Citric Acid Fermentation on Solid Materials F. J. CAHN, 4901 Glenwood Ave., Chicago, 111.
T
WO general methods of carrying out a mold fer-
Citric acid can be produced by fermentation with Aspergillus niger for 38 to 60 hours on solid materials containing sucrose or molasses. The citric acid yield is 45 per cent of the sugar content of the molasses and 55 per cent of the sucrose if pure sucrose is used, based on the materials introduced into the fermentation. The sugar solution is absorbed on beet p u l p or cane pulp which furnishes a large surface for the fungus growing in the spore-forming state. Sterilization of the mass to be fermented is not necessary, because the citric acid which is formed after one day suppresses the growth of the contaminating organisms.
mentation are possible. In the first method the fungus is grown on top of the solid or liquid medium which is to be fermented; in the second, the fungus is developed throughout the liquid. In the present paper the citric acid f e r m e n t b t i o n on a solid medium will be described. The first investigator t o try the commercial production of citric acid by fermentation was Wehmer (9). He used Citromyces as his organism in 1892 and in 1912 investigated the suitabilits of Aspsrg&s niger for citric"acid pioduction. Currie (Q fermented sucrose to citric acid by several Aspergillus niger strains and carefully investigated the nutritional conditions under which his special strains, growing on the surface of the liquid culture medium, produced the best yields of citric acid from sucrose. This work was recently confirmed by Doelger and Prescott (3) and was extended to glucose fermentation by Moyer (6). All these fermentations take a comparatively long time. With Currie's conditions the fermentation requires 6 to 8 days. During this long period the danger of contamination is always present thus making necessary careful sterilization of the medium prior t o the fermentation and sterile protection during the fermentation. In the experiments of Currie, of Moyer, May, and Herrick, and of Doelger and Prescott, high yields of citric acid on the sucrose or the glucose introduced were obtained. The present author and other investigators have found that Currie's procedure makes necessary an exceedingly fine control of the medium. The organism must be kept on a starvation diet as far as nutrient salts or vitamin-like substances are concerned. Under such conditions the fungus produces a few spores only after about 8 days. During the first 3 or 4 days after inoculation on liquid media the citric acid production on Currie's or similar poor media is only approximately 0.5 to 1 per cent of the sucrose introduced. FERMENTATION O N SOLID
CULTURES
The author's procedure, which consists in fermenting finely shredded carbohydrate-containing materials (I), allows the composition of the medium to be varied over wide ranges, from pure sucrose to blackstrap molasses. The fermentation time is from less than 2 days (38 hours) up to 4 days. Sterilization of the medium and special protection of the fermenting mass against invading organisms is not necessary. The good yield and rapid fermentation are due to the use of a very large surface of the culture medium on which the fungus can develop in contact with the air. The air provides the oxygen necessary for the aerobic citric acid fermentation. Falck and Kingma a t an earlier date (5) pointed out the importance of the ratio of surface area to volume of medium to be fermented, and Falck (4) described in a patent, simultaneously with the present author , fermentation procedures on lines similar to those to be described.
In the procedures of the author and of Falck the carbohydrate is
oxidized t o citric acid before sporulation begins. Since sporulation occurs under the favorable nutritional conditions that are offered to the developing f u n g u s , it takes place on the s e c o n d , third, or fourth day. When the fungus develops on top of a liquid layer about 13 mm. in depth and the solution is rich in nutrient salts, the fungus film produces such a large quantity of citric acid during the first 2 or 3 days that the citric acid concentration in the liquid will be about 0.5 per cent. When the fungus is allowed to develop on very thin liquid layers or thin solid layers of equally good nutritional value, the actual amount of citric acid produced in the first 2 or 3 days will be the same per area of fungus films as is produced with thick films or layers, but the concentration of citric acid will be 7 t o 8 per cent on the thin solid film covered with fungus growth on both sides and about 4 per cent on the liquid film covered with fungus growth on the surface only. At the same time the yield of citric acid based on the sucrose introduced into the fermentation becomes 50 per cent with solid films of 2 mm. thickness and 25 per cent with liquid films of 2 mm. thickness.
