production of fumaric acid by rhizopus arrhizus1 · rhodes, moyer, smith, andkelley[v duction of...

RHODES, MOYER, SMITH, AND KELLEY

REFERENCES

BARRON, E. S. G. 1951 Thiol groups of biological impor-tance. In Advances in euzzymiiology, pp. 201-266. Inter-science Publishers, Ltd., London.

DAVIS, B. D. AND MIINGIOLI, E. S. 1950 MIutanits of Escher-ichia coli requiring methionine or vitamin B,2. J. Bac-teriol., 60, 17-28.

DUBNOFF, J. W. AND BARTON, E. 1956 The activation of

protein sulfhydryl grouip by vitamin B12. Arch. Biochem.and Biophys., 62, 86-90.

KNOX, W. E., STUMPF, P. K., GREEN, D. E., AND AUERBACH,V. H. 1948 The inhibition of sulfhydryl enzymes as thebasis of the bactericidal action of chlorine. J. Bacteriol.,55, 451-458.

ROBERTS, R. B., COWIE, D. B., ABELSON, P. H., BOITON,E. T., AND BRITTEN, R. J. 1955 Studies of biosynthesisin Escherichia coli., p. 292. Carnegie Institution of Wash-ington Publication 607.

Production of Fumaric Acid by Rhizopus arrhizus1IT. A. RHODES, A. J. MIOYER, MABEL L. SMITH, AND SINAH E. KELLEY

Fermentation Laboratory, Northern Utilization Research and Development Division,2 Peoria, Illinois

Received for publication July 21, 1958

The formation of appreciable quanitities of fumaricacid by fermentation is largely confined to organisms ofthe order Mucorales and in particular to the genusRhizopus. Production of fumaric acid by these fungi hasbeen reviewed by Foster (1949, 1954), whose papersmay be consulted for prior references. Fumaric acidproduction is not strictly a one-product fermentation.Under limited conditions selected strains of Rhizopusproduce primarily fumaric acid, but usually ethanol ispresent together with a substantial proportion of acidsother than fumaric. In general, weight yields of fumaricacid have been reported as high as 40 to 50 per cent ofthe sugar fermented, glucose being the most commonlyemployed carbohydrate. Optimal conditions for theproduction of fumaric acid have not previously beenclearly defined for either surface or submerged fer-mentations. According to published data, the level ofvarious components of the fermentation medium suchas carbohydrate, potassium, iron, magnesium, zinc, andcopper can greatly influence the yield. The purpose ofthis paper is to describe the optimal conditions for theproduction of fumaric acid in shaken flask culture.

MATERIALS AND METHODS

The compositions of the media employed in this workare summarized in table 1. The two media (C and D)are optimum for the laboratory-scale production offumaric acid by the two strains of molds used.The organisms were cultured on slants of medium A

for 5 to 7 days at 33 C to obtain a massive crop ofspores. The spores were washed from slants of medium

1 Presented at the 58th General Meeting of the Society ofAmerican Bacteriologists, Chicago, Illinois, April 27 to May 1,1958.

2 Agricultural Research Service, U. S. Department of Ag-riculture.

A with 30 ml of a dilute detergent solution into 90 ml ofmedium B contained in a 300-ml indented Erlenmeyerflask. The spores were germinated in medium B byincubation for 18 hr at 33 C on a rotary shaker. Three-to five-ml quantities of medium B containing germi-nated spores were used as inocula for each 100 ml of thefermentation medium. The quantity of germinated

TABLE 1Summary of mtiedia uised for fuinaric acid production by

Rhizopuis species

Ingredient(g/1000 ml final vol)

Commercial glucose*High-test molasses....Crude lactose.......Glycerol .............Urea ...........Peptone ..........Corn steep liquor ..KH2PO4 ...........MgSO4 7H20........ZnSO4-7H20 ..........Ferric tartrate......FeSO4 7H20.........Cl$S04 ............MInSO4.4H20 ...KCl ............NaCl .............Agar ..... ....

Corn starcht.Methanolt ............

CaCO3 ...............

