CITRIC ACID FERMENTATION OF BEET MOLASSES'PHILIPP GERHARDT, W. W. DORRELL, AND I. L. BALDWIN

Department of Agricultural Bacteriology, University of Wisconsin, Madison, WisconsinReceived for publication August 3, 1946

Beet molasses constitutes an economical source of carbohydrate for industrialprocesses. Successful utilization of it in citric acid fermentation by molds ishighly desirable. Although beet molasses presumably is now being used indus-trially, such commercial methods are closely guarded trade secrets. Moreover,its use may be limited. Thus, von Loesecke (1945) states, "It is believed that. .. in present commercial practice it is necessary to use beet molases from acertain locality to achieve efficient operation." The literature concerning thefermentation apparently contains no fully satisfactory procedure for utilizationof beet molasses in existing plant conditions, although applicable studies havebeen reported. The ultimate object of this research, therefore, was to develop amethod by which beet molasses may be improved for use as a substrate in myco-logical citric acid production, giving yields comparable to those obtained withsynthetic media.The literature has been adequately reviewed by Wells and Herrick (1938),

Prescott and Dunn (1940), and von Loesecke (1945). Relatively few studieshave been concerned with beet molasses or other unrefined sugar sources. Ap-parently the only specific reference to the use of beet molasses was reported byRoberts and Murphy (1944); they developed a method in which a beet molassesmedium, impregnated on sphagnum moss, was fermented by a strain of Asper-gillus niger to yield 25 to 30 g of calcium citrate per 200 g of molasses in 2 days.Previously, Cahn (1935) had reported a similar process, using cane molasses.Chatterjee (1942), Palei and Frantzuzova (1936), Bernhauer, Iglauer, andKnobloch (1941), Das Gupta, Saha, and Guha (1940) have also reported studiesdealing with the fermentation of various molasses or raw sugars.

METHODSThree types of containers were employed. Preliminary experiments were

conducted in 6-oz bottles, the bottle being laid on the side to provide a large sur-faoe area with approximately 1-cm depth of medium. This furnished maximumcontact and utilization of the nutrients of the substrate by the mold mycelium;the fermentation would thus be expected to respond more markedly to experi-mental modification in the substrate. Smooth-walled glass tumblers were usedsubsequently to simulate shallow pan conditions. In some cases, plant con-ditions were more closely duplicated by the use of shallow aluminum pans36 x 25.5 x 7.5 cm in size, containing 5 liters of medium to give a depth of 5

1 Published with the permission of the Director of the Wisconsin Agricultural ExperimentStation.

555

P. GERHARDT, W. DORRELL, AND I. BALDWINcM. The bottles were plugged with cotton, the tumblers were covered with"airmat," and the pans were loosely covered with a piece of sheet aluminum.The mold used was Aspergillws niger. Cultures were stored as soil suspensions,

according to the recommendations of Greene and Fred (1934). Seed cultures forthe fermentation were prepared by inoculation from the soil suspension to bottleplates of sucrose nutrient agar. The plates thus inoculated were incubated for6 to 25 days. A standard suspension of spores was prepared by washing off thesecultures with distilled water to approximate 50,000,000 spores per ml. In thecase of bottles or tumblers, 1 ml of the spore suspension was used as inoculum;for the aluminum pans, 20 ml were used The containers were then gentlyswirled or agitated to ensure an even distribution of spores. The inoculated con-tainers were incubated at 30 C; analyses were usually made at 8, 10, and 12 days.Unless otherwise specified, the results given are for a 10-day fermentation period.The loss in volume at this time was measured directly by determining the differ-ence between the volume of the fermented liquor, including the mycelium mat,and the original volume; this loss was expressed as percentage of loss in volume.The fermented liquor and mat were then diluted and steamed for 15 minutes, inorder to kill the organism and to extract residual acid from the mat. After cool-ing, the liquor and mat were diluted to a given volume, thoroughly mixed, andaliquots taken for analyse.

Residual sugar in the fermented liquor, as well as sugar in the original medium,was determined by the method of Somogyi (1937).

