Gluconic Acid Production by Submerged Mold Growths under Increased Air Pressure 0. E. MAY,H. T. HERRICK,A. J. MOYER,AND P. A. WELLS,Bureau of Chemistry and Soils, Washington, D. C.
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growth cultures had no influence, UNGI, such as species of The production of gluconic acid f r o m comqualitatively or quantitatively, Aspergillus and Penicilmercial glucose by submerged growth cultures of on the acid formed. lium, are strongly aerobic Penicillium chrysogenum is greatly improved by I n 1928 Schreyer (8) reported organisms and therefore carrying out the process under increased air presthe r e s u l t s of a d e t a i l e d inn o r m a l l y grow and carry out sures. The addition of calcium carbonate to the vestigation of A . jumaricus, an their biological functions on the organism which produced mixsurface of liquid or solid subculture solutions causes a marked increase in the tures of g l u c o n i c a n d citric strates where they have access to yields. At and above a total air pressure of 3 acids from glucose. It was found a plentiful supply of o x y g e n . atmospheres and with a ratio of 1 gram of calcium that aeration and agitation of Under such conditions they are carbonate to 4 grams of glucose in the culture cultures to which calcium carcapable of i n d u c i n g complex bonate had been added caused solution, yields of gluconic acid f r o m 80 to 87 per chemical reactions of consideran increase i n g l u c o n i c a c i d able diversity, the n e c e s s a r y cent based o n sugar originally present, have been p r o d u c t i o n f r o m four to six energy being supplied almost enobtained in a culture period of 8 days f r o m the times that obtained with nontirely by oxidation of available time of inoculation with spores of the organism. aerated surface control cultures. carbohydrates to c e r t a i n end C i t r i c acid formation was Droducts. The Darticular substance or substances which finally appear depend on a number not affected. Two years later Thies (9) reported similar reof factors and may range in degree of oxidation from gluconic sults in experimenh with the same organism in which oxygen acid to carbon dioxide. Some of these biological oxidations, instead of air was bubbled through culture solutions consuch as those involved in the formation of gluconic and citric taining calcium carbonate. The best yields reported were acids, are remarkably efficient and have been utilized in in- around 64 per cent of theory in a 15-day culture period. Redustry. Many difficulties, however, are encountered which are cently Currie, Kane, and Finlay (6),by the use of submerged not present in vat fermentations in which yeasts and bacteria mold growths, claimed yields of gulconic acid as high as 90 are commonly used owing to the fact that the oxidations are per cent of theory in 48 to 60 hours. By maintaining the culcarried out with comparatively shallow layers of solution in ture liquid in a state of great agitation by means of a highwhich the ratio of surface area to volume (square centimeters speed stirrer, and at the same time drawing large volumes of to cubic centimeters) is rarely less than 0.2. If conditions air into the solution, a foamy mass of liquid was obtained, could be established under which molds could accomplish their and the organism was kept submerged and dispersed throughnormal oxidizing functions while in a submerged state, vats out the solution. might be employed instead of shallow pans, with a consequent Consideration of the work of Shreyer and of Thies pointed simplification of the utilization of these organisms. to the possibility of establishing the production of gluconic The ability of molds to produce hydrolytic enzymes under acid by submerged mold growths on a practical basis. Incertain conditions when grown submerged rather than as sur- creased oxygen concentration throughout a submerged growth face mycelia has been utilized for centuries in the Orient, and culture of a vigorous gluconic-acid-producing organism the modern amylo process for the manufacture of ethanol seemed to be a factor which might improve t h e efficiency of from starch has been evolved from old methods used in the the oxidation. With this in mind, a study has been made to preparation of alcoholic beverages in the Far East (11). The determine the effect of a n increase in the total air pressure ease with which submerged growths hydrolyze starch has also over the solution on gluconic acid production by a &rain of found application in the manufacture of lactic acid by fer- Penicillium chrysogenum. mentation (3). Another example of the utilization of hydrolytic enzymes of certain molds is found in the transformation EXPERIMENTAL PROCEDURE of tannin to gallic acid (4). Some investigation has also been made of the oxidative activities of submerged mold