FERMENTATIONS ON COMMERCIALLY AVAILABLE MEDIA The solid material on which the fermentation is conducted is composed of an absorbent solid material and of an absorbed carbohydrate solution. Since many different materials can be used as absorbent, and different types of carbohydrate solutions can be introduced in the form of absorbed solutions, a great variety of combinations can be made. Furthermore, naturally occurring solid materials which contain carbohydrates can be included-namely, sugar beet or sugar cane, potato, steamed rice, and Jerusalem artichoke; some of these are sliced or disintegrated before the fermentation, and some are available during a certain season of the year only. From a commercial point of view the materials to be used for a citric acid production should be abundant and comparatively cheap; they must also have good keeping qualities. The single particle to be fermented must have a large surface as compared to the volume. ABSORBENTS.As an absorbent beet pulp was found very suitable. It does not contain materials that can be leached out by water, it occupies a comparatively small space in its dry commercial form, and it swells readily in the carbohydrate solution with which it is impregnated; but its cost is somewhat high. Bagasse from sugar cane does not absorb so large a proportion of carbohydrate solutions as beet pulp, but i t is cheaper and is available in baled form. When used fresh as an absorbent, the yields of citric acid obtained with bagasse are always about 20 per cent smaller than those with beet pulp. Bagasse can be re-used as an absorbent, whereas beet pulp is attacked to such an extent that it is useless for more than one fermentation.
201
202
INDUSTRIAL AND ENGINEERING
CHEMISTRY
Vol. 27. No. 2
A sterilization of the mass prior to inoculation is not necessary, because the acid which develops after 24 hours suppresses the growth of bacteria to such an extent that the Aspergillus niger fermentation can proceed properly. INOCULATION. The inoculum is preferably a sporulated pure culture of Aspergillus niger on sucrose containing beet pulp. The subsequent fermentation has the same course whether this inoculum is 3 days or 1 month old. The inoculation is made by mixing the sporulated culture into the nonsterile mass. The weight of the solid culture used for the inoculation is preferably 0.5 per cent of the mass to be fermented. FERXENTATION PERIOD.The experiments show that the citric acid production by molds on solid material is not hindered by sporulation. In spite of the sporulation, good yields are obtained in 38, 40, and 50 hours after inoculation. In general the yields are the highest when the fermentation time is the shortest (less than 2 days). The fermentation is interrupted preferably shortly after sporulation sets in. If the temperature on the first day is below 20" C., the fermentation proceeds somewhat slower. No difference in the citric acid yield is obtained when the temperature on the first day is 20' or 35' C. in the fermenting mass. In order to insure a short fermentation period, the inoculation has to be heavy, as is indicated in the foregoing. FERMENTATION. The heat of oxidation of sucrose to citric acid is considerable. But when the layer to be fermented on the wire screen is not thicker than 2 inches, no difficulty is experienced on account of the heat liberated during the fermentation; this is the case even when the fermentation is completed iri 38 hours and the temperature of the fermenting mass rises to 40" C. At the same time the temperature of the air which sweeps over the fermenting mass is 30' t o 35" C. The drying-out effect, even a t 40', is not harmful on a 2-day fermentation. The fermentation of sucrose to citric acid on beet pulp as a carrier cannot be exposed to any motion by which one fermenting beet pulp particle will come into friction with another. The fine surface film of the fungus would in that case be damaged. Thus, beet pulp impregnated with sucrose gives unsatisfactory yields when it is fermented in a slowly rotating drum. This is contrary to the good results which FERMENTATION PROCEDURE Takamine ('7) obtained with his Rhizopus fermentation on FERMENTATION CHAMBER.Citric acid fermentation with wheat bran in rotating drums. Takamine explains his good solid materials is accomplished in trays with false bottoms results by the fact that the concave side of the single bran of iron wire screen; thus free access of air is assured from particle offers a surface to the growing organism that does above and below. The wire screen is painted with a Bakelite not come in contact with the other moving particles. Takavarnish that gives protection against corrosion, If sucrose- mine's process furnishes