Total acidity was determined by titration with 0.1 N sodium hydroxide, to thephenophthalein end point. Yields were calculated as the percentage of conver-sion of the sugar supplied to citric acid monohydrate, assuming it to be the onlyacid product present. By this method, a theoretical yield of 123 per cent wouldbe possible, if all the sugar carbon were converted to citric acid carbon. Al-though this representation of yields was not necessarily accurate, it nonethelesspermitted rapid gross differentiation of results.Under the conditions used, citric and oxalic acid made up virtually all of the

acid produced by the fermentation. Thus, it was possible to determine theamount of oxalic acid produced, and then merely to calculate, by difference, theamount of citric acid produced This procedure was carried out when differentialevaluations of citric and oxalic acid yields were desired. An oxalic acid micro-analysis was standardized for accurate use with molasses media; the method con-sisted of the usual precipitation with calcium chloride, centrifuging, decanting,and titrating the calcium oxalate while hot with 0.1 N ceric sulfate solution.

PERDIENTALStrains. In the preliminary work a strain of Aspergillus niger was selected

which would produce a maximum amount of citric acid in the shortest time andbe adapted to fermentation of a beet molasses substrate. A comparison of 20strains contained in our culture collection showed two strains to be markedlysuperior in their fermentative ability. Subsequent work was largely restrictedto use of these two strains. Strain 62 is the culture no. 67 employed by Wells,

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CITRIC ACID FERMENTATION OF BEET MOLASSES

Moyer, and May (1936); no. 72 was presumably A. niger 1015 of the AmericanType Culture Collection. Although no. 62 was superior on synthetic medium,it gave inferior results on beet molasses medium (see table 6). On the otherhand, strain 72, although inferior on synthetic medium, proved to be superior onbeet molasses media and subsequently was used routinely in these studies. Al-though no. 72 formed relatively large proportions of oxalic acid, it nonethelessgave the highest citric acid yields and its deeply invaginated mycelium mat madeit adaptable to deep layers of medium. In general, strain 72 could be expected

TABLE 1Variation in fermentative ability of single-spore-derived cultures of A. niger no. 62 on

synthetic medium

STRAIN NO.

62103105106107108111113116117120124131132135143156158160161165

PERCENTAGE YIELDS, TOTAL ACIDITY

7 days

42.549.163.452.651.857.248.958.456.955.248.955.256.344.257.652.849.147.652.853.254.2

8 days

44.051.158.957.660.858.748.361.961.961.754.656.653.142.254.262.147.455.255.862.461.6

9 days

52.251.163.359.161.558.447.461.460.463.456.053.253.142.655.865.847.655.063.260.058.5

10 days

46.750.461.459.362.158.740.962.361.058.255.753.353.537.160.063.652.859.764.658.060.8

to produce citric acid yields of approximately 50 per cent with properly preparedbeet molasses medium over a 10-day fermentation period (see table 6).No newly isolated strains of Aspergillus niger from natural sources gave yields

consistently better than no. 62 or no. 72. In some instances, an apparentlysuperior strain was isolated, but on continued cultivation the property wasrapidly lost.

It was hoped that an existing strain might be improved in its fermentativeability through a process of selection, using a single spore isolation procedure.Twenty single conidiospore isolations were made, using a modification of theChambers (1922) technique with the micromanipulator modification of Wrightand McCoy (1927). In isolating spores, the suspension of spores was allowed to

1946] 557

P. GERHARDT, W. DORRELL, AND I. BALDWIN

incubate for about 3 hours, and the swollen but not yet germinated spores werepicked for isolation; by this method a maximum percentage of germinated sporeswas obtained. The single-spore-derived cultures (from parent strain no. 62)were studied on the synthetic medium of Doelger and Prescott (1934), with theaddition of 0.16 mg of iron and 0.003 mg of manganese per liter of medium.The results in table 1 indicate that some stimulation, apparently due to the

isolation procedure, was observed in almost all the cultures; no marked increasein fermentative ability was achieved and maintained in subsequent studies,however.

Unless otherwise specified, strain 72 of Aspergillus niger was employed in thesubsequent studies on beet molasses media.