Inoculum

ASporula-

tion

4.0

6.010.0 ml0.61.61.0 ml0.40.30.088

0.250.0050.050.440.030.0

BGermi-nation

15.010.0

1.0

3.0 ml0.30.250.0660.01

1.030.0

Fermentation

cGlucose

Varied

1. t

0.5 ml0.30.40.0440.01

1.5%Varied

DMolasses

Varied

0.9t

0.10.150.044

1 .5%Varied

* Corresponds closely to glucose monohydrate.t Sterilized separately and added aseptically.t Thick boiling corn starch made up separately and added

to basal salts medium before autoclaving.

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PRODUCTION OF FU.MARIC ACID BY R. ARRHIZUS

spores used as inoculum was varied within this range.

Less than 3 ml caused slower fermentations and more

than 5 ml resulted in excessive mycelial growth andattendant reduced yields of fumaric acid from the sugar

utilized. The germinated spores were examinedmicroscopically for purity and extent of germinationbefore transfer to the fermentation flasks. Under theconditions used, nearly 100 per cent of the spores

germinated and formed a loose network of germ tubesand rudimentary, slightly branched hyphae.

Fermentations were conducted in 300-ml indentedErlenmeyer flasks, each containing 100 ml of the ap-

propriate fermentation medium. The flasks were in-cubated on a rotary shaker. Preliminary experimentssometimes were conducted utilizing these media in con-

ventional unagitated Erlenmeyer flasks. It is agreedgenerally that complete neutralization of the acidformed during the fermentation is necessary formaximal yields. Hence, sufficient CaCO3 routinely was

added to the medium before autoclaving to neutralizeacid formed during the projected experiments.

Fermentations were harvested according to thefollowing procedure. Culture broths were first diluted toa volume sufficient to allow complete solution of thecalcium fumarate present. The diluted broths were

steamed for 15 min, the culture liquor filtered off, themycelial material pressed and washed with boilingwater, and the combined liquid phase made to an

appropriate volume. Sugar was measured by themethod of Shaffer and Hartmann (1921). The dryweight of cell mass was obtained after collecting theacid-washed mycelium on cheesecloth and drying themycelium at 80 C for 5 days.

Initially, fumaric acid was determined gravimetri-cally as mercurous fumarate according to the method ofOlander (1929); total acids were calculated from thetitration of excess acid after neutralization of theCaCO3. In later wvork, both total fermentation acidsand fumaric acid were determined by an ion-exchangechromatography technique (Van Etten and McGrew,1957). For the determination of total acids by thismethod, a small aliquot (<0.5 milliequivalent) of thefermentation broth was passed through a small (7-cm)cation exchange column (Dowex3 50, H+ form); theacid was eluted with water and titrated with 0.01 N

NaOH. Duplicate samples of the eluate from the cationcolumn were passed through 6-cm anion exchangecolumns of Dowex 1 (OH- form). The anion columnsthen were washed successively with water and 0.35N acetic acid to remove acids such as malic, succinic,gluconic, 2- or 5-ketogluconic, itaconic, and glycolic.Fumaric acid was eluted with 0.1 N HCl and the eluatetwice evaporated to dryness on a steam bath. The

I Dow Chemical Co., Midland, Michigan. The mention ofproducts does not imply endorsement by the Department ofAgriculture over other products of a similar nature.

residue was dissolved with water and titrated with 0.01N NaOH. Fumaric acid was the only acid present in theevaporated 0.1 N HCl eluate from the anion resin.Ninety-seven to 98 per cent recovery of fumaric acidadded to fermentation broths was obtained with thismethod. The results of analyses made on fermentationliquids were comparable to those obtained with thegravimetric method employed in earlier phases of thiswork, as well as with a polarographic procedure. Thegravimetric determination tended to give resultsslightly higher than those obtained with either chro-matography or polarography.The production of fumaric acid by two strains of

Rhizopus arrhizus was studied during the course of thework: R. arrhizus Fisher strain NRRL 1526 and R.arrhizus Fisher strain NRRL 2582. The two organismswere selected on the basis of a survey of molds of thegenus Rhizopus contained in the ARS Culture Collec-tion at the North Utilization Research and Develop-ment Division. R. arrhizus 1526 previously had beenshown by Ward (1940) to produce 25 to 30 per centyields of fumaric acid from glucose. Rhizopus nigricansstrains described in previously published reports lackinvertase (Foster, 1954), as does R. arrhizus 1526.R. arrhizus 2582 differs from the other organismsmentioned by its capacity to utilize sucrose withoutprior hydrolysis. The apparent advantage of strainR. arrhizus 2582 was considered in emphasizing thestudy of the conditions for production of fumaric acidby this organism.