the commercial product Takadiaimpregnated beet pulp is used, the thickness of the loose layer stase, a mixture of enzymes. RECOVERY OF CITRICACJD. Extraction of the acid from must not be allowed to exceed 2 inches ( 5 cm.); otherwise there is not enough air circulation. If the inner parts of the the pulp for demonstration purposes was made according fermenting mass become too hot (above 43" C.), the acid to the systematic diffusion process of the beet sugar factories, and the temperature was kept a t 80" C. The acid liquor obproduction becomes low. tained from a fermented pulp of 6.96 per cent total acidity PREPARATION O F MATERIAL BEFORE FERMENTATION. The sliced fresh sugar beet cannot be fermented directly. had a concentration of 5.85 per cent total acidity. The acid The cellular structure of the beet must be broken down first; liquor contained 91.6 per cent of the acid introduced into the otherwise the hyphae of the growing fungus cannot reach the extraction. Citric acid was separated from the raw citric acid liquor as sucrose-containing cell sap. This breaking down of the cellular structure can be accomplished by steaming a t at- calcium citrate by the following process: mospheric pressure. When a certain type of beet was The liquor was neutralized in the cold with an amount of steamed for the short period of 20 minutes, the subsequent fermentation furnished citric acid; on the other hand, when milk of lime or calcium carbonate corres onding to 90 per cent of the total acidity, After standing for Ealf an hour the liquor the beet was autoclaved a t a pressure of 15 pounds per which contained precipitated calcium oxalate was filtered with the square inch (0.9 kg. per sq. cm.) for the same length of time addition of kieselguhr and the filtrate was heated for half an hour or longer, the end product from the author's fungus strain in an autoclave at 115' C. in order t o precipitate the calcium citconsisted only of carbon dioxide, This is peculiar since the rate. By this method of precipitation at 115' the yield of calcium citrate obtained from a molasses fermentation was sucrose content in the beet slices does not change by this heat approximately 10 per cent higher than the yield found analytitreatment a t 115" C. The inversion of the sucrose is ex- cally according t o Warrington's method (8),in the following ara graph. The presence of the unfermented constituents o r the tremely slight as can be shown analytically. Experiments were also made with pineapple pulp and the sweet pineapple juice free from its natural citric acid content. This combination was tried because the pineapple pulp and the pineapple juice, which has a higher ratio of sucrose to total solids than molasses, are available as by-products a t the same location in pineapple canneries. In one particular cannery a citric acid refinery is in operation. It is essential that the absorbent as well as the absorbed solution should not contain any material toxic to the fungus. CARBOHYDRATE MATERIALS.The carbohydrates used were primarily sucrose-containing substances. The Aspergillus niger strain with which the author worked is capable of fermenting fructose and sucrose, but not glucose, to citric acid. An abundant fungus growth, however, is developed on glucose, but practically no acid besides carbon dioxide is produced. The solid medium is attacked to some extent by the fungus, and the products obtained appear in the liquor produced by leaching. For this reason a pure citric acid solution from which the acid can be recovered by direct crystallization after concentration is not obtained, even if pure sucrose is used for impregnation. Since, therefore, pure sucrose has no special advantage, impure sucrose in the form of blackstrap molasses is more suitable from an economic point of view. The fermentation of a pure carbohydrate solution (sucrose or glucose) in a liquid state furnishes a liquor from which citric acid can be crystallized directly, whereas with the liquor obtained from fermentation on solid materials the citric acid has to be isolated first as the calcium salt. All the samples of the blackstrap molasses of the cane sugar manufacturer could be easily fermented, but some of the samples of the commercially available sugar beet molasses were toxic to the strain of Aspergillus niger with which the author was working. This toxic material was added or produced during the beet sugar manufacturing process since the sugar beet does not show any toxic material. With sugar cane the situation is reversed. The original sugar cane often contains material toxic to the fungus. This is removed during the cane sugar manufacturing process because the blackstrap molasses does not contain the toxic substances.
February, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
molasses hindered the precipitation of the calcium citrate when the precipitation was made a t boiling temperature and atmospheric pressure. ANALYTICAL PROCEDURE. Before and after fermentation the mass was weighed t o determine the loss of weight during the fermentation. For total acidity a representative sample of 2 to 3 grams, wei hed to the second decimal place, was titrated in a mortar wit% an excess of 0.1 N sodium hydroxide and phenolphthalein and 0.1 N hydrochloric acid. A pestle was used to disintegrate the mass during the titration. About, 10 to 12 rams of the fermented mass were leached out with hot water or the determination of the ratio of citric acid to total acidity. Of this filtered liquor, an amount was used whose total acidity was equivalent to about 1.00cc. of 0.1 N sodium hydroxide. The exact figure was noted. A normal calcium chloride solution in 10 per cent excess was added to the neutralized liquor. The precipitate of calcium oxalate was allowed to settle out for half an hour. It was filtered off, and the filtrate was boiled for half an hour to reduce the volume to 15 cc. The calcium cit'rate was collected on an ashless filter paper and washed with hot water. The calcium citrate was ashed, and the mixture of calcium oxide and calcium carbonate obtained was transferred to a beaker and titrated with 0.1 N hydrochloric acid and phenol hthalein. The ratio of the number of cubic centimeters from tge 0.1 N hydrochloric acid titration and from the 0.1 N sodium hydroxide titration represents the ratio of citric acid to total acidity expressed in equivalents.
citric acid was smaller in the subsequent fermentation. After the fermentation the leached-out pulp was used for another fermentation (experiment 7) which gave a still smaller yield (15.3 per cent) of citric acid on the basis of molasses. The total yield of the combined experiments 6 and 7 in actual recovery as calcium citrate was 328 grams of citric acid from 1875 grams of molasses (17.5 per cent yield) and 900 grams of beet pulp.
f
DISCUSSION OF EXPERIMEKTS Experiments 1 and 2 (Table I) show the yield obtained on a sucrose solution containing Currie's salt mixture and on beet pulp impregnated with pure sucrose solution. With the addition of Currie's salt mixture the yield did not change. Heavy inoculations were always made. The results represent the average of ten fermentations. With a fermentation on beet pulp impregnated with sugar solution the question arises as to how much of t'he acidity produced is derived from the beet pulp itself. The wet beet pulp of the beet sugar manufacturing process contains small amounts of sucrose which are inverted through the action of high heat during the drying process. Another source for the acid produced by the Aspergillus fermentation might be the pectin substances of the beet pulp. A fennentation of beet pulp which was impregnated with water and not with a 15 per cent sugar solution yielded acidity calculated as citric acid of around 4 per cent on the weight of the dry beet pulp. In beet pulp impregnated with 15 per cent sugar solution, about twice as much dry pulp as sugar had to be used. If, therefore, the yield of citric acid is 55 per cent on the sucrose introduced, it is certain that a t least 47 parts of citric acid are derived from 100 parts of sucrose, whereas 8 parts or less are derived from the 200 parts of dried beet pulp which are introduced simultaneously into the fermentation. In experiments 3 and 4 the material was prepared by placing a certain amount of beet pulp into the molasses solution, which consisted of 24.7 parts of molasses and 75.3 parts of additional water, and was just brought to the boiling point. The supernatant liquor was decanted after 10 minutes, a new batch of pulp was impregnated with it, and this was continued until all the molasses solution was used. The yields of citric acid, 23.4 and 24.6 per cent, respectively, based on the weight of the molasses introduced, are calculated from the large-scale calcium citrate precipitation where 210 is taken as the molecular weight of citric acid (with one molecule of water of crystallization). In experiment 5 the impregnation was similar to those of experiments 3 and 4, but was carried out a t 30" C. without heat application. The yield in the experiment in which no sterilization or even pasteurization was used was nearly as good as in the experiments with pasteurization (22.7 per cent). In experiments 3, 4, and 5, 4.4 square feet of tray area were used for the fermentation of 1250 grams of cane molasses. In experiment 6 a higher molasses concentration and consequently a smaller amount of pulp were used. The yield of
203
TABLEI. YIELD OF CITRICACID UNDER VARIOUS CONDITIONS OF FERMENTATION CITRIC
i
Beet pulp (23%) Currie's sucrose soln. (77%) 2 Beet pulp (23%) Pure sucrose soln. (77%) 3 Beet pulp (1150 grams) Cane molasses (1250 grams) 4 Beet pulp (1400 grams) Cane molasses (1250 grams) 5 Beet pulp (1500 grams) Cane molasses (1250 grams) 6 Beet pulp (900 grams) Cane molasses (1250 grams) 7 Beet pulp (900 grams) Cane molasses (625 grams)
1 1 J.