Development of a Beet Molasses MediumProximate chemical analyses of beet molasses revealed a comparatively high

percentage of metals, particularly iron. Since the fundamental research of Currie(1917), it has been apparent that excess quantities of many metals may markedlyinhibit the formation of citric acid by A. niger strains. Preliminary studies con-firmed this fact: when untreated beet molasses was tested in the fermentation, aheavily sporulated mycelium mat was formed by the mold and relatively pooryields of citric acid were obtained. It was concluded, therefore, that the inhibit-ing factor in beet molasses might well be an excess quantity or an unfavorablebalance of metallic ions, and the problem was approached from this point of view.

In the wine industry, potassium ferro- and ferricyanide frequently have beenused to remove iron from the product (Creuss, 1934). Ferrocyanide was used forsev-cral years by Mr. William Eisenman of the Heyden Chemical Corporation forthe processing of molasses in citric acid fermentations, and the authors wish toacknowledge their indebtedness to him for a statement of the procedure whichhe employed. A systematic study of varioqs factors which might affect thetreatment of molasses with ferro- and ferricyanide was undertaken.

Preliminary development of the medium was made using a single sample ofbeet molasses, no. 1. Optimum conditions for treatment of this molasses sample,which in many respects is similar to the procedure of Eisenman, were foundto be as follows: To 340 g of beet molasses partially diluted were added 0.60g of potassium ferrocyanide (in solution), and the whole was made to 1 literwith distilled water. Ten g of diatomaceous earth were added and mixedthoroughly. The medium thus prepared was allowed to stand overnight in agraduated cylinder or similar container at approximately 6 C. The medium wasplaced in the fermentation containers and autoclaved at 120 C for 15 minutes.The final medium thus prepared contained approximately 15 per cent sugar.A total of four samples of Straighthouse beet molasses from different localities

were treated with ferrocyanide. All of the samples gave satisfactory citric acidyields, although optimum conditions were established for only one sample, no. 1.The ferrocyanide level, pH, and sterilization conditions required for maximumyields with the different samples vary considerably. Thus, table 2 gives yieldsobtained with the four samples of molasses sterilized and unsterilized, in each

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1946] CTmRIC ACID FERMENTATION OF BEET MOLASSES 559

case with the pH unadjusted and the ferrocyanide treatment optimum for theno. 1 molasses. Under these conditions, molasses no. 1 gave better results whensterilized, the other three apparently givng better results in an unsterile condi-tion. No trouble was encountered from contamination of the unsterile media inthe course of the fermentation, presumably because of the high concentration ofacid formed. Further evidence of the necessity for determining the optimumtreatment for a given lot of molasses in large-scale production became apparentin the case of molasses no. 2. The pH of this sample was 8.4, as compared to thenearly neutral reactions of the other samples. By adjusting the pH of this sam-ple before ferrocyanide treatment to an optimum, pH 6.0, and sterilizing the me-dium, total acidity yields as citric were increased from 90.5 to 101.5 per cent, andcitric acid yields, from 44.8 to 54.0 per cent.

TABLE 2Effect of sterilization in the preparation of beet molasses media using different

molasses samplesPERCENTAGE YIELDS, 10 DAYS

SMPLE NO. STERILIZATIONSA3PINO.ASS | SERZATIO Total acidity C Citric acid:as citric Ctiacd Oacaid total acidity

1 + 102.1 58.8 39.1 60.095.5 45.4 45.1 50.2

2 + 90.5 44.8 41.0 52.269.5 47.8 19.6 70.9

3 + 37.586.6 53.6 29.6 64.4

4 + 30.7 18.5 11.0 62.777.5 44.2 29.3 60.2

citric acid* Citric acid: total acidity =citric acid + oxalic acid X 100.

The relationship between total acidity yields and the amounts of potassiumferro- and ferricyanide added as precipitants to molasses no. 1 is illustrated infigure 1. An excess of the ferrocyanide apparently was somewhat less seriousthan an inadequate quantity, although the range of the optimum level was rathernarrow. The optimum amount for 340 g of molasses sample no. 1 was about 0.60g; the percentage of the citric acid yield as well as the total acidity yield was foundto be maximum at this level. Although slightly higher yields were obtained withferrocyanide, the ferricyanide treatment gave nearly equal yields and did notexhibit such a narrow optimum level.

