Effect of Alcohols on the Mycological Production of Citric Acid in Surface and Submerged Culture I. Nature of the Alcohol Effect ANDREW J. MOYER Northern Regional Research Laboratory,1 Peoria, Illinois Received for publication July 7, 1952 There has been a steady increase in the consumption of citric acid during the past 25 years. An estimate by Cochrane (1948) places annual manufacture at approxi- mately 35 million pounds. Commercial production of citric acid is reputedly based on fermentation by As- pergillus niger of a suitable grade of beet syrup in shal- low pans. A submerged process appears to be highly desirable, and many articles and patents (Perquin, 1938; Karow, 1942; Szucs, 1944; Waksman and Karow, 1946; Karow and Waksman, 1947; Shu and Johnson, 1948a, b; Perlman, 1949; Schweiger and Snell, 1949; and Snell and Schweiger, 1949)'have appeared in this field. Proposed procedures all require a source of highly purified carbohydrate and in most cases also the use of oxygen for aeration. Large-scale production of citric acid in submerged culture has not been reported. Sev- eral improvements in the submerged culture process are needed. These include the use of less inoculum, greater fermentation speed, and the use of crude, cheap carbohydrate sources. Investigations on these problems have been in prog- ress for several years in the Fermentation Division of this laboratory. It was discovered in the course of the work that the use of low molecular weight alcohols- methanol, ethanol or isopropanol-as adjuncts to the culture medium greatly increased citric acid produc- tion in both surface and submerged culture.2 Such use has made it possible to ferment directly crude carbo- hydrate substrates which other investigators have found necessary to purify, especially for use in sub- merged culture. This paper deals with the nature of the alcohol stimulation as related to the presence of trace elements as well as to initial acidity of the medium and to the quantity of inoculum used. GENERAL METHODS The strains of the Aspergillus niger group of molds used were obtained from the Northern Regional Re- search Laboratory's Collection. 1 One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 2 The April 10 issue of Chemical Abstracts, 1951, carries an abstract of a paper by Sakaguchi and Baba showing that ethanol and methanol slightly increased citric acid yields. Surface cultures consisted of 50 ml portions of me- dium inoculated with dry ungerminated spores in 200 ml Pyrex Erlenmeyer flasks. Submerged cultures consisted of 100 ml of medium including alcohol adjuncts and inoculum in 300 ml flasks. The given alcohol was added to the cool sterile medium just prior to inoculation. Inoculations were made with a suspension of germinated spores. Flask cultures were shaken either on a Ross-Kirshaw machine at 150 rpm or on a Gump shaker at 200 rpm. All cul- tures were incubated at 30 C unless otherwise indicated. Nutrient salts were of C. P. grade. The corn steep liquor was- a commercial grade containing about 50 per cent solids. The glucose'was the ordinary commer- cial variety known as Cerelose3 or Clintose3 containing about 10 per cent water and considered to be glucose monohydrate. The sucrose was a commercial grade of high purity. Absolute ethanol and a synthetic grade of methanol were used. Measurements of pH were made with a glass elec- trode. The cultures were harvested by pouring the mycelium and fernented liquor onto a muslin cloth strainer. The mycelium was squeezed by hand and then placed back into the flask with 50 or 100 ml of distilled water for surface or submerged cultures, re- spectively. This mixture of water and mycelium was heated to boiling and again squeezed through the cloth strainer. The two lots of liquid were combined and made up to the desired volume. Aliquots were taken for volumetric titration with 0.1 N alkali. Phenolphtha- lein was used as indicator. The mycelium was dried to constant weight at 90 C. Citric acid was determined by the methods employed by Wells et al. (1936), and oxalic acid was determined as calcium oxalate precipi- tated with CaCI2. Glucose was determined by the Shaffer-Hartmann (1921) method and sugars reported on an anhydrous basis. Sporulation was estimated under a scoring system where a value of 5 represents a uni- form heavy crop of spores. 3 The mention of products does not imply endorsement or recommendation by the Department of Agriculture over other products of a similar nature not mentioned. 1 on May 8, 2020 by guest http://aem.asm.org/ Downloaded from
Transcript
Effect of Alcohols on the Mycological Production of Citric Acidin Surface and Submerged Culture
I. Nature of the Alcohol Effect
ANDREW J. MOYER
Northern Regional Research Laboratory,1 Peoria, Illinois
Received for publication July 7, 1952
There has been a steady increase in the consumptionof citric acid during the past 25 years. An estimate byCochrane (1948) places annual manufacture at approxi-mately 35 million pounds. Commercial production ofcitric acid is reputedly based on fermentation by As-pergillus niger of a suitable grade of beet syrup in shal-low pans. A submerged process appears to be highlydesirable, and many articles and patents (Perquin,1938; Karow, 1942; Szucs, 1944; Waksman and Karow,1946; Karow and Waksman, 1947; Shu and Johnson,1948a, b; Perlman, 1949; Schweiger and Snell, 1949;and Snell and Schweiger, 1949)'have appeared in thisfield. Proposed procedures all require a source of highlypurified carbohydrate and in most cases also the use ofoxygen for aeration. Large-scale production of citricacid in submerged culture has not been reported. Sev-eral improvements in the submerged culture processare needed. These include the use of less inoculum,greater fermentation speed, and the use of crude, cheapcarbohydrate sources.