t
8 9
CARBO-
OF
HYDRATE"
FERMEN-
MEDIUM 1
ACID
PERIOD
Freah bagasse (13.15%) Pineapple juice (66.9%)
10 Fresh bagasse (22.8%) Pineapple juice (40.8%)
1
11 Fresh bagasse (15.3%) Molaeses (21.0%) 12 Baled bagasse (17.6%) Molasses (23.3%)
1
1
TATION
IN SOLN
Hours
55
BA0ED ON ADDED CARBOHYDRATE
RATIO^
%
CC.
42
15
55.0
98
42
15
55.0
96
51
24.7
23.4
77.9
46
24.3
24.6
79.0
43
24.7
22.7
77.1
45
30.5
18.5
74.2
42
30.5
15.3
70.8
60
82.5
6.6
84.0
60
66.9
8.0
91.2
60
40.8
9.4
78.5
60
21.0
19.0
81.8
66
23.3
15.0
78.3
13 Baled bagasse re-used 89 22.2 17.8 (moist) (29.6%) 78.8 Molasses (22.2%) . .. 96 5.2C 74.6 14 Sliced sugar beet 97 5.8C 93.1 15 Sliced suear cane a Sucrose, molasses, or pinea ple juice. ( c c , N citric aciB b Ratio = cc. N total acid) 'O0' 6 Citric acid based on sugar beet or sugar cane brought into fermentation.
-
The combined experiments 6 and 7 show that one pound (0.45 kg.) of citric acid can be obtained from 5.7 pounds (2.6 kg.) of molasses and 2.75 pounds (1.25 kg.) of beet pulp which cost (3.43 2.75 =) 6.18 cents whereby molasses is figured at 0.6 cent and pulp at 1.0 cent a pound. The cane molasses contains 46.5 per cent total sugar of which 15.5 per cent is invert sugar and 31.0 per cent sucrose. The protein content is 4.2 per cent. The acid part in the calcium citrate obtained from a molasses fermentation was found to contain a t least 97 per cent citric acid. By decomposing a certain amount of calcium citrate with sulfuric acid, an acid liquor was obtained from which by evaporation citric acid was crystallized by seven consecutive crystallizations. The final mother liquor, with which further crystallizations were not made, contained only 3 per cent acidity on the basis of the acidity brought into this crystallization experiment. In experiments 8 to 11 bagasse, concentrated pineapple juice, and cane molasses %-ere used. The pineapple juice had a Brix content of 29" and an acidity of 0.4 per cent calculated as citric acid. It was obtained by concentrating the original juice which had a 10 to 12 per cent Brix and from which the natural citric acid was precipitated as calcium
+
20 I
I \ 1) li S 'l II I A 1.