growths. Surface cultures of the organism used have been previously Wehmer (10) and Elving (6) reported experiments in which investigated here and have given excellent yields (65 per cent) citric-acid-producing organisms were forced to grow sub- of gluconic acid from glucose with no formation of oxalic or merged in aerated substrates, but the yields of citric acid ob- citric acids. A report of this work will appear elsewhere. tained under such conditions were low as compared with those I n the following work, commercial glucose containing apgiven by normal surface cultures. Bleyer (2) has described proximately 91.5 per cent of glucose and 8.0 per cent of water a process for the production of citric acid from sugars by was used in all experiments. Culture solutions were made up molds in which the solution, contained in vats, is aerated with to contain approximately the equivalent of 20 per cent of pure sterile air with occasional mechanical agitation of the liquid. glucose, and the actual quantity present was then determined Amelung (I) in 1930 reported the results of aeration experi- by the Schafer-Hartmann method. The following nutrient ments with submerged growths of Aspergillus japonicus, salts were used : a typical surface-growing, citric-acid-producing organism. While the mold grew well under such conditions, acid producSalt Grame/liter 3.0 tion was considerably less than that obtained with surface 0.15 cultures. Addition of calcium carbonate to the submerged 0.125 575
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Gas-washing bottles (500 cc.) constructed with sintered-glass false bottoms and fitted with ground-glass stoppers were used as culture flasks. Two hundred cubic centimeters of culture solution were used in each bottle. The cultures were sterilized by autoclaving a t 15 pounds per square inch (1.05 kg. per sq. cm.) steam pressure for 15 minutes, cooled, and inoculated with spores of the organism. When calcium carbonate was used, it was sterilized s e p a r a t e l y and added to the culture solutions after inoculation. The culture bottles were then placed in a constant-temperature autoclave, the details of which are shown in Figure 1. After making the connections with rubber tubing from the b o t t l e s t o the proper needlevalve outlets, the autoclave was closed and the desired air pressure built up. The pressure was controlled by valve A . From A the air passed through a humidifier, B, where it was saturated with moisture and then freed from s u s p e n d e d matter by passage through cotton in filter C. Upon opening the needle valves to the atmosphere, air passed through the culture solutions from the bottom (by way of their sintered-glass false bottoms, which broke up the air stream into fine b u b b l e s ) , aerating and agitating them. The rate of air flow was controlled accurately by adjustment of the needle valves and was measured by means of a calibrated flowmeter. Constant temperature was maintained by heating coil D, operated through a relay controlled by a mercury column in H . Provision was made for the sterilization of the autoclave by means of steam admitted through pipe L and exhausted through pipe M . At the termination of the e x p e r i m e n t the culture liquid was filtered from the submerged mycelial growth, which was thoroughly washed and pressed out. The washings were combined with the filtrate, and the volume was measured. Aliquots were then taken for estimation of reducing sugars and gluconic acid. After preliminary tests for oxalic and citric acids were made, gluconic acid was estimated by titration. In experiments in which calcium carbonate was added to the culture s o l u t i o n s , soluble calcium was determined by precipitation as calcium oxalate and titration with potassium p e r m a n g a n a t e and calculated back to gluconic acid. Yields were calculated as the ratio of glucose used to produce gluconic acid to glucose originally present in the culture solution. All results tabulated are averages of a t least four cultures, except when stated otherwise. GLUCONIC ACIDYIELDS Extremely variable yields of gluconic acid were obtained in the first experiments. This condition was remedied to a large extent by the addition of various inert materials to the culture solutions to serve as supports for the mold growth. These included chips of porous plate, asbestos, lump pumice, c h a r c o a 1, Filter-Cel, and r i c e hulls. The effect of increased air pressure in such experiments is indicated in Table I. While yields were improved in some measure, in no case did they exceed 50 per cent of theory. The most pron o u n c e d effect noted was a decided stabilization of acid production. When the pressure mas increased above that of the atmosphere, better checks were
obtained within a given group of culturea aid rims carried out under similar conditions.
