ms
FERMEN
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HERBERT E. SILCOX and S Y L V A N B. LEE MERCK & C O M P A N Y , INC., R A H W A Y , N. J.
F
ERMEXT-4TIOK has been .the subject of several rcccnt reviews. Johnson discussed individual fermentation processes of industrial significance (48),Boruff and Van Lanen reviewed the production by the fermentation industry during World War I1 (14),Porter presented a paper on recent advances in fermentation (78), and I'ulmer discussed the physical-chemical approach to problems of fermentation (SO). The intent of this review is t o present fermentation as a unit process. Industrial fermentation may be defined as the production of chemicals by a series of enzyme-catalyzed chemical reactions with bacteria, yeasts, or molds under aerobic or anaerobic conditions. Although fermentation is one of the oldest processes and has important economic significance due t o its widespread use in industry, i t must still be classified as a n a r t rather than a science. This is in part due to the complex nature of fermentation and the large number of independent and dependent variables that affect the evaluation of results. Secondly, alt,hough fermentation involves difficult interrelated problems in biology and engineering, men i n these two fields have worked independently until recent years. It is increasingly becoming the practice to have biologists and chemical engineers working on the same problems, and some men are now being trained i n both biology and engineering as biochemical or biological engineers. This trend has led tjo the increased recognition and expansion of the engineering phases of fermentation and the application of tho physical-chemical approach t o the solution of fermentation problems. The five basic prerequisites of a good fermentation process arc: 1. A microorganism t h a t forms a desired end product. This organism must be readily propagated and be capable of maintaini ng biological uniformity, thereby giving predictable yields. 2 . Economical raw materials for the substrate. 3. Acceptable yields. 4. Rapid fermentation. 5 . A product t h a t is readily recovered and purified.
The development of a process t h a t meets all these conditions is complex and time-consuming. It is felt, however, that such research and development problems would be simplified by a better understanding of fermentation as a unit process. It is rccognized at the outmt that the information currently reported does not allow the establishment of a firm unit process. However, a n attempt will be made to establish the pattern of such a process fist by a discussion of individual processes, emphasizing those data t h a t pertain t o unit process information, and secondly by summarizing the factors involved in fermentation.
ETHYL ALCOHOL The production of ethyl alcohol is fast becoming a science. I n this, the oldest and largest of the fermentation industries, many new developments have been brought about largely because of the wartime importance of alcohol. The most important factor in the production of industrial ethyl alcohol is raw material economics. During the war new plants were built t o produce alcohol from carbohydrate sources other than cane blackstrap molasses, notably grain, wood, and waste sulfite liquors from the paper industry. A discussion of the economic features of alcohol production has been presented by Tousley (93) and Jacobs (46). The past few years have seen the development of processes
for the production of alcohol from wood products. D a t a on the saccharification of wood were presented a t a symposium on sugars from wood (43). The U. S. Forest Products Laboratory has developed a modification of the Scholler process of wood hydrolysis which has become known as the Madison wood sugar process (38, 39). A plant using this process has been in operation a t Springfield, Ore. A second development has been the production of alcohol from sulfite waste liquors. Processes of this type have been installed at Thorold, Ontario, Canada (21, @), and a t Bellingham, Wash. (25). I n bot,h plants the fermentation process (20 hours) is continuous and the yeast from fermentation is re-used. The Nadison wood sugar process also operates with yeast re-use. The advantage of reusing yeast is the ability to begin product,ivc: fermentation immediately on ro-enlry of the yeast into the fermentation system and the essential elimination of sugar losscs due to the development of yeast cells. Barley malt has long becn uscd in this country for the saccharification of starch. The 17-artinie malt shortage brought about interest in other amylase sources. The use of moldy braii as a source of amylase has been summarized by Underkofler, Severson, Gocring, and Christcnsen (95,96). Mold bran is prepared by growing mold on moist acidified wheat bran. Following inoculation with A . oTgzlzae, incubation is performed on trays in a specially ventilated room. Such a process requires a largc tray area and tho mechanical diificultics of handling large numbcrs of trays become apparent. E r b and Hildebrant (24) have successfully used mycelium of a11 amylolytic mold for saccliarilying starch from wheat flour for the production of ethg;l alcohol. The mold was prepared by submerged growth in tanks with aeration i n a medium containing grain stillage. However, it was not, possible to eliminate completely the use of malt in the subsequent alcohol fermentation. Adams, Balankura, Andreasin, and Stark ( 4 ) showed bhat substituting dry distillers' solublcs for stillage resulted i n superior and more consistent performance in the production of amylase and did not require the addition of malt. The production of amylase by submerged fermentation under aseptic conditions contributes one more step toward pure culture alcohol fermentation, in that the contaminat'ion introduced by the addition of ma.lt has been eliminated. The Seagram group (st?, 97) has presented the development and. design of a continuous cooking and mashing system for cereal grains. The advantages of such units for the preparat,ion of substrates are obvious in the simplicity of the unit and the controlled time-ternperaturc conditions to which the substrate is subjected. The Seagram group (5, 81) has also presented a design of an experimental continuous alcohol unit. Although still in the experimental stage, this step indicates the increasing knowledge and control of ferniontation that are being achieved.
ACETONE-BUTYL ALCOHOL Little has been published recently on the technology of the acetone-butanol fermentation. The change from molasses to grain as a carbohydrate source during the war years involved no difficulties, a s the techniques for fermenting grain were known before acceptable molasses-fermenting organisms were discovered.