Investigations on these problems have been in prog-ress for several years in the Fermentation Division ofthis laboratory. It was discovered in the course of thework that the use of low molecular weight alcohols-methanol, ethanol or isopropanol-as adjuncts to theculture medium greatly increased citric acid produc-tion in both surface and submerged culture.2 Such usehas made it possible to ferment directly crude carbo-hydrate substrates which other investigators havefound necessary to purify, especially for use in sub-merged culture. This paper deals with the nature ofthe alcohol stimulation as related to the presence oftrace elements as well as to initial acidity of the mediumand to the quantity of inoculum used.
GENERAL METHODS
The strains of the Aspergillus niger group of moldsused were obtained from the Northern Regional Re-search Laboratory's Collection.
1 One of the laboratories of the Bureau of Agricultural andIndustrial Chemistry, Agricultural Research Administration,U. S. Department of Agriculture.
2 The April 10 issue of Chemical Abstracts, 1951, carries anabstract of a paper by Sakaguchi and Baba showing thatethanol and methanol slightly increased citric acid yields.
Surface cultures consisted of 50 ml portions of me-dium inoculated with dry ungerminated spores in 200ml Pyrex Erlenmeyer flasks.Submerged cultures consisted of 100 ml of medium
including alcohol adjuncts and inoculum in 300 mlflasks. The given alcohol was added to the cool sterilemedium just prior to inoculation. Inoculations weremade with a suspension of germinated spores. Flaskcultures were shaken either on a Ross-Kirshaw machineat 150 rpm or on a Gump shaker at 200 rpm. All cul-tures were incubated at 30 C unless otherwise indicated.
Nutrient salts were of C. P. grade. The corn steepliquor was-a commercial grade containing about 50per cent solids. The glucose'was the ordinary commer-cial variety known as Cerelose3 or Clintose3 containingabout 10 per cent water and considered to be glucosemonohydrate. The sucrose was a commercial grade ofhigh purity. Absolute ethanol and a synthetic gradeof methanol were used.Measurements of pH were made with a glass elec-
trode. The cultures were harvested by pouring themycelium and fernented liquor onto a muslin clothstrainer. The mycelium was squeezed by hand andthen placed back into the flask with 50 or 100 ml ofdistilled water for surface or submerged cultures, re-spectively. This mixture of water and mycelium washeated to boiling and again squeezed through the clothstrainer. The two lots of liquid were combined andmade up to the desired volume. Aliquots were takenfor volumetric titration with 0.1 N alkali. Phenolphtha-lein was used as indicator. The mycelium was driedto constant weight at 90 C. Citric acid was determinedby the methods employed by Wells et al. (1936), andoxalic acid was determined as calcium oxalate precipi-tated with CaCI2. Glucose was determined by theShaffer-Hartmann (1921) method and sugars reportedon an anhydrous basis. Sporulation was estimated undera scoring system where a value of 5 represents a uni-form heavy crop of spores.
3 The mention of products does not imply endorsement orrecommendation by the Department of Agriculture over otherproducts of a similar nature not mentioned.