EXPERIMENTAL RESULTS

Of the factors tested, the type and concentration ofnitrogen source employed in the medium exerted thegreatest effect on growth and acid production. The mostsuitable sources of nitrogen for the fermentative pro-

TABLE 2Effect of nitrogen sources on funiaric acid production from

glucose by Rhizopus arrhizus strain NRRL 2582

Sugar Fumaric Yield FumaricNitrogen Source* Fermented AAcid Fumaric Acid ofFemnetFormed Acidt Total Acid

g/10O nil g/100 ml %

(NH4)2S()4 ............ 12.1 8.0 66 77NH4Cl ................ 12.0 7.8 65 80(NH4)2HP04 .......... 12.0 7.8 65 82NH4NO3 ............ 12.0 8.2 68 83N114Acetate .......... 12.0 8.1 67 81Urea ................. 12.0 8.5 71 80Asparagine ........... 12.0 7.7 64 76NZ AmineB... 12.0 7.5 62 80

Five-day fermentation of medium C.* Equiivalent to 0.047 g ammonium nitrogen per 100 ml

fermentation medium.t Initial sugar concentration: 12 g glucose/100 ml.t Per cent yield = (g fumaric acid produced)/(g sug:ar

fermented) X 100.

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RHODES, MOYER, SMITH, AND KELLEY[v

duction of fumaric acid were ammonium salts and urea(tables 2 and 3). The most efficient fermentations wereobtained with urea, and unless otherwise noted, ureawas used in the experiments reported in this paper.The yields of fumaric acid produced, when various

concentrations of glucose were fermented with R.arrhizus 2582 and 1526, are presented in tables 4 and 5.The usual pattern of fumaric acid production by theseorganisms is clearly demonstrated. Optimal yields offumaric acid were obtained between 4 and 7 days,depending upon the concentration of sugar initiallypresent. The fermentation can be conducted successfuillyusing any of the sugar concentrations tabulated;16 per cent is the greatest concentration fermented togive maximal yield at 7 days or less. Culture R. arrhizus2582 was more efficient than R. arrhizus 1526, par-ticularly at higher concentrations of sugar. The amountof mycelium tended to increase when higher concentra-tions of urea were provided in the medium (table 3).Approximately 37 mg of nitrogen per 100 ml of mediumwere required for the fermentation of glucose by bothorganisms when glucose was the carbohydrate used.This may partially account for decreased yields offumaric acid usually obtained when the medium con-tained more than 47 mg of nitrogen (0.1 g urea) per100 ml of medium. The per cent fumaric acid of totalacid also tended to decrease with increased sugar andnitrogen concentrations. Apparently other fermenta-

TABLE 3

Effect of nitrogen sources and levels on fumaric acid productionfrom molasses by Rhizopus arrhizus strain NRRL 2582

Age atHarvest

days

3

5

7

Nitrogen Supplied

Amount Source

mng/100 ml

37

47

37

47

37

47

UreaNH4Cl(NH4)2SO4UreaNH4C1(NH4)2SO4UreaNH4CI(NH4)2SO4UreaNH4CI(NH4)2SO4UreaNH4Cl(NH4)2SO4UreaNH4Cl(NH4)2SO4

Sugar Fumaric Yield Dry WtFermen- Acid Fumaric of

ted* Formed Acidf Mycelium

g/100 ml

8.17.87.89.38.89.211.210.811.413.112.513.213.712.813.213.813.713.7

g/lOO ml

3.73.54.04.33.84.27.36.87.48.67.97.89.78.79.09.69.08.8

464551464346656365666359716868706664

g/100 ml

0.340.340.340.490.460.510.420.380.420.670.610.620.560.470.540.760.680.71

tion acids, which usually represent about 20 per cent ofthe total, tended to replace fumaric acid when thefermentation conditions were not optimum. The ratioof fumaric acid to total acid varied less than did theyield of fumaric acid; usually between 70 and 80 percent of the total acid formed during the fermentationwas fumaric acid.