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However, several publications have shed more light on various aspects of this fermentation. Oxford, Lampen, and Peterson (70) showed t h a t two growth factors, biotin and a factor present in yeast extract, are required by Cl. acetobutylicum in the acetone-butanol fermentation. Leonard and Peterson (56) presented data for the fermentation of wood hydrolyzates with Cl. butylicum No. 39 t o butanol and acetone. Waste sulfite liquor has also proved fairly acceptable as a carbohydrate source in this fermentation (107). Wood, Brown, and Werkman (109) used C1a added in the form of CHaC1300H t o cultures of Cl. acetobutylicum and then located the 0 3 containing materials i n the products of fermentation. The application of isotopic techniques may be of increasing significance i n the development of a more completo understanding of fermentation mechanisms in general. h/lcCoy (60) has discovered a new organism for acetone-butanol fermentation and also described techniques for varying the ratio of acetone to butanol in the completed fermentation. One of the more interesting developments in the acetone butanol industry is the production and recovery of by-products high in riboflavin and other growth factors, which have proved to be valuable feed adjuncts. Microbiological production of riboflavin is discussed in more detail in the following part of chis review.
MICROBIOLOGICAL PRODUCTION
OF RIBOFLAVIN
During the past 8 years considerable attention has been given t o the production of riboflavin and riboflavin-rich concentrates by various microbiological methods. One of the first patents issued (64) described a process for obtaining a riboflavin-rich concentrate from the spent liquors of the molasses acetone-butanol fermentation and such products are now on the market as feed adjuncts. Later it was found that corn plus unpolished rice (103) fermented with C1. acetobutylicum (Weizmann) in limited concentrations of iron, cobalt, copper, lead, and zinc (6) will give vastly superior riboflavin yields. Iron was found detrimental t o riboflavin production in the acetone-butanol fermentation of corn mashes, and only small yields could be obtained in carbon steel fermenters. To overcome this, 2,2'-bipyridiyl was added t o the fermenting mash to tie up the excess of free iron in the medium (41). Sulfite as sodium sulfite has also been effective in increasing the yields of riboflavin in rice-corn mashes fermented by tho acetonebutanol organism (65). Whey has now been used for the production of riboflavin concentrates (62). Leviton (57) has presented a theory to explain the relationship of sulfite and iron to the inhibition of the microbiological synthesis of riboflavin by Cl. acetobutylicurn. One of the most interesting riboflavin-producing organisms is Eremothecium ashbyilii (89). This organism is probably used for the greatest portion of the riboflavin produced by niicroorganisms at the present time (76, 80). Ashbya gossypii (106) and several Candida species (I?, 18, 88) have been reported to produce large quantities of riboflavin and appear to have industrial possibilities (19, 89). Several methods involving chemical agents have been proposed for separating the riboflavin from the fermented mash (36, 44, 61, 61). However, from a fermentation viewpoint the method of most interest involves the use of bacteria such as E. coli and 8. faecalis to reduce the riboflavin, thereby causing it t o precipitate (45).
ANTIBIOTICS Antibiotics have achieved recent importance in the fermentation field. The antibiotics, of which penicillin and streptomycin are the outstanding examples, may be defined as chemical compounds produced by living organisms and capable of inhibiting
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the life processes of other living organisnis. A review on antibiotics has been prepared by Benedict and Langlykke (13). Generally, in fermentation, it is possible to visualize the production of the desired product from one of the raw materials although the mechanism of product formation may not be completely understood. I n this manner a fermentation yield mav be defined. However, in the production of antibiotics, the mechanism of formation of the desired product is more complex and thus far yields based on starting materials have not been possible. By the same reasoning, maximum yields in antibiotic fermentation cannot as yet be defined. Although operating conditions and fermenter design have played a n important role in the maintenance of large scale maximum productivity, the more important increases in the level of productivity have been brought about by the microbiologists through use of improved media and selection of high yielding strains PENICILLIN
I n the period covered by this review, the penicillin process has grown from one yielding 2 units of activity per milliliter produced in bottles t o one where activities of several hundred units per milliliter are obtained in large volume tanks and in less fermentation time, Obviously mention of all the advances t h a t broughi about this rapid development is not possible. Production media based on cornsteep liquor, mineral salts, and lactose, and adaptation of the submerged method of culture were reported by Moyer and Coghill (66, 67). Cottonseed meal has been reported as a n alternate for cornsteep liquor (68). Strain selection and improvement have been very effective in* increasing fermentation productivity. Advances were made by Raper (79) through the selection of high yielding members of the Penicillium notatum-chrysogenum group. By irradiation of these high yielding strains, still higher producing mutant strains have been isolated ( 7 ) . The subject of mutations in microbiology has been reviewed by Tatum (90) and Beadle (8-11). Mutations may be introduced by irradiation with x-rays, ultraviolet light, or neutrons. T h e existence of several compounds, all exhibiting so-called penicillin biological properties, was recognized early. One of these compounds, penicillin G, was characterized by the benzyl radical and yielded phenylacetic acid on degradation. Moyer and Coghill (68) added phenylacetic acid as part of the fermentation medium in order t o evaluate possible increase in yield and increase in the ratio of penicillin G to other penicillins present. They reported a n increase in yield and also increased ratio OS benzyl penicillih in surface cultures, but not in submerged cultures with the exception of cultures in a medium containing wheat bran. Higuchi, Jarvis, Peterson, and Johnson (42) made a similar study using derivatives of phenylacetic acid in conjunction with Penicillin chrysogenum Q176. These studies established t h a t properly selected adjuvants resulted in increased yields and increased ratios of penicillin G. The use of adjuvants is now accepted practice in penicillin fermentation. When penicillin is produced by submerged fermentation in tanks, many factors influence the course of the fermentation and the penicillin yield. The construction, operation, and performance of pilot plant penicillin fermentation equipment have been described (37, 84, 85). D a t a concerning the metabolism of penicillin-producing molds have been presented by a number of investigators (67, 34, 47, 66, 86). The penicillin yield, with a given culture, is a function of such variables as pH, temperature, trace nutrients and toxicants, rate of carbohydrate oxidation, aeration, agitation, foam control, rigorous protection from contamination, composition and preparation of medium and methods of development and use of inoculum. It has been the interpretation and integration of these labora-
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Lory and pilot plant studies that have gradually evolved the efficient fermenter designs and operating conditions that have made possible the economic large scale fermentation of penicillin.