The best visible characteristic of a surface cultureproducing high yields of citric acid is little or nosporulation. Clark (1899) found that a medium con-taining 0.25 N ethanol stimulated mycelial develop-ment and greatly inhibited sporulation in cultures ofA. niger. He made no fermentation studies and of-fered no suggestion that ethanol might have an effecton citric acid production.The effect of 1, 2, and 3 per cent methyl, ethyl, and
isopropyl alcohols on the production of citric acid byA. niger NRRL 567 in surface culture is shown in table1. With methanol, marked stimulation of citric acid
TABLE 1. Effect of methyl, ethyl and isopropyl alcohols on theproduction of citric acid by A. niger NRRL 667
in surface cultures
ALCOHOLADDED
None
Isopropyl
Ethyl
Methyl
AMOUNTor
ALCOHOLADDED
0
123
123
123
0.1 N ACIDPRODUCZD
PERCULTURZ
ml
10
641168
9250333
106374630
WEIGHTOP
MYCELIALGROWTH
g1.22
1.101.040.41
1.201.161.03
1.170.990.84
GLUCOSECONSUMED
PERCULTURE
85.10
4.943.731.24
5.085.143.99
5.905.986.10
WEIGT YIEW
ACID ONGLUCOSECONSUMD
*8.220.0
31.152.9
11.540.166.1
SPORECROP
score
5
4.540
551
550
Production medium, amounts per 1 liter: Glucose by analy-sis 126.0 g (6.3 g per culture); NH4NO3, 1.75 g; K2S04, 0.23 g;NH4H2P04, 0.40 g; corn steep, 0.15 ml; ZnSO4 7H20, 0.044 g.
Initial pH: 2.9 by adjustment with HCI.Age at harvest: 6 days.
production was obtained at a 3 per cent and somewhatless at a 2 per cent level of the alcohol. Substantialincreases were found with ethanol at 2 and 3 per cent.With methanol and ethanol at 3 per cent there was alsoa reduction in mycelial growth, a marked inhibitionof sporulation, and an increase in the efficiency of thefermentation as judged from the amounts of sugar con-sumed. The least stimulating effect on acid productionwas obtained with isopropyl alcohol where pronouncedtoxicity was encountered.A large number of strains of the A. niger group were
employed in a survey to determine the effect of variousconcentrations of methanol on the production of citricacid from sucrose. A portion of this survey is shown intable 2. Most of the strains showed a marked increasein total acid production and in fermentation efficiency.
A few strains gave only slight increases in yield; thesestrains appeared to have a very low tolerance for meth-anol as indicated by weak and slow vegetative growth.All strains produced a heavy crop of spores in cultureswithout methanol, whereas sporulation was completelyinhibited by 3 per cent methanol during the 8-dayfermentation period. There was less mycelium, on the
TABLE 2. Effect of three per cent methanol on the production ofcitric acid from sucrose by various A. niger strains in
surface cultures
MOLD STRAINS
NRRL 67
NRRL 321
NRRL 326
NRRL 335
NRRL 340
NRRL 350
NRRL 372
NRRL 567
NRRL 604
NRRL 1736
NRRL2001*
XT0.1 N ACID
ANOL PRlADDED ULR
03
03
03
03
03
03
03
03
03
03
0
ml
334582
452740
290496
388818
250740
202722
314800
268734
170722
184776
204574
DRYWEIIGHT
OFMYCRLUALGROWTH
1.381.24
1.521.29
1.381.37
1.691.34
1.721.41
1.641.50
1.591.10
1.080.95
1.151.01
2.251.03
1.821.31
WRZIGHTYID OPcrraicACID ONSUCROSECONSUMD
3749
4364.7
8.346.0
3565.5
27.463.9
2860.1
2666.5
2158.5
1464.6
1665.8
1645
TOP OPrMKUCOLOR
blackwhite
blackwhite
blackwhite
blackwhite
blackwhite
blackyellow
blackwhite
blackyellow
blackyellow
tanwhite
tanwhite
SPORECROP
score
50
50
40
50
50
&0
50
50
50
50
50
* Aspergillus wentii supplied by S. A. Waksman.Production medium, amounts per 1 liter: Sucrose, 152.0
g (7.6 g per culture); MgSO4.7H20, 0.50 g; KHP0O4, 0.15 g;NH4NO3, 1.5 g; corn steep, 6.0 ml; ZnSO4c7H20, 0.044 g.
Initial pH: 4.1.Age at harvest: 8 days.
dry basis, in the alcohol than in the control cultures.The color of the non-spore-bearing mycelium variedfrom white to a deep orange, depending upon the moldstrain used. This production of pigment was not cor-related with ability to produce citric acid.