TABLE 4Effect of urea and glucose levels on fumaric acid production by

Rhizopus arrhizus strain NRRL 2582

Age at InitialHarvest Glucose

days

3

5

7

g/looml

101214161012141610121416

Concentration of Urea Nitrogen (mg/100 ml)

Sugarfer-

mented

g/100ml

9.09.19.89.49.611.513.014.29.911.814.016.0

37

110

._

>4

575554466973626857697164

47 56

FA/ Sugar -0 FA/ Sugar .0TAt fer- -W TA fer- _P

mented mented >4

818179738185808274828481

gIlooml

9.810.611.410.89.611.513.515.19.911.814.015.9

554648406371676059636844

Medium C was used.* Per cent yield = (g fumaric acid

fermented) X 100.

807677707880817885828669

gIlooml

9.710.912.213.09.611.513.515.09.911.814.015.9

505231396262625938584030

FA/TA

767664688180816762817459

produced)/(g sugar

t Per cent of total acid (TA) which was fumaric acid (FA).

TABLE 5Effect of levels of urea and glucose on fumaric acid production by


Age atHarvest

days

3

5

7

InitialGlucose

g/1loml

101214161012141610121416

Concentration of Urea Nitrogen (mg/100 ml)

37

>4

Sugarfer-

mented

g/100ml

8.67.97.88.29.610.911.19.59.611.613.612.3

555352476567485067655955

FA/TAt

807475777877757281787672

Sugarfer-

mented

g/100ml9.810.610.811.79.611.513.514.69.611.613.715.7

47

0.@

464843486062575561616162

FA/TA

747574797778767482767881

56

Sugar .fer- X

mented >

g/100O on

9.8 5110.4 4511.8 4012.8 429.6 5411.5 5713.5 4815.2 489.6 5011.6 6013.7 5415.7 57

FA/TA

807875757479747170787476

Medium C was used.* Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.t Per cent of total acid (TA) which was fumaric acid (FA).

Medium D was used.* Initial sugar concentration: 14 g/100 ml (invert value).t Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.

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PRODUCTION OF FUMARIC ACID BY R. ARRHIZUS

If it is assumed that 1 mole of fumaric acid is formedfrom 1 mole of hexose sugar fermented, then thetheoretical weight yield of fumaric acid is 64.4 per centof the sugar fermented. Most rapid utilization of thecarbohydrate occurs early in the course of the fermen-tation and the rate of utilization decreases as the fer-mentation continues.The fermentation of high-test molasses by strain

R. arrhizus 2582, shown in table 6, usually occurs at a

more rapid rate than does the fermentation of glucose.This difference may reflect the stimulation of accessory

mineral and other factors present in molasses. StrainR. arrhizus 1526 also will ferment invert molassesutilizing both moieties of the hydrolyzed sucrose.

Suitable neutralization of the fumaric acid formedduring the fermentation is necessary for the optimalproduction of acid (table 7). Because of the low solu-bility of fumaric acid (0.7 g per 100 ml water), fumaricacid crystals rapidly accumulate in the fermentationmedium and cause thickening or gel formation unless a

salt of the acid, such as calcium fumarate, is formed inthe culture medium by neutralization. Calcium car-

bonate in excess can be used in laboratory shake-flaskfermentations to provide the continuous neutralizationwhich otherwise could not be readily accomplished. Anexcess of calcium carbonate beyond that required forneutralization of acid seemed to stimulate acid produc-tion.