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growth on beet molasses medium, has been described by the British Intelligence Objectives Sub-Committee (16, 16).
LACTIC ACID The production of lactic acid by fermentation processes has increased appreciably in recent years, most likely because of The development of the streptomycin fermentation process the relative convenience of adapting this process t o efficiently has been similar to that for penicillin. The techniques and proutilize carbohydrate by-products such as glucose, hydrol, whey, cedures used in penicillin development have been qualitatively molasses, and sulfite maste liquor from other industries. The if not quantitatively followed and in general the same limitation? recovery process used And the purity of product obtained is deand restrictions have been encountered. Although considerable pendent on the source of ,sugar clioscn for fernicntation. Dework has been reported on the chemistry of steptomycin, verj scriptions of two such commercial processes have been presented little published information is available on factors necessary Cor (20*7 1 ) . production fermentation. The general fermentation proceduresused by Merck& C O I P I ~ ~ I I J , , This fernientatioii is nor ruii under strictly aseptic Conditions jince the bacteria (bacilli and lactobacilli) used will perform at, Inc., have been described by Porter ( 7 7 ) . Gottlieb and Anderson higher temperatures and fairly low pH levels, thus reducing conreported on some of the morphological and physiological factors tamination difficulties. in streptomycin production (55), and presented. information on Sulfite waste liquor appears to be a feasible substrate for large oxygen consumpt,ion. and its rehtion to antibiotic productioa scale production of lactic acid (48)but the process has not been (36)e proved in large scale operation. Details of fermentation and recovery procedures have been d.escribed by Peckham ( 7 1 ) and 2,3-BWTANEDIBh h y Filachione and Fisher (25). During the recent war the amount of effortexpended 011 laburaCory and pilot plant development of the 2,3-butanediol fermentaOTHER ACIDS tion was extensive. The reason Por the attention vias the posNuIiierous other organic acids have been or can be produced sible use of 2,3-butanediol in the production of butadiene for on a commercial scale by fermentation processes. synthetic rubber manufacture. However, ample production Gluconic acid production has been described by Williams (108). of butadiene from alcohol and petroleum products lessened the Ispergillus niger grown on a glucose-nutrient salts medium in the wartime interest in 2,3-butanediol and today this chemical is presence of calcium carbonate gives gluconic acid i a 95% yie1.d not in large scale production because of its lack of large scale based on the carbohydrates consumed. utility. The technology of the fermentation both by members The itaconic acid fermentation has received considerable atof the genus Aerobacter and by Aerobacillus polymysa has been tention during the last few years (50, 58, 69, 66). Superior well developed and the economics of the fermentation are favoryields of itaconic acid are obtained with Aspergillus terreus on. able. glucose nutrient media. Aerobacle,, will give as high as 40% yields based on tho weighr, Fumaric acid can be produced on a large scale according to the of sugar ferinented. Carbohydrate sources used are wood process described by Waksman (99) using molds of the order sugar, acid hydrolyzed starch, and grains and molasses (69, Jlucorales on a carbohydrate medium having a zinc salt present 76, 105). during the growth of the mycelium and then replacing the original Aerobacillus poEyinyxa will ferment starch directly. The culture fiuid with a carbohydrate solution containing an ircjr, yields of 2,3-butanediol are lower than with the Aerobacter fermend t and completing the fermentation at pH 5.0 to 6.5. tation (1, 6, 3, I S , 29). This fermentation has some ad2-Ketogluconic and 5-ketogluconic acids can be produced f r o x vantages, however, in that only the I-2,3-buCanediol is formed, glucose by bacteria of the genus Pseudomonas and Acetobucter, This product may have potential use as a n antifreeze compound. respectively, by aerating and agitating the inoculated masherAcetoin is readily obtained by bacterial oxidation of 2,slander pressure (86, 104). butanediol (31,85,94). STREPTOMYCIN
CITRSC ACID
At the present time i t is believed that essentially all the citric: acid prepared by fermentation is produced by surface culture processes. However, much of the recent uvorlc has been directed toward submerged citric acid production. ‘The submerged growth procedures described by Waksmetn and Xarow (100, 101) and by Szucs (87) involve growth of the mold m j d i u m on one medium, followed by citric acid production on another. Considerable work must still be done on cultures, media, and techniques of operat,ion before submerged fermeiitation for citric acid becomes economically feasible. Citric acid fermentation yields appear to be sensitive to trace metallic constituents in the medium (73)and the effect of these metals varies widely with different st’rains. A large part of the material in cane molasses responsible for low citric acid yields is present in the ash. Pretreatment of the cane molasses with cation exchange materials or by precipitation with potassium ferrocyanide removes some of the inhibitory material and results i n higher yields of citric acid ( 7 4 ) . Von Loesecke has presented a n extensive review on m logical citric acid production (98). The operation of a German citric acid plant, employing surface
YEAST Recent yeast proauctfciii results :i.ith baker’s and food yea.;t,a have been covered in a review by Johnson (48). Thaysen and his co-workers (01, 9%) developed e, process for the continuous production of Torulopsis utilis (var. major) in which the yields obtained were as high as 6006 dry yeast based on -the augar utilized. Food yeast, produetion from a variery uf substrates has alsc received much attention. A strain of Torula yeast has beer: found l o grow readily in sulfite waste liquor. This process not only gives a vahablc yeast product but aids in solving the waste disposal problem of the paper industry. The techniques of producing a fodder yeast from- waste sulfite liquor have beer: presented (40). The sulfur dioxide vas removed by steam stripping or lime precipitation and excessive foaming was overcome by using fermenting apparatus having a central draft tube. Wood hydrolyzates have beera found t o be a suitable substrate for Torula utilis and Candida tropicalis (75) and suitable production techniques have been developed for this substrate as w!l as for using Torula on wood sugar or ethyl alcohol stillage (63,54) D e Becse and Liebmann (23) have revieved the aeration problems in yeast production. I n this review, based largely on the prewar German yeavt industry, ihe writers d.escribe in detail ~