Since the work of previous investigators (Bernhauerand B6ckl, 1932; Foster, 1949) had shown that ethanol
could be assimilated and even converted to citric acid,while methanol was not usually assimilated, compari-son of the two alcohols was extended. Varying amountsof either methanol or ethanol were added to a 1 per centglucose-nutrient salt medium. The various concentra-tions of methanol resulted in an increase in acid produc-tion, but there was no increase in mycelial growth. Incontrast, there was a marked increase in growth in thecultures receiving up to 4 per cent ethanol. With bothalcohols there was an increase in acid production onlyafter a definite toxic condition had developed as indi-cated by lack of sporulation and retarded mycelialgrowth. These facts indicate that ethanol but notmethanol can be utilized as a carbon source for mycelialgrowth.The favorable effect on citric acid production of a
low initial pH of the medium has been reported by otherinvestigators (Foster, 1949). In table 2 it is shown thatgood citric acid yields resulted with an initial pH as highas 4.1 when 3 per cent methanol was added to theproduction medium. A series of experiments was con-ducted to determine whether correlation exists betweeninitial pH and the concentration of methanol requiredto stimulate citric acid production. Data presented intable 3 show that acidification alone had little effecttoward increasing citric acid production or decreasingsporulation.'In contrast, the stimulating action of meth-anol on citric acid production was very marked overa pH range of 1.95 to 3.1. At the higher pH value, 3per cent methanol was more effective than 2 per cent,whereas 2 per cent was more effective at the lower pHvalue. The lowered rate of acid production obtainedwith 3 per cent methanol at pH 1.95 appeared to bedue to an unfavorable degree of toxicity. It is of in-terest to note that a substance giving a deep blueprecipitate with iodine was detected in cultures havingvery low initial pH. This substance may be identicalwith that reported by Boas (1916) and by Steinberg(1940).Fermentation of glucose by A. niger in the presence
of CaCO3 at an initial pH of 6.5 ordinarily leads togluconic acid production (Moyer et al., 1940). The addi-tion of 3 to 4 per cent methanol to such a medium re-sulted in the complete inhibition of gluconic acid pro-duction while high yields of citric acid were produced.With 1 or 2 per cent methanol, a mixture of gluconicand citric acids was obtained.
Submerged CulturesCulture maintenance and inoculum preparation. The
stock cultures were maintained on Czapek's agar slants.Rapid and heavy sporulation at 32 C was obtainedwith an agar medium containing beet molasses asfollows: Commercial glucose, 30.0 g.; beet molasses,20.0 g; (NH4)2HP04, 0.6 g; urea, 0.3 g; MgSO4-7H20,0.13 g; KH2PO4, 0.15 g; KCI, 0.2 g; Fe-tartrate, 0.005
g; MnSO4-4H20, 0.02 g; CuSO4 5H20, 0.005 g; cornsteep liquor 3.0 g; tap water, 200 ml; CaCO3, 0.5 g;agar, 40.0 g; and distilled water to make 1 liter. Thismedium can be used in the conventional manner inslant tubes, Petri dishes, flasks, flat bottles, etc., forgrowing spores. Successive generations of stock cul-tures when grown on this medium showed an apparentslow but effective loss of fermentation ability.A germinated spore inoculum was prepared in the
following manner: Ten grams of dry, sharp, sterile,white sand was poured onto a 7-day old agar slantculture (about 15 cm2 of spore-bearing mycelium) con-taining the foregoing medium. The sand was shakenand stirred with a sterile needle to break up the sporechains. The spores were washed off with 25 ml of asterile soap solution (0.1 g of Ivory soap in 300 ml of
TABLE 3. Effect of methanol concentration and medium acidi-fication on citric acid production by A. niger NRRL 567
in surface cultures
INITIALACIDITY
pH
3.102.481.95
3.102.481.95
3.102.481.95
METHA-NOL
ADDED
00
222
333
0.1 N ACIDPRODUCED
PERCULTURE
"l112114156
628736870
824816726
DRF WEIGHT GLUCOSEOF CONSUMED
MYCELIAL PERGROWTH CULTURE
g1.111.151.35
0.991.031.01
0.860.880.85
g5.925.806.27
8.118.318.87
7.797.777.58
WEIGHT YIELDOF CITRICACID ONGLUCOSECONSUMED
12.112.515.9
49.556.162.7
67.767.264.5
SPORECROP
score
555
431
000
* Corrected for HCl added in medium acidification.Production medium, amounts per 1 liter: Glucose by analy-
sis, 184.4 g (9.22 g per culture); MgSO4c7H20, 0.4 g; NH4H2PO4,0.4 g; K2S04, 0.23 g; NH4NO3, 1.5 g; corn steep, 0.5 ml;ZnSO4-7H20, 0.044 g; Fe-tartrate, 0.010 g.