Effect of pH. The optimum pH level for the ferrocyanide treatment was in-vestigated in the next series of fermentations and was found to be approximately7.0. This result is in marked contrast to the effect of pH on the production ofcitric acid in synthetic media; in this case, the yields were maximum at low pH

P. GE W. DORRELL, AND I. BALDWIN

P-

4c

14

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a;

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IL

FIG. 1. EFFECT OF

0.5 1.0 1.5GRAMS PRECIPITANT PER 340 GRAMS MOLASES

AmOuNTs oi POTAssIum FEzRO AND FERmCYANIDE USEDIN TREATMENT OF BEET MOLASSES

49

0-I..

a

0

a.

&o 4.0 so ao TO topH ADJUSTMENT

FIG. 2. EFFECT OF pEI ADJUSTMENT IN FEROCYANIDEITREATMENT OF BET MOLASSES

50 [VOL. 52

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CITRIC ACID FERMENTATION OF BEET MOLASSESlevels, often below 3.0. In the beet molasses media, yields of citric acid as wellas total acidity were markedly better at the higher pH range; moreover, the moldspores failed even to germinate at a range below pH 4.0. Figure 2 shows theeffect of varying the pH on total acidity yields with beet molasses no. 1. Varia-tion in pH before ferrocyanide treatment of the medium, after treatment butbefore sterilization, and after both treatment and sterilization gave highest yieldsat a neutral reaction. The pH was routinely adjusted before ferrocyanide treat-ment and sterilization. In no case was p1 adjustment made after the fermenta-tion was started, nor was possible change in pH checked after the original adjust--ment. The germination of spores and mycelium formation were markedlyinhibited at the lower pH values; there were no apparent differences in morpho-logical characteristics of the mycelial mats between pH 5.0 and 8.0. Most of theother samples of beet molasses gave satisfactory results with the pH unadjustedthroughout.

TABLE 3Gross effect of different 80 C incubators and of different temperature levels within one incubator

PERjCENTAGE YIELs, 10 DAYSINCUBATOR NO. TElPERATURG LOSS IN VOLUME, Total a Cra |_d_lcdAVERAGE 'C 10 DAYS Total acidity Citric acid Oxalic acid Citric acid:total acidity

1 33 29 87.0 52.8 30.8 63.130 22 85.428 19 83.0 47.0 31.8 59.626 19 60.625 19 50.3

2 30 44 79.0 39.7 35.2 53.13 30 54 73.84 30 45 89.0 46.2 38.4 54.65 32 44 85.4 45.8 35.3 56.5

Room temp. 23 24 47.5

Effect of temperature. Since various temperatures have been reported as opti-mum for the fermentation, it seemed desirable to check the influence of incuba-tion temperature upon the fermentation of strain 72 on beet molasses medium.Composite results of the study are given in tables 3 and 4. A temperature of 30C appeared to be optimal, and this temperature has been employed throughoutthe work. However, it was also indicated that marked differences in yields mayoccur using different incubators of apparently the same temperature, pointing touncontrolled factors in incubation such as humidity, circuliation, etc. An at-tempt to shorten the incubation period for the fermentation was made by usinga preliminary incubation period of 4 days at a higher temperature followed by asecondary period at 30 C. Although spore germination and formation of themycelium mat was accelerated by this method, no appreciable effects on yieldswere apparent (table 4).

Effect of inoculum. The influence of the size of spore inoculum upon the fer-mentation of strain 72 on beet molaes medium was checked. The results are

1946] 561

P. GERHARDT, W. DORRELL, AND I. BALDWIN

given in table 5. A comparatively heavy seeding of spores was found to be neces-sary for satisfactory spore germination and mycelium formation. The heavierinocula served primarily to shorten the time of incubation necesary. The no. 2suspension was employed as the standard inoculum for experimental work, withapproximately 1 ml of the suspension per 50 cm2 surface area of medium.