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Streptomycin Fermentation Unit of Merck 81 Company, Inc., Elkton, Va.
several types of aerating systems from the perforated pipe type to very fine porous aerators with and without agitation. Air requirements for yeast production with these various systems are given along with power consumption figures for the compressed air. The Germans have developed the Waldhof fermenter (82) which contains a central draft tube. Below the draft tube the air enters through a revolving aerator and rises through the medium on the outside of the draft tube to the top of the fermenter. The fermenter is so designed, with regard to spacing arrangements of the draft tube and aerator, that foam, which forms a t the top of the batch, falls through the draft tube and is recirculated by the centrifugal action of the aerator. This fermenter has several advantages with regard t o low air requirements and lack of necessity for defoamers but power costs are believed to be somewhat high. Fermenters of this type should have rather wide application in the fermentation as well as the yeast industry.
MICROBIAL ENZYMES There are a large number of commercial enzyme preparations which are produced by microorganisms. No attempt will be made here to give a detailed review of microbial enzyme production. A fairly recent review is available (10.2). Some preparations are impure concentrated enzyme eomplexes whereas other preparations contain somewhat purified enzymes for specific purposes. One aspect of mold enzyme production
and use was discussed previously in connection with the ethyl alcohol fermentation and this is potentially a large scale use for mold amylase.
BIOLOGICAL WARFARE During World War 11, the United States, in cooperation with Canada and Great Britain, carried out an extensive research and development program on biological warfare. The Merck report (63)defines biological warfare as the use, and defense against the use, of bacteria, fungi, viruses, rickettsias, and toxic agents derived from living organisms to produce death or disease in men, animals, and plants. A vast amount of information was gained with regard to production, properties, and use of biologically active agents. Although details of’production techniques are not available (63)i t is obvious that the development of methods and facilities for mass production of pathogenic microorganisms and their products and prevention of contamination of air, water, and land surrounding the experimental station have made large contributions which will be applicable in the fermentation industry.
FERMENTATION, U N I T PROCESS
A fermentation process may be said to be made up of two interrelated and usually overlapping parts. The first part is the development of cells in which the enzyme system may function, and the second is the production of the product by a series of enzyme-catalyzed reactions. Different fermentations are characterized by properties and conditions surrounding these two
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steps of the process. Under the present state of knowledge, it is not always easy to differentiate between these properties and conditions with respect to cause and effect. This is largely due to incomplete information relative to the mechanism by which the enzyme system is developed and the exact part it plays in the conversion of raw materials into products. Because of this lack of understanding, most fermentation processes have been initiated through screening a large number of combinations between various microorganisms and substrates. Once a potential combination has been found, the factors surrounding the fermentation have been evaluated by trial and error, which leads to the development of increasingly efficient processes. T o date it has not been possible to define quantitative relationships for the prediction of fermentation performance. Thus the discussion of fermentation as a unit process resolves itself to a qualitative discussion of those factor2 which experience has shown to be important in the various fermentation processes. MICROORGANISM
The microorganism chosen should be stable, rugged, and capable of giving uniform yields under standard operating conditions. The protection of this microorganism and the elimination of other interfering organisms are among the more inipor tant requirements of a good fermentation process. The stability of microorganisms, while they are being used in the fermentation system, is one of the properties that limits the length of time a given fermentation system can be said t o function properly and one of the reasons why the development of continuous fermentation processes has been retarded. On the other hand, induced mutation has been a technique used in the laboratory for the development of higher yielding and more efficient organisms. I n many fermentations there is a period of adaptation of the organism to the substrate chosen for the fermentation and usually the medium used for inoculum development is the same as t h a t in the production fermenter. However, there are known exceptions to this procedure and it is not uncommon to attain superior production yields using inoculum developed in subsl iates which differ from t h a t of the production medium. SUBSTRATE
The substrate is made up of those materials found necessary for the proper development of the cell system and those t h a t will be converted to product. The materials going into the substrate normally cannot be clearly defined, because usually a portion of the substrate comes from natural sources and undoubtedly rontains some of the necessary trace materials required for a good fermentation. rllthough some progress has been made on the use of synthetic media, in general, poorer results are obtained. This i s probably due to the fact that insufficient information ie available on the trace materials required for the fermentation. TRACE NUTRIENTS AND T O X I C A N T S
Trace materials, both organic and inorganir, are known maIked1y to affect ferinentation processes. Certain of thesc materials act as nutrients or growth factors for the microorganism, while others are toxicants and tend to inhibit the normal couise of fermentatioii. Although it 1s possible in some cases to knolv and control the effect of these materials, often they are apparently present, but undefined, in the raw materials used for fermentation. I n such cases, the raw materials must be chosen by trial and error and evaluated by fermentation methods. As equipment is always subject to some corrosion, the choice of materials of construction is made, at least in part, through consideration of the nutrient or toxjc effects that it may have on the fermentation.