Age at harvest: 8 days.
distilled water). This wetted spore,suspension was di-vided equally between two 300-ml Erlenmeyer flaskscontaining 90 ml of the following medium: Commercialglucose, 50.0 g; NH4NO3, 1.0 g; KH2PO4, 0.25 g;MgSO4c7H20, 0.1 g; ZnSO4 7H20, 0.044 g; Fe-tartrate,0.005 g; corn steep liquor, 0.8 ml and distilled waterto make 900 ml.Each flask also received 0.4 g of agar before steriliza-
tion. Immediately after removal from the autoclave,each flask was shaken vigorously to obtain a good dis-persion of the agar. This dispersed agar greatly in-hibited clumping or pellet formation by the germinatedspores. Pellet formation has been found undesirablein either germination flasks or production cultures.After inoculation, the flask cultures were incubated at30-32 C on a shaker for approximately 22 hours. At
this stage usually two germ tubes have grown fromeach spore, but with little, if any, branching of eithertube. Of this suspension of germinated spores, 1 to 3ml portions were employed to inoculate each produc-tion culture.
Use of the gerninated inoculum. The use of a ger-minated inoculum as contrasted with an inoculum ofungerminated spores results in an economy of originalspore-bearing mycelium and in a reduction by at least1 day of the fermentation period in the productioncultures. The use of a 1 to 2 per cent by volume of agerminated spore inoculum has advantages over theprocedures of Perquin (1938), Sziics (1944), and Karowand Waksman (1947) in which the mycelium from 1volume of inoculum medium was required to seed 1volume of production medium. Their procedures ap-pear to be both difficult and expensive to follow, under
TABLE 4. Effect of amount of inoculum and methanolconcentration on citric acid production by A. niger
Production medium, amounts per 1 liter: Glucose by analy-sis, 112.2 g (11.22 g per culture); MgCl2-6H20, 0.15 g; NaH2PO-H20, 0.15 g; KCI, 0.2 g; NaNO3, 1.62 g; NH4Cl, 0.27 g; ZnSO4-7H20, 0.044 g; Fe-tartrate, 0.005 g; MnSO4c4H20, 0.1 g; cornsteep liquor, 0.15 ml.
Initial pH: 4.05.Age of inoculum: 20 hours.Age at harvest: 9 days.
aseptic conditions, in large tanks for citric acid produc-tion.
It was found that the amount of the germinatedinoculum is correlated to some extent with the conc n-tration of methanol that is optimal for citric acid pro-duction in submerged culture. The data presented intable 4 show that 1.5 to 2 per cent methanol is far moreeffective with 1 than with 2 or 3 per cent inoculum byvolume. The greatest total acid production was ob-tained with 3 per cent methanol and 3 per cent byvolume of inoculum. However, the optimal amounts ofmethanol and inoculum can be expected to be differentwhen the medium is more acid than pH 4.05.The optimal concentration of methanol has been
found to vary with the age and size of the individualunits of mold growth from each spore. In data notpresented here it has been shown that in productioncultures containing 3 per cent methanol, 4 per cent of
a 12-hour old inoculum was required to give a fermenta-tion equal to that obtained with 1 per cent of an 18-hour old inoculum. It appears that the tiny units ofgrowth in a 1 per cent inoculum at 12 hours found a 3per cent methanol concentration too toxic to overcome.
Acidification with HCI of. submerged cultures sub-sequently inoculated with a suspension of germinatedspores gave results in most cases similar to those ob-tained with the surface cultures (table 3). However,some difficulties were encountered, in part due to pHchanges resulting from medium sterilization. More con-sistent results were obtained by making the initialacidification with citric acid. Table 5 shows that acidifi-
TABLE 5. Effect of initial acidification with citric acid andmethanol on the production of citric acid by A. niger NRRL
372 in submerged culture
UTETANOLADDED
1.52.02.53.01.52.02.53.01.52.02.52.0
0.5 N CITIaCACID ADDEDPER CULTURE
ml
0000555510101015
0.1 N CITRICACID PRODUCEDPER CULTURz*
ml-
6689511140112068910481383967810113014061269
DRYWEIGHT ORMYCELIALGROWTH
8
1.931.801.641.592.141.651.591.301.981.681.401.57
WEIGHT YIELDOF CITRIC ACIDBASED ON GLU-COSE CONSUMED
40.153.061.673.5
61.773.5
74.867.6
INITIALACIDITY
pH4.14:14.14.12.62.62.62.62.352.352.352.30
* By titration with correction for citric acid added; nodetectable oxalic or gluconic acid present.