.\
> If
15 .\ ( i 1 S E E I< I N C: C li E IMI S T R Y
citrate Sur ita cun\-cr*ioii t,o citric :id. T l i c j i i i c c tiad II w q t r content OS f r ~ m10 t i 1 12 per cetit, coriiposed of about 4 to 5 per cent sucrose and 6 t u 7 per cciit iiivert sugar. Tlie bagasse used in erperilneliti. 8 to 11 \vas obtained by crllsiiing fresh sugar cane under a gressiirc uf 12,000 pounds per square inch (844 kg. per ' 4 . ern.), resoaking it, in w-ater, anti repressing and ed pulp a t 80" C. SOT one day. cornparatire esimiriicnts were 1113dein which bagasse impregnated wit11 i i d a x s e s \yas fermented; stcrilization by autoclaving was used in soiiie eases and no steriliaation or pasteurizatiuir in the otliers. S o iniprovement ciiiild be shown by sterilization. A rleeirleii advantage of bagssse >is its clieapiress a i d the f compared with beet pul the bagasse CAD bc used era1 tiiries over without lowering the yield. The yield of 19 per ccut citric acid witlr molasces, introduced as in experiment 11 on the cornhimation of bagasse and molasses (experiment 11 is typieal of ten experiments) was ohtaincd when Sresli sugar cane was used for the greparation of the bagasse. Instead of bagasse from fresh dugar cane, haled bagasse (a raw material for the rni~irufactureof insulating boards) was used. The carbohydrates left in this baled bagasse from the cane sugar manufactiiring prueess undergo spontaneous fermentation. Tlie yields of citric acid from the combination of baled bagasse and cane molasses were smaller than the yields on fresh bagasse irmpreqated with cane molasses. Also a longer period of fermentation was necessary.
Val. 27, No. 2
Table I iiicludes data ou the fermentations of sliced sugar beet and sugar cane having a cross section of 0.5 X 0.25 inch (1.27 x 0.635 cm.) (experiments 14 and 15). The materials were steamed for 30 minutes a t atmosplieric pressure. The values given are an average of ten experiments in the ease of su6a.r beets and of three in the case of sugar cane. Tlie experimente with sugar beets showed clearly that too long an exposure of the sliced beets to steam, especially to highpressure steam [15 pounds per square inch (0.9 kg. per sq. ern.)], lowem the yield of citric acid. The best method to prepare the sugar beets for ferrnentatioii is to steam them at. 100" C. for haLf an hour.
LITERATURE CITED J., U.S. Patents 1,809,797(June9, 19dl) and 1,812,136 (June 30, 1931). (2) Currie, J. N.,J . Biol. Chem.,31,15-37(1917). (3) Dodger. W. P.. and Prescott. 8. C.. END. EKG. Ctrnu.. 26. 1142-9 (1934) (4)Falok, R.,German Patent 473,727(Maroh 20,1Y29). (5) Falck, R.,and Kingma. van Beyma thoe, Be?.. 57,915(1934). (6) Moyer, A. J., May, 0.E., and Herriok, H. T . . papep presented before Diviyion of Biological Chemistry at 85th Meeting OT A. C. S.. Washington. D. C.. March 20 to 31,1933. (7) Takamine, Jokiohi, J. IND.ENQ.CBEM.,6,824-8 (1914). (8) Warrington, R..J . Chem. Soc., 28,934(1875). (9) Wehmsr, C., Chem.-Zlg.. 57,313(1933). (1) Calm, F.
K.rcsrrso Norsmbs. 30, 1934; origin*
rubmitted Nmombar 24. 1983.
THEALCHEMIST BY Thomar W$ck (W.vck) (1617-1677)
No. 50 in the Berolzheimer series of AIchemical and Historical Re roductions is by the same artist who painties Nos. 5, 35, 39, and 45. The original painting is in The Louvre in Paris. Like all of Wijck's work this shows the dusky, curtained mom, indicative of mystery. In other respects the composition resembles that of the younger Teniers, and shows more laboratory apparatus than do the other paintings of this artist.
. . *
A detailed liat oi the Brst thirtyazr reproduotiom in the -rim, together with iuU particdm for obtsinins photographic copiea of the o~isinBil8.appaared *a ow issue for January, 1934, D B ~ B112. A supplementary list of Nos. 37 to 98 is in our 3an"Bry. 1935,isaue. P W b 102. No. 49 is in the January, 1935, isme. pas8 80.