Previous workers (Waksman and Foster, 1938;Foster and Waksman, 1939) have emphasized the roleof metal salts in fumaric acid production by R. nigricansand Rhizopus oryzae. In particular, the amount of zincpresent in the medium was shown to influence greatlythie amount of acid or growth produced during thefermentation. The amount of growth was reported tobe proportional, and fumaric acid production inverselyproportional, to the quantity of zinc present. Iron hadnearly the opposite effect. The influence of variousconcentrations of salts in the medium on the formationof fumaric acid by R. arrhizus 2582 is shown in table8. No attempt was made to purify the salts or otheringredients of the medium. Under the conditions used,the elements which exerted greatest effects upon growthwere zinc, magnesium, and phosphorus. Fumaric acidproduction was severely limited by less than optimallevels of magnesium, and both growth and acid pro-

duction were restricted completely in the absence ofphosphorus. About 10 ppm Zn+ appeared to beoptimum for fumaric acid production by R. arrhizus2582, although the effect of different concentrations ofzinc was not as marked as that demonstrated with otherorganisms by previous workers. Magnesium ion con-

centrations between 20 and 40 ppm were most ef-fective. Phosphorus, present as the phosphate ion, was

required in moderately high concentration (200 ppm

P04) for optimal production of fumaric acid. Iron,

added as iron tartrate, did not greatly alter the fer-mentation. The addition of corn steep liquor did notenhance acid production.

Results of similar experiments indicate that lesseramounts of phosphate (75 ppm P04) and magnesium(15 ppm) need to be added for optimal production offumaric acid from high-test molasses as compared withglucose. Corn steep liquor and iron are not added to themedium when molasses is used as the source of fer-mentable carbohydrate, but the addition of 10 ppm

or less of zinc ion may be beneficial.

TABLE 6Effect of urea and molasses levels on fumaric acid production by


Age atHar-vest

days

3

5

7

8

InitialSugar*

g/100ml

1012141510121415161214151616

Concentration of Urea Nitrogen (mg/1OO ml)

28

Sugarfer- Yieldt

mented.-10

g/100ml

5.66.85.66.88.08.88.39.49.111.412.011.611.613.3

6154454567646257557569626164

37

Sugarfer- Yield

mented.-G

gIlooml

7.58.27.48.49.611.011.611.911.311.613.413.514.415.4

6156484873706460557269676264

47

Sugarfer- Yield

mented,/1.gIlooml

8.78.88.79.39.811.612.213.013.511.813.814.515.415.6

6051444868695859546866616062

56

Sugarfer- Yield

mented

gIlooml

9.19.89.7

9.811.713.3

14.611.813.8

15.515.6

544740

646153

526263

5255

Medium D was used.* Initial sugar concentration (invert value).t Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.

TABLE 7Effect of carbonate and urea levels on fumaric acid production by


Concentra- S Fumaric Yield Fumariction Urea CaCO3 ugarm * Acid Fumaric Acid ofNitrogen ermente Formed Acidt Total Acid

mg/100 mtl g/1O "ml g/100 ml g/100 ml % %

28 3 10.2 5.0 49 766 11.2 7.0 62 7810 12.6 7.7 61 75

42 3 11.0 5.2 47 786 12.8 7.2 56 7810 14.0 9.1 65 80

56 3 12.3 5.3 43 806 13.8 7.4 54 7810 14.0 9.0 64 82

Seven-day fermentation of medium D.* Initial sugar concentration: 14 g/100 ml (invert value).t Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.

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RHODES, MOYER, SMITH, AND KELLEY

Moyer (1953) has reported that the addition of 1 to3 per cent (v/v) methanol to the medium aids theformation of citric acid by Aspergillus niger. Methanolapparently increased the tolerance of the mold forcertain salts and was slightly toxic to the mold at thelevels which were effective in increasing the yield ofcitric acid. Addition of methanol to fermentationsbrought about by either of the two strains of R. arrhizuscaused increased yields of fumaric acid, compared tothose obtained in fermentations conducted withoutmethanol. The data in table 9 indicate that between10 and 25 per cent greater yields of fumaric acid mayaccompany the addition of methanol to the fermenta-tion of molasses by R. arrhizus 2582. Methanol had agreater effect on the yield of fumaric acid from molassesby R. arrhizus 2582 than it had on the fermentation ofglucose brought about by either R. arrhizus 2582 orR. arrhizus 1526. Efficiency of fumaric acid productionby R. arrhizus 1526, based on sugar used, was notaffected by the presence of methanol. However, theamount of sugar used and the amount of fumaric acidproduced were increased, particularly during the earlystages of fermentation. In contrast, methanol increasedmarkedly the yield of fumaric acid per unit of sugarfermented by R. arrhizus 2582, but did not stimulatethe rate at which the sugar from molasses was utilized.