Production medium, amounts per 1 liter: Glucose by analy-sis, 121.6 g (12.16 g per culture); NH4NO3, 1.80 g; NH4Cl,0.134 g; MgCl2-6H20, 0.20 g; RCd, 0.10 g; KH2PO4, 0.20 g;NasSO4, 0.06 g; Fe-tartrate, 0.01 g; ZnSO4.7H20, 0.044 g; cornsteep liquor, 0.2 ml.
Inoculum: 2 per cent by volume of a 22-hour old germinatedspore suspension.
Age at harvest: 7 days.Incubation temperature: 32 C.
cation with 0.5 N citric acid to give initial pH's of 2.35or 2.6 gave increased acid production in the presenceof 2 or 2.5 per cent methanol.
Effect of methanol on tolerance to trace elements. Theextreme sensitivity of A. niger strains to manganese,iron, and zinc among the elements for citric acid pro-duction has been demonstrated by numerous investiga-tors (Bernhauer et al., 1941; Perlman et al., 1946; Shuand Johnson, 1948a; Foster, 1949; Tomlinson et al.,1950). Our experiments showed that many A. nigerstrains will produce good yields of citric acid whenmethanol is added to the medium containing highlevels of these trace elements. The effect of methanolin a commercial glucose medium containing various
levels of zinc sulfate in submerged cultures of A. nigerNRRL 372 is shown in table 6. As the level of zinc wasincreased above 10 ppm there was a small increase inmycelial growth and a slight decrease in acid produc-tion and fermentation eflficiency. These results are incontrast with those of Shu and Johnson (1948a) show-ing a marked decrease in acid production when zincwas increased above 5 ppm in the usual fermentationwithout methanol.The effect of various combinations of iron, supplied
as ferrous sulfate, and methanol on the production ofcitric acid from commercial glucose is shown in table7. Under the conditions used, 5 ppm of iron decreasedthe citric acid yield with 1.5 per cent methanol, butdid not prevent greatly increased citric acid yields with2.5 per cent methanol. It is of interest to note that 2and 2.5 per cent methanol added to the cultures with-out iron proved too toxic and resulted in sharply de-creased yields of citric acid. These results indicate thatiron has an effect on methanol tolerance. In experi-ments not reported here, three levels of inoculum, 1,2, and 3 ml of germinated spore suspension, were
tested in the medium without added iron in the presenceof 2 per cent methanol. There was no significant toxicityin cultures receiving the 3 ml size inoculum, and thecitric acid yield was high. It is not known whether thisincrease in methanol tolerance is due to more myceliumor to trace elements added along with the inoculum.The data in table 7 show that the addition of man-
ganese (10 ppm) also caused a marked increase in acidproduction by the cultures containing methanol (2 per
cent). The addition of iron and manganese togetherresulted in only a slight decrease in the citric acid pro-
duced in cultures containing 2.5 per cent methanol.Similar increases in methanol tolerance and increasedacid production, due to added iron and manganese,have been obtained when sucrose of high purity was
employed as the carbon source. The data in table 8demonstrate that corn steep liquor supplied at lowlevels of 0.1 to 0.2 ml per liter of medium caused an
increased production of citric acid in the presence ofmethanol. The value of corn steep liquor is believed tobe due, at least in part, to its trace element content.These observations that high levels of trace elementscould be tolerated when methanol was used as an ad-junct to the medium have led to further investigationson the use of such crude carbohydrate sources as molas-ses and starches in submerged cultures.