iii
successive
%
%
%
%
%
1
52.5 52.3 84.2
13.t 20.6
52.4 56.6
:is.4 40.4
65.4
2
% a0.i
16.8
57.5
38.2
t6.2
43.5 41~7
2.6
pi
Addition of calcium carbonate to the culture solut,ioiis wns found t,o increase greatly the yield of gluconic acid and a1 avoided t.Jie necessity for any other supporting mcdiuirr the solutions. In the preseirce of ealcirirn carbonate the usc of increased air pressures exerted a ia\~orableeffect on acid prodiict.ion and induced a11 appreciable acceleratioii in the rate of oxidation, as sliown in Table if. Thur, lrliile tlie yields given in Table I were baaed on a culture period o f IO days, excellent yields of acid witli practically complet,e utilization of the glucose were obtaiired resularly in the presence o f calcium carbonate in a culture period of 8 day.;. However, when the experiroents were allou-ed to run as long as 8 days, increases in air pressure above 2 atniosplieres were apparently without advantnge, as practically all of the glucose was ut,ilized in that time at the lower pressures. If cultures were harvested a t 6 days after inoculation, the effect of increased air pressure iii accelerating the oxidation became noticeable, but greater variation in yield was observed within a given series. TABLE11. EFFECT OF INCREASED AIH PREYSUREY ON GLUCONIC ACID PRODUCTION IN PRESENCE OF CALCIUM CARBONATE
quaritit,y of calciuin carbormte eensed the calciiini glueonate t o crystallize io tile eulture solutions so tlrat in almost all of the experiiiient,s the solutiolis set t.o a stiff musli at 7 to 8 days after inoeulation. Such precipitation is objectionable only if at.tempts are made to re-use the mold growth for subsequent oxidat,ions, for the heating of t,lic solution to dissolve tile calcium gluconate has an adverse effect on the enzyme complex of tlie organism. The gluconate separating during the course of a run WRS of good quality, and one recrystallization to free it from hits of mycelium sufficed to yield a pure product. When 7 . 5 grams of calcium carbonate were used per culture,
FrGURE
2. THREE STAGES OF A SUHMERQED FERMENTATION; SURFACE CULTUREAT LEFI
separstioil took place only at infrequent intervals, but a slight diiniiiution in yield occurred. The three bottles shown iii Figure 2 illustrate three stages of a submerged fermentation. Calciurn gluconate has separated from the solution a t the right. A surface culture is included for purposes of coniparison. Preliiiiinary experiments liave been made at a pressure of :+ as:? 4i:2 8916 r6:4 :3.5 atmospheres to ascertain whether the mold growtli de4 70.8 61.0 94.0 82.4 veloped during the coursc of a run could be used again for a 5 74.3 66.1 83.0 81.3 6 74.5' 72.2" 99.0 86.1 secondary oxidatiotl by draining off the spent liquor and sup94.0 87.1 plying fresh glucose solution. The cultures were eacli made ,a .ayerage "1 t w o culturea up of 200 cc. of 20.8 per cent glucose nutrient solution to The quantity of cnlciuiri carbonate present in the culture which ouly 5 grams or calcium carbonate were added in order iriedium had a noticeable effect on tlie quantity of glucose to avoid crystallization of calcium gluwnate. They were utilized by the organism and on tire yield of gluconic acid. harvested 10 days after inoculation. The liquid was filtered The results present,ed in Table 111 were obtained at a pres- rapidly through clieesecloth attached to the neck of the culsure of 6 atmospheres, but values of the same relative order ture bottle and was subsequently analyzed for gluconic acid have been found at lower pressures. and reducing sugar. The mold growth was returned to tlie culture bottle, 200 cc. of sterile 20 per cent glucose solution TABLE111. EFFWPOF QUANTITY OF CALCIUM CAHBONATE containing no niineral nutrients were added, and the bottle ON GLUCONIC ACID PRODUCTION was replaced in the autoclave. Four days later the culture solution was removed and analyzed, Two runs were thus riiado in a period of 14 days wit,h one inoculation. A total of 74.4 graou of gluconic acid vas produced which was equiva% % lent to a yield of 83.8 per cent on the glucose supplied. This 0 40.8 28.8 is practically t.he same yield as would be obtained from two 2.5 70.9 68.3 5.0 83.3 71.2 ordinary runs requiring a total time of 16 days. Aii addi1o.o 95.0 88.5 tional inoculation and cleaning of equipment were eliminated, 12.0 96.8 86.8 as well as 2 days of culture. The saving in time could uriIn ailditioii, Iiumerous experiments carried out a,t different douhtedly be extended st.ill farther by additional experimentimes liave established that the ratio of 10 grams of calcium tation. carbonate to 40 grams of glucose is sufficient to neutralize all Once the irriiriiiiurii rate of aeration nec of the gluconic acid produced and that sucli cultures give 80 agitation of the solution liad been reached, it was found that to 87 per cent yields consistently. However, tlie use of this furtlier increase in the rate was without pronounced effect.