TABLE 4Effect of preliminary incubation at a higher temperature with a secondary incubation at a

lower temperaturePERCENTAG YELDS, CNTAGE YIELDS, 10 DAYS

PERCENTAGE TOTAL ACIDITYPRELIMINARY SECONDARY OSS I VOLUE,INCUBATION, C NCUDATION, C 10 DAYSrCitricad:8 days 10 days 12 days Citric acid Oxalic acid totalacidity

30 30 42 84.0 91.4 99.9 45.0 41.9 51.833 33 57 81.5 84.0 86.5 43.6 36.4 54.537 37 65 74.6 74.6 73.3 39.0 31.8 55.1

33 30 46 86.0 89.7 96.6 47.1 38.3 55.137 30 49 83.2 91.8 96.6 48.6 38.9 55.6

TABLE 5Effect of amount of spore inoculum

SPORE SUSPENSION PEQRCNTAGZ YIELDS, TOTAL ACmITY

Dilution Density Number of spores 6 days 8 days 10 danumber (2-log G)* per ml 6das 8ay 0as

1 4 X 1.824 108,800,000 69.2 80.6 84.92 2 X 1.824 54,400,000 56.2 68.8 73.13 1.824 27,200,000 46.4 60.2 77.14 0.930 13,600,000 43.3 62.2 68.45 0.542 6,800,000 44.8 64.9 57.06 0.274 3,400,000 31.8 56.2 58.6

* Evelyn photometer, 420 ms filter.

12 days

84.2

76.362.869.5

Fermeznttion in ShaUlow PansPlant fermentation conditions were simuh*ted by the employment of shaiow

aluminum pans (36 x 25.5 x 7.5 cm) containing 5 liters of medium to give a 5-cmdepth. The composite results of a number of experiments conducted in thesepans are given in table 6. In all cases the fermentation period was 10 days.The yields in general appeared to be fairly uniform. In one instance, unusuallyhigh yields of citric acid, as well as a high proportion of citric acid to total acidity,were obtained; however, these results were not consistent and pointed to uncon-trolled factors. Yields of approximately 45 to 50 per cent citric acid were ob-tained consistently on a 5-cm depth of beet molasses medium with a moderatelyhigh concentration of sugar in the substrate. When high yields were obtained

[VOL. 523562

1946] crruic ACM EBMETATION OF BEET XOIL5BE 563in pans with 'strain 72 on beet molasses medium, large amounts of a crystallinematerial were deposited. Chemical analysis indicated this material to be potas-sium tetraoxalate, KH3(C204)2.2H20. Adequate dilution and heating of thefermented liquor containing these crystals was necessary before sampling foranalyses.

TABLE 6Fermentation in shallow aluminum panw

PERCENTAGE SUGAR PRZTG US 0DYIN MEDIUM PRCENTAGE PEAG S, 10 DAYSNOP. PAN NO. STRLAIN NO. i,'sI _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _NO. ~~~~~Before After VOLUME, Cti cdfermen- fermen- 10 DAYS Total Citric Oxalic Citrcacidtation" tation acidity acid add addity1 1 62 15.0 4.8 24 40.8 30.6 9.3 76.8

2 72 15.0 2.9 22 94.6 65.2 26.4 71.33 72 15.0 2.3 32 103.1 72.0 27.9 72.0

2 1 72 14.8 50 91.6 45.7 41.2 52.62 72 14.8 44 92.8 47.3 40.9 53.6

3 1 72 14.3 2.7 30 89.6 43.8 41.3 51.32 72 14.3 2.1 93.9 46.8 42.5 52.4

4 1 72 15.0 1.5 43 94.2 45.6 43.3 51.32 72 15.0 1.6 42 95.6 47.4 43.1 52.4

5 1 72 13.6 1.2 49 100.2 50.0 45.3 52.42* 72 13.6 4.6 31.6 20.7 9.8 67.8

6 1 72 16.0 34 95.3 49.8 40.8 54.9* Mycelium heavily sporulated.

ACKNOWLEDGMENTThe authors wish to expres their appreciation to Dr. W. W. Umbreit, Dr. W.