Composition of the production medium. The kind andconcentrations of the nutrient salts supplying mag-nesium, sulfur, potassium, and phosphorus have beenstudied. Considerable variations in the various saltscan be made without significantly influencing the citricacid yield. In the utilization of inorganic nitrogen suchas NH4Cl, the chloride ions are not assimilated as
rapidly as the ammonium ions, thereby leading to
TABLE 6. Effect of zinc sulfate on production of citric acid byA. niger NRRL 372 in submerged shaker cultures with
methanol
TOTAL 0.1 N ~~~~WEIGHT YIEWJ OFZINC TACID P0R DRY WEIGHT OF GLUCOSE CITRIC ACID BASEDADDED CUIDURPE MYCELIUM CONSUMED ON GLUCOSE
acidification of the medium. There appears to be someadvantage in using a mixture of NH4Cl and NH4NO3;NH4Cl alone is not satisfactory. The composition ofthe production medium given with table 5 is as nearlyoptimal as presently known for A. niger NRRL 372,the strain studied most thoroughly.
DIscussIONThe exact role of methanol or ethanol in stimulating
the production of citric acid by A. niger strains is notclear. These alcohols divert mold metabolism in slightlyacidic (pH 6.3) media from gluconic to citric acidproduction, and enhance yields in the more acidic mediausually used in citric acid fermentation. It may beconcluded that both the presence of the alcohol andthe acidity of the medium bring about an alteration inthe normal carbohydrate metabolic pathway so thatcitric acid accumulates. Simultaneous use of both con-ditions for bringing about the desired interference re-sults in greater citric acid yields than use of eitheralone.
Current theories (Stern and Ochoa, 1951; Erkamaet al., 1949) postulate that citric acid arises by con-densation of acetate with another intermediate com-pound (oxalacetate), and it may seem that ethanolmight act by providing a source of acetate. However,the alcohol effect is best brought about by methanol,and it is obvious that acetate cannot arise directlyfrom this alcohol. Formaldehyde, formic acid, andhexamethylene tetrtnine did not stimulate citric acidyields. Metabolic interference by methanol is also mani-fested by the slowing of mycelial growth at optimalmethanol concentrations.One of the effects of methanol or ethanol is to in-
crease greatly the tolerance levels of manganese, iron,and zinc far above those required for mycelial growth.This permits use of media of improved nutritionalbalance, and the increased citric acid yields realizedmay result in part from this fact. The increased toler-ance toward trace elements thus permits use of crudecarbohydrate sources (such as blackstrap molasses) forcitric acid production. This will be described in a sub-sequent paper.As can be seen from the data, the stimulation of
citric acid production by methanol in synthetic mediais affected by cultural conditions, and especially by themold strain used. The age and the amount of mycelialinoculum which is probably a reflection of its surfacearea may be critical. These factors must be investigatedin applying the effect of the alcohol in any individualcase
ACKNOWLEDGMENTRecognition is given for the valuable assistance of
Mabel Smith and Sinah E. Kelley in culture harvestsand analyses of fermented liquors.
SUMMARY
It has been demonstrated that the addition of meth-anol or ethanol greatly stimulates the production ofcitric acid by Aspergillus niger. Methanol on a volumebasis is more effective than ethanol, which itself can beassimilated and converted to citric acid. The effective-ness of these alcohols is increased by medium acidifica-tion. Higher levels of zinc, iron, and manganese can betolerated in either surface or submerged culture forcitric acid fermentation if a slightly toxic concentrationof alcohol is present. The use of methanol to stimulatecitric acid production should find application in thecommercial production of this acid.
REFERENCESBERNHAUER, K., AND B6CKL, N. 1932 Zum Chemismus der
durch Aspergillus niger bewirkten Saurebildungsvorgange.VII. tlber die Umwandlung von Alkohol in Citronensaure.Biochem. Z., 253, 16-24.
BERNHAUER, K., KNOBLOCK, H., AND IGLAUER, A. 1941 tUberdie Siurebildung aus Zucher durch Aspergillus niger.XI. Faktoren der Citronensaurenhaufung. Biochem. Z.,309, 151-178.
BOAS, F. 1916 StArkebildung bei Schimmelpilzen. BiochemZ., 78, a08-312.
CLARK, J. F. 1899 On the toxic effect of deleterious agentson the germination and growth of certain filamentousfungi. Botan. Gaz., XXVIII, 289-327 and 378-404.
COCHRANE, V. W. 1948 Commercial production of acids byfungi. Econ. Botany, 2, 145-157.
ERKAMA, J. H. AND H.^AGERSTRAND, B. 1949 The effect ofaeration on the formation of citric acid in surface moldcultures. Acta Chem. Scand., 3, 858-861.