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Thus, increasing the flow of air through the solution from 40 to 160 cc. per minute brought about only slight improvement in acid production. TABLE IV. DISTRIBUTION OF GLUCOSE AT TERMINATION OF EXPERIMBNT
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of calcium carbonate represents an average of 6 per cent of the sugar consumed, while in those cultures containing 10 grams of calcium carbonate it is equivalent to 4 per cent. The portion of the sugar consumed appearing as mycelium and unknown by-products ranges from 5.7 to 8.1 per cent. The yields of gluconic acid resulting from the use of sub(200 cc. nutrient solution containing 40.6 grams glucose per flask. rate of airation through Bolution, 40 DC.per minute: preasure, 3 atmbspheres; merged growth cultures under pressure were uniformly sutemperature, 30" C.) perior to those obtained with control surface cultures grown at UTILIZATION OF GLUCOSE-Mycelium atmospheric pressure. It was found that increased air presCaCOs Gluconic and coz PmB CULTURS acid Respiration by-productem Unchanged PRODUCJ~D sure over undisturbed surface cultures exerted an inhibiting Grama Gram8 Grams Grams Grams Grams rather than an accelerating effect on acid production, as yields 2.37 2.1 2.5 32.0 4.13 3.47 obtained under pressure were always slightly (10 per cent) 2.5 27.8 1.80 2.6 8.40 2.64 10.0 35.6 1.58 2.6 0.92 2.32 but consistently lower than those obtained with controls a t 2.7 34.3 1.55 10.0 2.05 2.28 atmospheric pressure. Furthermore, the submerged growth e Calculated by difference. cultures seemed to be less sensitive than surface cultures to Some carbon dioxide is produced in the biological oxidation changes in culture conditions, as tap water was substituted of glucose to gluconic acid as a result of side reactions, prob- for distilled water in several runs and variations were made ably connected with the synthesis of living matter. The in the concentration of nutrient salts without appreciably quantity of carbon dioxide formed was determined experi- disturbing the yield of gluconic acid. mentally by absorption in 17 per cent potassium hydroxide contained in gas-washing bottles similar to those used as ACKNOWLEDQMENT culture vessels. After passage through the absorption flasks, The authors wish to express their appreciation to G. E. the gases were led through barium hydroxide solution in order Ward and L. B. Lockwood for assistance rendered in the to detect any unabsorbed carbon dioxide. The same quantity course of this investigation. of air passing through the culture vessels was led through control absorption flasks to obtain blank corrections on carbon LITERATURE CITED dioxide in the air. Determination of absorbed carbon dioxide Amelung, H., Chem.-Ztg., 54, 118 (1930). was made according to the modified method of Winkler (7). Bleyer, B., German Patent 434,729 (1926). Deductions from total carbon dioxide found were made for Boullanger, E.,British Patent 13,439 (1900). Calmette, A,, German Patent 129,164 (1902). carbon dioxide in the air and for that evolved as a result of the Currie, J. N.,Kane, J. H., and Finlay, A., U. S.Patent 1,893,819 action of gluconic acid formed on the calcium carbonate pres(1933). ent in the culture solutions. The value for the latter was Elving, F., Ofersigt Finska Vatenskap SOC.Fard. (in German), calculated from the quantity of soluble calcium found in the 61,No. 15 (1918-19). Kuster, F. W., 2.anorg. Chem., 13, 127-50 (1897). culture solutions a t the termination of the experiment. The Schreyer, R.,Biochem. Z., 202, 131-56 (1928). results are presented in Table IV. The organism must deThies, W.,Zentr. Bakt. Parasitenk., I I , 8 2 , 321-47 (1930). rive almost all of its energy from the oxidation of glucose Wehmer, C., Chem.-Ztg.,36, 1106-7 (1912). to gluconic acid rather than to carbon dioxide. A greater Wehmer, C., in Lafar's Handbuoh der technisohen Mykologie, Vol. 5, pp. 319-42, Jena, 1905-14. part of the sugar consumed goes to carbon dioxide when the oxidation is carried out in the presence of smaller quantities R ~ C E I VJanuary ~D 17, 1934. Preaented before the Division of Biological of calcium carbonate. This general trend is also indicated Chemistry at the 85th Meeting of the American Chemical Society, Washingby the results given in Table 111. The sugar disappearing ton, D. C., March 26 to 31, 1933. This paper is Contribution 232 from the to form carbon dioxide in the cultures containing 2.5 grams Color and Farm Waste Division, Bureau of Chemistry and Soila.
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