The optimal level of methanol in the medium was 1.5per cent. This amount of methanol did not affect con-sistently the amount of mycelium produced during thegrowth of either strain of R. arrhizus. The ratio offumaric acid to total acid produced in the fermentationwas not altered by the presence of methanol in themedium.

TABLE 9Effect of methanol on fumaric acid production from molasses

by Rhizopus arrhizus strain NRRL 2582

Age atHarvest

days

3

5

7

TABLE 8Effect of salts on the formation of fumaric acid from glucose by


Element

Zn++(ZnSO4- 7H20)

Mg++(MgSO4- 7H20)

Fe+++ (iron tar-trate)

P04m (KH2PO4)

Corn steep liquor

Amount

ppm

051015020406002.55.58.00

105210315mg/10O0

50100150

SugarFer-

mented*

g/100 til

13.613.413.713.710.413.613.713.713.713.713.713.70.813.613.713.7

13.713.713.713.7

FumaricAcid

Formed

g/100 ml

7.27.08.8.7.83.77.58.87.38.18.88.48.2

7.38.87.9

8.48.87.88.1

YieldFumaricAcidt

535264573655645359646160

546458

61645759

FumaricAcid ofTotalAcid

767482776078827680828178

798275

80827576

Concen-trationUrea

Nitrogen

mg/100 ml

37

47

56

37

47

56

37

47

56

Methanol SugarIFermented*

01.501.501.501.501.501.501.501.501.5

g/100 mtl

8.17.98.89.09.89.910.310.911.511.711.811.811.711.711.811.811.811.8

FumaricAcid For-mation

g/100 ml

3.54.73.64.83.64.65.47.85.87.65.77.26.99.16.68.66.27.5

YieldFumaricAcidt

435941533746527250654861597756735263

Dry WtMycelium

g/loo ml

0.320.380.560.430.670.750.440.450.630.660.790.850.580.640.730.720.830.84

Medium D was used.* Initial molasses sugar concentration: 12 g/100 ml (invert

value).t Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.

TABLE 10Effect of temperature on the production of fumaric acid by


Temp

C

30

33

35

38

Concentra -tion UreaNitrogen

mzg/JOO ?ml

80100120801001208010012080100120

Sugar Fumaric AcidFermented* Formed

g/100 ml

7.78.58.99.39.89.89.810.010.09.39.910.0

g/l0O ml

4.64.44.65.55.34.95.65.64.54.85.04.8

YieldFumaricAcidt

595252595450575645525048

FumaricAcid of

Total Acid

807679827976797972787880

Three-day fermentation of medium C.* Initial sugar concentration: 10 g glucose/100 ml.t Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.

Five-day fermentation of medium C.* Initial sugar concentration: 14 g glucose/100 ml.t Per cent yield = (g fumaric acid produced)/(g sugar

fermented) X 100.

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1PRODUCTION OF FUMAARIC ACID BY R. ARRHIZUS

The optimal temperature for the production offumaric acid by the two strains of R. arrhizus is between33 and 35 C. The data obtained in table 10 illustrate theeffect of temperature on the production of fumaric acidfrom glucose by R. arrhizus 2582. Although greaterquantities of fumaric acid were obtained at 33 or 35 Cthan at other temperatures, yields based upon thesugar utilized during the fermentation were not greatlydifferent at 30, 33, or 35 C. Less sugar was fermented at30 C than at other temperatures and smaller quantitiesof acid resulted from the sugar utilized at temperaturesgreater than 35 C. At 41 C, growth and sugar titiliza-tion were severely limited and only traces of acid wereproduced.