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STERILIZATION
Sterilization requirement are an important part of every fermentation process. However, the degree of asepsis required depend7 upon the particular process being considered. T h e more rigorous the adherence t3 3terile operation, the more closely pure culture fermentation is achieved. Certain fermentations, such as the antibiotics, arc extremely sensitive to contamination, and in such processes every precaution is taken for the maintenance of pure culture fermentation. Pure culture fermentation is also a ncccssary requirement for any continuous process if uniformity of the organism is to be maintained. On the other hand, certain fermentations, such as batch fermentation for alcohol, are less sensitive to rontarnination and less rigorous conditions regarding asepsis aIe necessary for efficient pcrformance. Generally, sterilization of the substrate is accompliJhed by heat. However, the productivity from the substrate is often reduced by the use of excessive heating, possibly through degradation of some of the nutrient compounds in the substrate. Thus, thc sterilization should be done under conditions sufficient to bring about sterility but limited in order t o minimize degradation in the substrate. The application of continuous sterilization and mashing for tho preparation of the substrate has been discussed and the advantages of using such a process are obvious in the controlled timetemperature conditions to which the substrate is subjected. I n the case of aerobic fermentations, it is necessary to supply sterile air during the course of fermentation. Such air has been produced in a number of different ways, including passage of air through columns packed with carbon, cotton, or glass wool, subjecting the air to electronic precipitation or ultraviolet light, scrubbing with caustic, sulfuric acid, or disinfectant solutions, or by heat. The efficiency and the mechanism of sterilization by thesc methods have not been firmly established. However, t h e use of columns packed with carbon, cotton, or glass wool has been BYtensive and apparently satisfactory. T H E FERMENTER
The fermenter is fundamentally a tank, constructed of materials consistent with the fermentation for which it is intended, The prime requirement of the fermenter is that its design should be sufficient to maintain the required degree of asepsis during the fermentation. I n pure culture fermentation all inlets and outlets are protected against the possible entry of contamination into the fermenter. Temperature of fermentation is a n important variable and the fermenter should be designed with the ability to maintain tcmperature closely. I n the case of aerobic fermentations the agitation and aeration systems are also important. These are discussed below. The layout of fermenters in a fermentation procesb depends largely upon the method which is to be used for initiating fermentation. If the large scale fermentation is initiated by spores or small volume vegetative inoculum, the fermentation time in the production unit is cxtcnded appreciably. I n order to achieve maximum productivity per gallon of installed fermenter capacity, many fermentation processes are initiated from spores through soed fermenters o i progressively larger size so that more adequate percentage of inoculum may be added to the final. or production, fermenter. At times it is the practice t o cross-inoculate from one large fermcnter to another. The success of this type of inoculatioa depends in large measure on the stability of the organism, as successive fermentations are carried out n ith organisms further and further removed from the initial culture. T h e seed fermenters mentioned above, although smaller in size, are usually similar in design t o the main production fermenter.
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THE F E R M E N T A T I O N
Anaerobic fermentation is accomplished in a two-phase system (medium solution and cells). By the same reasoning, aerobic fermentation involves a three-phase system. The phases to be considered are medium solution, cells, and air, and the mechanism of fermentation undoubtedly involves the transfer of various materials from one phase t o another. Following this reasoning, it is probable t h a t the rate and efficiency of fermentation are affected, at least in part, by those factors which have been shown to affect the rate and efficiency of heterogeneous chemical reactions. A large nutnber of rate processes could be visualized for any particular fermentation. To date, little information is reported relative to the individual resistances which may be involved or the relationship of one resistance to another in evaluating possible controlling steps for the rate of fermentation. The effects of aeration, agitation, and temperature, as well as physical properties of the substrate and the cells, are the factors which affect these rate steps and fundamental work is needed in order to attain a better understanding of this phase of fermentation and consequently the development of more efficient designs for fermenters and fermentation procedures.
