of the many fermentation products have led to the development of a unique terminology involving such terms as “donators,” “acceptors,” “hydrogenations,” “dehydrogenations,” “hydrations,” “dehydrases,” “activators,” “dislocation,” “dismutation,” “catalytic protoplasmic groups,” “graded protoplasmic affinities,” and an imposing array of enzymes involved in various details of microbiological dissimilation. I n fact, the terminology threatens to become as imposing as that developed by Ehrlich and school with reference to immunology. Chemicals Produced by Action of Microorganisms on Carbohydrates
The complexity of the chemical problems involved may be illustrated by a brief survey or summary of the chemicals reported as produced by the action of microorganisms upon the carbohydrates and upon the intermediates. I n Table I are listed the products of the action of microorganisms on the hexoses and pentoses. It is of interest to note the similarity of the products from these monosaccharides, especially in view of the fact that the industrial fermentation of agricultural wastes involves a fermentation of the pentosans. Chart I summarizes the fermentative interrelationships of the microbiological dissimilation products of the carbohydrates. These include the products identified as being produced by the action of bacteria, yeasts, or molds. The dissimilation products are arranged alphabetically in the
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left-hand column as substrates. I n the right-hand column are the same materials classed as products. The lines connecting a member of the left-hand column with certain members of the right-hand column represent the products formed by the action of bacteria, yeasts, or molds on the given substrate. Conversely the lines connecting a given product with given substrates show at a glance the different substrates from which the product may be formed by microbiological action. Products of Action of Microb’rganisms o n Hexoses a n d Pentoses“ Acetaldehyde +Fumaric acid +Acetic acid +Gluconic acid +Acetone Glycerol Acetylmethyl carbinol Glycolic acid +Butyl alcohol +Hydrogen +2,3-Butylene glycol Ketogluconic acid +Butyric acid +Lactic a!id Caproic acid Malic acid Caprylic acid Methylglyoxal +Carbon dioxide +Oxalic acid +Citric acid +Propionic acid Pyruvic acid +Ethyl alcohol +Formic acid +Succinic acid a All the items in the table are produced from the hexoses and t blose marked are likewise produced from the pentoses. Table I-Reported
+
+
The alphabetical arrangement in the chart is wholly arbitrary. The ideal arrangement would be in the order of free energies of formation. However, such values are available for only a few of the chemicals listed. This at once emphasizes the need of the calculation of these values for all the compounds involved before these interrelationships can be interpreted on a strict basis. Other methods of arrangement might be on the basis of structure, intermediate products, or heats of combustion. The chart immediately shows the complexity of the chemical problems involved. One of the first needs is the development of adequate quantitative methods of analysis of such mixtures. Many methods now used are qualitative or semiquantitative a t best. Until such methods are developed, itis impossible to formulate a complete flow sheet for a given organism under given conditions. Moreover, adequate methods may show traces of compounds which are of fundamental importance in any complete theory of the mechanism of the fermentation involved. It is evident that the fermentation products are not simply degraded products of the substrate, but that in many instances there occur synthetic reactions. Reference to the products from acetaldehyde shows this point in striking fashion. I n fermentation processes the reagents (enzymes) are relatively mild agents, so that in effect the process from inception to completion-that is, to carbon dioxide and wateris magnified and spread over considerable periods of time. Thus, in microorganisms there are presented unique tools for the production of chemicals. Each item in the table and chart holds the possibility of commercial production by fermentation processes. It is evident that only B few of the possibilities have been exploited. The problem resolves itself into the bringing together of the right organism or organisms and the right medium under optimum conditions. Recent Reviews on Fermentation (1) Buchanan and Fqlmer, “The Physiology and Biochemistry of Bacteria,” Vols. I, 11, 111, Williams and Wilkins, 1928 to 1930. (2) Fred and Peterson, Chapter on Fermentation in “Annual Survey of American Chemistry,” Vol. IV, Chemical Catalog, 1930. (3) Fulmer and Werkman, Index to Chemical Action of MicroBrganisms on Non-h-itrogenous Organic Compounds. C. C. Thomas, Springfield, 1930. (4) Nord, “Mechanism of Enzyme Action and Associated Cell Phenomena,” Williams and Wilkins, 1929. (5) Schoen, “Le problPme des fermentations, les faits et les hypothher,” Masson. Paris, 1926. (6) Stephenson, “Bacterial Metabolism,” Longmans, 1930.