DISCUSSION

As is true of organic acid production by other fungi,the concentrations of carbohydrate and nitrogen in thefermentation medium are of crucial importance tofumaric acid production by R. arrhizus. The fermenta-tion of both glucose and molasses by R. arrhizas2582 and of glucose by R. arrhizus 1526 requiredabout 40 mg nitrogen per 100 ml, either as urea orammonium salts. Further, the same amount of nitrogenwas required for best conversion of carbohydrate atlevels of 10 to 16 per cent. The amount of mold my-celium formed was proportional to the amount ofnitrogen supplied in the medium.A characteristic of this fermentation at completion is

the nearly constant proportion of fumaric acid to totalacid. Initially, the ratio of fumaric acid to total acid islow, but increases as growth of the organism ceases andas acid production dominates the fermentative activity.The effects of the various factors tested indicate thatconditions optimum for the production of greatestamounts of fumaric acid also are optimum for thegreatest per cent of fumaric acid of total fermentativeacidity. Hence, conditions which exclude the utilizationof carbohydrate for the formation of other acids andmetabolic products, including cell substance, are es-sential for efficient fumarate formation.The continued formation of fumarate in fermenta-

tions in which the carbohydrate has been completelyutilized and the simultaneous increase in the ratio offumaric acid to total acid support the concept thatcertain initial products of the fermentation later areutilized by the mold to form additional fumaric acid.Such metabolic products as ethanol and carbon dioxidemay serve as substrates for fumarate production byR. arrhizus, as was demonstrated with the R. nigricanscultures studied by Foster et al. (1949). Convincingevidence that a carbon dioxide fixation mechanism maybe operative in the metabolism of these molds has beenobtained (Barinova, 1941; Foster et al., 1941; Fosterand Davis, 1948). The increased yields observed in the

presence of an abundance of carbonate may indicatethat carbon dioxide was utilized by R. arrhizus. Theseworkers concluded that under highly aerobic conditionsR. nigricans forms fumaric acid by a direct combinationof the methyl carbons of two moles of a two-carbonintermediate derived from ethanol. Evidence obtainedwith C"4 ethanol indicated that fumarate was formed bydirect condensation of a two-carbon fragment; inaddition, yields of fumarate from ethanol were so highthat the tricarboxylic acid cycle was excluded as anintermediate mechanism in the formation of fumarate.Kornberg and Krebs (1957) and Kornberg and Madsen(1957a, b) have demonstrated the occurrence inPseudomonas of a "glyoxylate cycle" variant of thetricarboxylic acid cycle which acts to replace inter-mediates drained from the tricarboxylic acid cycle.Kornberg and Krebs (1957) have postulated a cycle offumaric acid formation by Rhizoputs to fit the data ofFoster and co-workers. According to their postulate,glyoxylate and succinate are formed from isocitrate bymeans of the isocitrase reaction initially demonstratedto occur in various genera of bacteria by Smith andGunsalus (1955) and Wong and Ajl (1955). Theglyoxylate thus formed condenses with acetate in theform of acetyl-coenzyme A to form malate which thenis oxidized to generate oxalacetate. The oxalacetatecombines with acetate ultimately to form succinatethrough the usual tricarboxylic acid series of reactions.Summation of these reactions provides for the forma-tion under aerobic conditions of 1 mole of fumarate from2 moles of acetate. The accumulation of fumarate maybe attributed to low activity or absence of fumarase.

SUMMARY

Optimal conditions were determined for the shaken-flask production of fumaric acid by two strains ofRhizopus arrhizus. The strains differed chiefly in theirability to utilize sucrose.

Fumaric acid was produced by the fermentation of10 to 16 per cent concentrations of glucose, sucrose, orthe partially inverted sucrose of high-test molasses.Under suitable conditions 60 to 70 per cent of the sugarutilized was converted to fumaric acid in 3 to 8 days,depending upon the amount of sugar employed. Of thetotal acids produced, 75 to 80 per cent was fumaricacid. The kind of nitrogen source and the amountsupplied influenced the yield of fumaric acid. The mostefficient fermentations were obtained with appropriatelevels of either urea or ammonium sulfate. Underspecified conditions the addition of zinc, magnesium,phosphorus, iron, and corn steep liquor to the mediumwas necessary to secure optimal yields of fumaric acid.Up to 25 per cent greater yields of fumaric acid wereobtained by the addition of methanol to fermentationsconducted with molasses.