LITERATURE CITED Adams, G. A., Can. J . Research, F24, 1 (1946). Adams. G. A.. and Leslie, J. D., Ibid., p . 12. Ibid., p. 107. Adams. S. L.. Balankura. B.. Andreasin. A. A , , and Stark, W. H., IND.'ENG.'cHEM., 39, i 6 i 5 (1947). Altsheler, W. B., Mollet, H. W., Brown, E. H. C., Stark, W. H., and Smith, L. A,, Chem. Eng. Progress, 43,467 (1947). Arsberger, C. F., U. S. Patent 2,326,425 (Aug. 10, 1943). Backus, iM.P., Stauffer, J. F., and Johnson, M. J., .J. Am. Chem. SOC.,68, 152 (1946). Beadle, G. W., Am. Scienlist, 34, 31 (1946). Beadle, G. W., Chem. Eng. A-ews, 24, 1368 (1946). Beadle, G . W., Chem. Rev., 37, 15 (1945). Beadle, G. W., Physiol. Rev., 25, 643 (1945). Benedict, R. G., and Langlykke, A. F., Ann. Rev. Microbiol., 1, 193 (1947). Blackwood, A: C., and Ledingham, G. A., Can. J . Research, F25, 180 (1947). Boruff, C. S., and Van Lanen, J. AM., IND. ENG.CHEM.,39, 934 (1947). British Intelligence Objectives Sub-committee, H.M. Stationery Office, London, Final Rept. 220, Item 22. Ibid., 489, Item 22. Burkholder, P. R., Arch. Biochem., 3, 121 (1943). Burkholder, P. R., PTOC. Natl. Acad. Sei., 29, 166 (1943). Burkholder, P. R., U. S. Patent 2,363,227 (Nov. 21,1944). Burton, L. V., Food Inds., 9, 571, 617 (1937). Callaham, J. R., Chem. Be Met. Eng., 50, 104 (December 1943). Dale, J. K., U. S. Patent 2,421,142 (May 27, 1947). de Becse, G., and Liebmann, A. J., IND.ENG.CHEM.,36, 882 (1944). Erb, N. M., and Hildebrant, F. &I., Ibid., 38, 792 (1946). Ericsson, E . O., Chem. Eng. Progress, 43, 165 (1947). Filachione, E. M., and Fisher, C. H., IND.ENG. CKEM.,38, 228 (1946). Foster, J. W., Woodruff, H. B., and McDaniel, L. E., J . Bact., 51, 465 (1946). Foster, J. W., Woodruff, H. B., Perlman, D., MeDaniel, L. E., Wilker, B. L., and Hendlin, D., Ibid., 51, 695 (1946). Fratkin, S. B., and Adams, G. A , , Can. J . Research, F24, 29 (1946). Fulmer, E. I., Brewers Digest, 18, 37 (September 1943). Fulmer, E. I., Underkofler, L. A., and Bantz, A. C., J . Am. Chem. SOC.,65, 1425 (1943). Gallagher, F. H., Bilford, H. R., Stark, W. H., and Kolachov, P. J., IND. ENG.CHEW., 34, 1395 (1942). Giulliermond, A., Rev. mycol., 1, 115 (1936). Gordon, J . J., Grenfell, E., Knowles, E., Legge, E. J., McAlister, R. C. A., and White, T., J.Gen. Microbiol., 1, 187 (1947).
1607
(35) Gottlieb, D., and Anderson, H. W., Bull. Torreg Botark. Club, 74, 293 (1947). (36) Gottlieb, D., and Anderson, H. W., Science, 107, 172 (1948). (37) Grenfell, E., Legge, B. J., and White, T., J . Gen. Microbiol., 1, 171 (1947). (38) Harris, E. E., and Beglinger, E., IND.ENG. CHEM.,38, 890 11946). (39) Harris, E. E., Hajny, G. J., Hannan, M., and Rogers, 8. C., Ibid., 38, 896 (1946). (40) Harris, E. E., Hannan, M., and Marquardt, P. R., Paper Trade J.,125, 34-37 (1947). (41) Hickey, R. J., U. S. Patent 2,425,280 (bug. 5, 1947). (42) Hiauchi, K.. Jarvis. F. G.. Peterson, W. H.. and Johnson, M. J.. 2. Am. Chem. Soc., 68, 1669 (1946). (43) Hilbert. G. E.. et al.. IND.ENG.CHEM..37. 4 (1945). (44j Hines, G. E., U. S. Patent 2,367,644 (Jan. 16, 1945). (45) Ibid., 2,387,023 (Oct. 16, 1945). (46) Jacobs, P. B., "241cohol from Agricultural Commodities," Agr. Research Adm., U. S. Dept. Agr.,1945. (47) Johnson, M. J., Ann. N . Y . Acad. Sei., 48,57 (1946). (48) Johnson, M. J., Ann. Rev. MicrobioE., 1, 159 (1947). (49) Joseph, H. G., Sewage Work J.,19,60 (1947). (50) Kane, J. H., Finlay, A. C., and Amann, P. F., U. S. Patent 2,385,283 (Sept. 18, 1945). (51) Kereszetesy, J. C., eta!., U. S. Patent 2,355,220 (Aug. 8, 1944). (52) Koffler, H., Knight, S. G., Frasier, W. C., and