H. Peterson, and Dr. M. L. Johnson of the University staff for their numeroussuggestions and criticisms during the progress of this research project.The investigation was supported in part by grants from the Heyden Chemical

Corporation, New York, N. Y.ADDENDUM

Since this manuscript was submitted, it has been brought to our attentionthat a similar process for the production of citric acid from beet molasseswas in use in Germany during the war. A detailed account may be found intwo reports by the British Intelligence Sub-Committee, 32 Bryanston Square,W.1, London: Chemische Fabrik Joh. A. Benckiser, G.M.B.H., Manufactureof calcium citrate, B.I.O.S. Final Report No. 489; and Production of citricacid at the factory of Joh. A. Benckiser, .Ladenburg, near Heidelberg, B.I.O.S.Final Report No. 220.

P. GEREARDT, W. DORRELL, AND I. BALDWIN

SUMMARYA study was made of beet molasses treated with potassium ferro- or ferricyanide

for use as a substrate in mycological citric acid production. A selected strain ofAspergillus niger was used. Optimum conditions of precipitation, pH, incuba-tion temperature, and inoculum were established for one beet molasses sample,although samples from several other sources also gave satisfactory results withthis method. In shallow pans, simulating conditions in industry, yields of citricacid of approximately 50 per cent of the available sugax (calculated as sucrose)were attained.

REFERENCESBERNHAUER, K., IGLAUER, A., AND KNOBLOCH, H. 1941 tUber die SLurebildung aus Zucker

durch A. niger. Biochem. Z., 307, 298-306.CAHN, F. J. 1935 Citric acid fermentation on solid materials. Ind. Eng. Chem., Ind.

Ed., 27, 201-204.CHAMBERS, R. J. 1922 Isolation of bacteria and dissection of cells. J. Bact., 8, 1-5.CHATTERJEE, N. P. 1942 Citric acid from molasses by A. niger. J. Indian Chem. Soc.,

5, 201-205.CREUSS, W. V. 1934 The principles and practice of wine making. Avi Publishing Co.,

New York.CURRIE, J. N. 1917 The citric acid fermentation of A. niger. J. Biol. Chem., 31, 15-37.DAS GUPTA, G. C., SAHA, K. C., AND GuHA, B. C. 1940 The fermentative production of

citric and oxalic acids from molases. J. Indian Chem. Soc., 8, 64-74.DOELGER, W. P., AND PREsSCOTr, S. C. 1934 Citric acid fermentation. Ind. Eng. Chem.,

Ind. Ed., 26, 1142-1149.GREENE, H. C., AND FRED, E. B. 1934 Maintenance of vigorous mold stock cultures.

Ind. Eng. Chem., Ind. Ed., 26, 1297-1299.PALEI, T. Y., AND FRANTZuZOVA, M. A. 1936 Biochemical citric acid production. Proc.

Inst. Sci. Research Food Ind. U. S. S. R., 3, No. 4, 7-30.PRESCOTT, S. C., AND DUNN, C. G. 1940 Industrial microbiology. McGraw-Hill Publish-

ing Co., New York.ROBERTS, O., AND MURPHY, D. 1944 A rapid fermentation method for the production of

calcium citrate and calcium gluconate from beet molasses. Sci. Proc. Roy. Dublin Soc.,23, 307-314.

SOMOGYI, M. 1937 A reagent for the copper-iodometric determination of very smallamounts of sugar. J. Biol. Chem., 117, 771-776.

VON LOESECKE, H. W. 1945 A review of information on mycological citric acid production.Chem. Eng. News, 23, 1952-1959.

WELIS, P. A., AND HERRICK, H. T. 1938 Citric acid industry. Ind. Eng. Chem., Ind. Ed.,30, 255-262.

WXELIS, P. A., MOYER, A. J., AND MAY, O. E. 1936 The chemistry of the citric acid fermen-tation. J. Am. Chem. Soc., 58, 555-558.

WRIGhIT, W. H., AND MCCOY, E. F. 1927 An accessory to the Chambers apparatus for theisolation of single bacterial cells. J. Lab. Clin. Med., 12, 795-00.

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