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REFERENCES

BARINOVA, S. A. 1941 Uber die bildung von fumar-undbernsteinsaure in den kulturen von Rhizoputs nigricans.MIikrobiologiva, 10, 716-728.

FoSTER, J. W. 1949 Chemttical activities of fuingi. AcademicPress, Inc., New York, New York.

FOSTER, J. W. 1954 Fumaric acid. In Indutstrial fermenta-tions Vol. I, Chap. 15. Edited by L. A. Underkofler andR. J. Hickey. Chemical Publishing Co., Inc., New York,New York.

FoSTER, J. W. AND WAKSMIAN, S. A. 1939 The specific effectof zinc and other heavy metals on growth and fumaricacid production bv Ihizopuis. J. Bacteriol., 37, 599-617.

FOSTER, J. W., CARSON, S. F., RITBEN, S., AND KAMEN, M. 1).1941 Radioactive carbon as an indicator of carbon di-oxide utilization. VII. The assimilation of carbon dioxideby molds. Proc. Natl. Acad. Sci. U. S., 27, 590-596.

FOSTER, J. W. AND DAVIS, J. B. 1948 Anaerobic formation offumaric acid by the mold Rhizopuis nigricans. J. Bac-teriol., 56, 329-338.

FOSTER, J. W., CARSON, S. F., ANTHONY, D. S., DAVIS, J. B.,JEFFERSON, W. E., AND LONG, M. V. 1949 Aerobic for-mation of furmaric acid in the mold Rhizoputs nigricans:synthesis by direct C2 condensation. Proc. Natl. Acad.Sci. U. S., 35, 663-672.

KORNBERG, H. L. AND KREBS, H. A. 1957 Synthesis of cellconstituents from C2-units by a modified tricarboxylicacid cycle. Nature, 179, 988-991.

KORNBERG, H. L. AND MADSEN, N. B. 1957a Synthesis ofC4-dicarboxylic acids from acetate by a "glyoxylate by-

pass" of the tricarboxylic acid cycle. Biochim. et Bio-phys. Acta, 24, 651-653.

KORNBERG, H. L. AND MADSEN, N. B. 1957b Formation ofC4-dicarboxylic acids from acetate by Pseuidomlonas KB1.Biochem. J., 66, 13P.

AIOYER, A. J. 1953 Effect of alcohols on the mycologicalproduction of citric acid in suirface and submerged culture.I. Nature of the alcohol effect. Appl. Alicrobiol., 1, 1-7.

OLANDER, A. 1929 Studien uiber Brombernsteinsaure. Z.Physik. Chem. (Leipzig), Abt. A, 144, 49-72.

SHAFFER, P. A. AND HARTMIANN, A. F. 1921 The iodometricdetermination of copper and its use in sugar analy sis.II. Mlethods for the determination of redtucing stugars inblood, urine, milk and other soluitions. J. Biol. Chem.,45, 365-390.

SMITH, R. A. AND GUNSALIUS, I. C. 1955 D)istribution andformation of isocitrase. Nature, 175, 774-775.

VAN ETTEN, C. H. AND 1IcGREW, CLARA E. 1957 Ion ex-change micromethods for separation of fermentation acids.Determination of fuimaric acid in fermentation broth.Anal. Chem., 29, 1506-1509.

WAKSMAN, S. A. AND FOSTER, J. W. 1938 Effet du ziniC surla vegetation de Rhizopuis nigricans et la productiond'acide par cet organisme. Compt. rend., 207, 483-486.

WARD, G. E. 1940 The biochemistry of the genus Rhizopuisw th special reference to the production of three-carbonaind four-carbon acids. Ph.D. thesis, Georgetown Univ.,Washington, D. C.

WONG, D. T. 0. AND AJL, S. J. 1955 Isocitrase in Escherichiacoli. Natture, 176, 970-971.

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