Burris, R. H.. J . Bact., 51, 385 (1946). (53) Kurth, E. F., IND. ENG.CHEM.,38,204 (1946). (54) Kurth, E. F., and Cheldelin, V. H., Ibid., 38, 617 (1946). (55) Legg, D. A., and Beesch, S.C , U. S. Patent 2,370,177 (Feb. 27, 1945). (56) Leonard, R. H., and Peterson, W. I€., IND.EXG. CHEM.,39, 1443 (1947). (57) Leviton, A,, J.Am. Chem. Sac., 68, 835-40 (1946). (58) Lockwood, L. B., and Reeves, M. D., Arch, Biochem., 6, 455 (1945). (59) Lockwood, L. B., and Ward, G. E . , IND.EN^. CHEM.,37, 405 (1945). (60) McCoy, E. F., U. S. Patent 2,358,837 (Apr. 23, 1946). (61) McMillan, G. W., Ibid., 2,367,646 (Jan. 16, 1945). (62) Meak, R. E., Pollard, H. L., and Rodgers, N. E., Ibid., 2,369,610 (Feb. 20, 1945). (63) illerck, G . W., Chem. Eng. News, 24, 1346 (1946). (64) Miner, C. S., U. S.Patent 2,202,161 (May28, 1940). (65) Moyer, A. J., and Coghill, R. D., Arch. Biochem., 7, 167 (1945). (66) Moyer, A. J., and Coghill, R. D., J.Bact., 51, 57 (1946). (67) Ibid., 51, 79 (1946). (68) Ibid., 53, 329 (1947). (60) Olsoii, R . H., and Johnson, M. J., Ibid., 55, 209 (1948). (70) Oxford, A. E., Lampen, J. O., and Peterson, W. H., Biochem. J . , 34, 1588 (1940). (71) Peckham, G. T., Chem. Eng. News,22, 440, 469 (1944). ENC.CHEM.,36, 803 (1944). (72) Perlman, D., IND. (73) Perlman, D., Dorrell, W. W., and Johnson, M. J., Arch. Biochem., 11, 131 (1946). (74) Perlman, U.,Kita, D. A., and Peterson, W. H., Ibid., 11, 123 (1946). (75) Peterson, W. H., Snell, J. F., and Fraeier, W. C., IND.ENG. CHEM..37. 30 11945). (76) Piersma,". O., U'. S. Patent 2,400,710 (May 21, 1946). (77) Porter, R. W., Chem. Eng., 53 (lo), 94 (1946). (78) Ibid., 54 (12), 141 (1947). (79) Raper, K. B., Ann. N . Y . Acad. Sci., 48, 41 (1946). (80) Rudert, J., U. 5. Patent 2,374,503 (April 24, 1945). (81) Ruf, E. W., Stark, W. H., Smith, L. A., and Allen, E. E., IND. ENG.CHEM.,40, 1154 (1948). (82) Saeman, J. F., Looke, E. G., and Dickermann, G. K., F I A T FinaZ Rept. 499, 117 (Joint Intelligence Objectives Agency, Washington, D. C.) (1945). (83) Sjolander, N., and Eisenman, W., U. S. Patent 2,401,778 (June 11, 1946). (84) Stefaniak, J. J., Gailey, F. B., Brown, C. S., and Johnson, M. J., IND.ENG.CHEM.,38,666 (1946). (85) Stefaniak, J. J., Gailey, F. B., Jarvis, F. G., and Johnson, M . J., J . Bact., 52, 119 (1946). (86) Stubbs, J. J., Lockwood, L. B., Roe, E. T., and Ward, G. E., U. S. Patent 2,318,641 (May 11, 1943). (87) Ssucs, J., Ibid., 2,353,771 (July 18, 1944). (88) Tanner, F. W., Jr., et al., Science, 101, 180 (1945). (89) Tanner, F. W., and Van Lanen, J. M., U. 5.Patent 2,424,003 (July 15, 1947). (90) Tatum, E . L., Am. Rev. Biochem., 13, 667 (1944). (91) Thaysen, A. C., Food, 14, 116 (1945). (92) Thaysen, A. C., and Morris, M. B., British Patent 560,800 (April 28. 1944). (93) Tousley, R. D., Chem. & Met. Eng., 50,120 (October 1945). \ - - - - I
INDUSTRIAL A N D ENGKNEERING CHEMISTRY
160%
Underkofler, L. A., Fulmer, E. I., Bantz, A . C., and Kooi, I?. I., Iowa State Coll., J. Sci., 18, 377 (1944) (95) Underkofler, L. A., Severson, GI M., and Goering, K, J., Iwn ENC.CHEM.,38, 980 (1946). (96) Underkofler, L. A., Severson, G. M., Goering, K. 9.. and Christensen, L. M., Cereal Chem., 24, 1 (1947). (97) 'Vnger, E. D., Wilkie, H. F . , and Blankmeyer, XI, O., T'r(zm.4 m (94)
a
Inst. Chem. Engrs., 40, 421 (1944). (98) von Loesecke, H. W., Chem. E%@. N e w s , 23, 1952 (1945). (99) Waksman, S. A., U.S. Patent 2,326,986 (Aug. 17, 1943). (100) Waksman, S.A , and Karow, E. O., IND. ENG.CHCN.,39, 82' (1947).
/109) Waksman, S. A., and Ksrow, E. O . , U. 8. Patent 2,394.03'' (Feb. 5, 1946).
RIEDEL and Crafts showed that, aluminurn chloride and other metal halides could be used as a condensing agent for alkylations, dealkylations, acylations, polymerizations, and her unit processes. Syntheses made possible through. the catalytic intervention of metal halides now include the preparat,ion of practically all classes of compounds. The more importanf of the syntheses include alkylation, acylation, halogenation, dehydration, dehydrogenation, isomerization, polymerization, and intra- and intermolecular rearrangements. The use of metal halides in syiithetic organic chemistry, like the employment of microorganisms (SO), is merely a convenient and important tool that can be used Cor the economic production of a wide varktjy of commercially useful compounds ( 8 , 14, bo). I n t'he foliowing review, however, the recent advances in alkylations, isomerizations, polymerizations, and Pries migrations t h a t are catalyzed by Friedel-Crafts catalysts Rave not been included hecause of assignment to other contributors.
MECHANISM OF REACTlON
The mechanisms of Friedcl-Crafts syntheses have long bcsrsubjects of experimentation and conjecture. K i t h the use of radioactive isotopes and new scientific tools, more light is beirig she(% on this phase of the reaction. Ulich ( S I ) has concluded t h a t tht. ketone synthesis takes place eit,her as a homogeneous reaction after aluminum chloride has gone into joliitioni in the _Form of at! addition compound, or as a surface reaction if an excess of a h aninum chloride is present. The hydrocarbon synthesis k autocatalytie and proceeds rapidly after a heavy oil phase has been formed by the addition of aluminum chloride to the reaction products. The use of gallium chloride, which is readily soluble irr many solvents, makes possible hydrocarbon syntheses as a purely homogeneous reaction. Its eEcienxy, however, decreases during alyst or disconlinuatior. the reaction owing to poisoning of t,be of the promoter effect. I n 1937 Fairbrother (11) suggested that in Friedel-Crafts reactions involving acyl and alkyl halides, the addition complexez bhat are formed may be regarded as coordination compounds containing the anion (AlC14)-: CH&OCl
+ MCI, --+ ;(:.HsCOj
-+
4 (AICh)
In the anion all the chlorine atonia concerned in the reactiorj ehould have a n equal chance of escaping as hydrogen chloride,
Vol. 40, No. 9
Wallerstein, L ~INO. ? Ewc;. C H E M 31, ~ , 1218 (1939). Walton, M . T., U. S. Patent 2,368,074 (Jan. 23,1946). Ward, G. E., Lock\wood, 1;. B., Tabonkin, B., Stubbs, 9. J,, and Roe,E. T., Can. Patent 416,593 (Nov. 23, 1943). (105) Ward, 6. E., Pettijohn, 0. G , , and Cophill, R.D., ISD. EN@. CHEM.,37, 1189 (1945). (106) Wickerbam, L. J., et al., Arch, Bbchem., 9 , 95 (1946). (107) Wileyp-4. J., Johnson. M. J., M c C o y , E., and Pe
(102) (103) (104)
IND. ENO.GIEM., 33, 606 (1941).
(108) (109)
Williams+A. E., Mfg. Chemist, 16, 239 (1945). Wood, R.G., Brown, R.V;'~~m d Werkman, C~
Arch. Bio-
chem., 6 , 213 (1945),
IVR:D
July 6, 4948.
T h i a wab confirmed eP;perirrrenically b y the w e of radioactive chlorine isotopes. Korshak and Kolesnikov (18) have addured further evidence in support of Fairbrother's hypothesis. I\ her. benzenp, ethyl chloroformate, and aluminum bromide ere permitted t o ieact under agitation, the evolved gases icjnraineu 132 26 mole yo hydrogen bromide and 17.74 mole 7, hydroger, chloride. Ailurninurnbromide and acetyl chloride yield a cornplex which, when sublecied t o distillation, yields a mixture of ace:) 1 chloride (83.6Sc6) and acetyl bromide (16.32%). Tha rpaetxon of the above complex with absolute ethanol or beneenr gave analogously mixturrs of hydrogen bromide and hydrogrlrr vhloride in similar ratio. Irl furthev studies, Korshak and Koleiriikov (19: ahowred t h a t che rraLtion of aluminum bromide with diverse albjl, aryl, and ari lalli-j1 chlorides resulted in the forniation of about 75% hydrogen bromide and 25% hydrogrn chloiide Similarly t h e use of aluniinuni chloride with bromo compounds gave approximately 7 5 c hydrogen chloride and 25m0 hydrogen bromide These resulk confirm the belief that. the mechanisrr, depends on the formation of a complex containing the ion (AlX,)rhat can react with equal fnrilltv through any of the halopri *,omt.
XHEWMODYNAMlCS
OF FRIEDELCRAFTS REACTIONS
'1he heals of formation of APC13 aluminum chloride comploxcs wth orgacic compounds are ir? eome inqtances large, and thi4 complex formation has a great effect on the courw as svpll x4 products of reaction.
In reactions such as shown below,
n:k~feCX(g)f CsHs(l)
A4.1CY*
--+
CGR6_.,Me,(l)
+ nRC1
(2:
Campbell and Elejr (9) found that the heat of formation of h!Brs(C&&CHO), is 30 kg.-cal. per mole of aluminum bromide. This compiex formation, by making total free energy of reactlion strongly negative, is fmport,ant in obt,aining the excellent no~-rnd yields. In Reaction 2 thermodynamic calculations, excluding complex formation, show no preference among toluene, CsH4(CH,),, and @e113(CH3)3. The heats of complex formation are hICla.@&L(CHa)z= 22 kg.-cal.; AIC13.CflHa(CHs)a= 8 kg.-cal.; and AICI3.GHbCH,= 0 kg.-oxl.