FOSTER, J. W. 1949 Chemical activities of fungi. Acad.Press, New York, N. Y., pp. 1-648.
KAROW, E. 0. 1942 The production of citric acid in sub-merged culture. Ph.D. Thesis, Rutgers University, NewBrunswick, N. J.
KAROW, E. O., AND WAKSMAN, S. A. 1947 Production ofcitric acid in submerged culture. Ind. Eng. Chem., 39,821-825.
MOYER, A. J., UMBERGER, E. J., AND STUBBS, J. J. 1940Fermentation of concentrated solutions of glucose togluconic acid: Improved process. Ind. Eng. Chem., 32,1379-1383.
PERLMAN, D. 1949 Mycological production of citric acid-The submerged culture method. Econ. Botany, 3, 360-374.
PERLMAN, D., DORRELL, W. W., AND JOHNSON, M. J. 1946Effect of metabolic ions on the production of citric acid byAspergillus niger. Arch. Biochem., 10, 131-143.
PERQUIN, L. H. 1938 Bijdrage tot de kennis der oxydativedissimilatie van Aspergillus niger von Tiegham. Dis-sertation, Delft.
SAKAGUCHI, K., AND BABA, S. 1942 On the citric acidfermentation by Aspergillus niger. J. Agr. Chem. Soc.Japan, 18, 1033-4; Bull. 85-6.
SCHWEIGER, L. B., AND SNELL, R. L. 1949 Fermentationprocess. U. S. Patent 2,476,159.
SHAFFER, P. A., AND HARTMANN, A. F. 1921 The iodometricdetermination of copper and its use in sugar analysis.II. Methods for the determination of reducing sugars inblood, urine, milk and other solutions. J. Biol. Chem.,45,36E-390.
SHU, P., AND JOHNSON, M. J. 1948a The interdependenceof medium constituents in citric acid production in sub-merged culture. J. Bact., 56, 577-585.
SHU, P., AND JOHNSON, M. J. 1948b Citric acid by submergedfermentation with Aspergillus niger. Ind. Eng. Chem.,40, 1202-1205.
SNELL, R. L., AND SCHWEIGER, L. B. 1949 Production ofcitric acid by fermentation. U. S. Patent 2,492,667.
STEINBERG, R. A. 1940 Action of some organic compoundson yield, sporulation and starch formation by Aspergillusniger. J. Agr. Research, 60, 765-773.
STERN, J. R., AND OCHOA, S. 1951 Enzymatic synthesis of
citric acid. I. Synthesis with soluble enzymes. J. Biol.Chem., 191, 101-172.
Sz ucs, J. 1944 Citric acid production by fermentation. U.S.Patent 2,353,771.
TOMLINSON, V., CAMPBELL, J. J. R., AND TRUSSELL, P. C.1950 The influence of zinc, iron, copper, and manganeseon the production of citric acid by Aspergillus niger. J.Bact., 59, 517-527.
WAKSMAN, S. A., AND KAROW, E. 0. 1946 Process for theproduction of citric acid. U. S. Patent 2,394,031.
WELLS, P. A., MOYER, A. J., AND MAY, 0. E. 1936 Thechemistry of the citric acid fermentation. I. The carbonbalance. J. Am. Chem. Soc., 58, 555-558.
Effect of Alcohols on the Mycological Production of Citric Acidin Surface and Submerged Culture
II. Fermentation of Crude Carbohydrates
ANDREW J. MOYER
Northern Regional Research Laboratory,' Peoria, Illinois
Received for publication July 7, 1952
The desirability of employing cheap and readily avail-able sources of carbohydrates in the mold fermentationprocess for production of citric acid is well recognized.Publications dealing with the use of cane, beet or"high-test" molasses, cellulose hydrolyzates and starchproducts in the surface culture process have been re-viewed by von Loesecke (1945). Some of these ma-terials have also been employed in a submerged cultureprocess (Karow, 1942; Sjolander, 1945; and Waksmanand Karow, 1946). Many of the difficulties in develop-ing a submerged procedure have been emphasized byPerlman (1949). Carbohydrate materials commonlyavailable have not been found satisfactory unless sub-jected to considerable purification. Recently the Na-tional Research Council of Canada (1952) reported asubmerged culture process whereby a special grade ofbeet molasses treated with potassium ferrocyanide gavea top yield of 72 per cent acid in 70 hours. Oxygen wasused for aeration during most of the fermentation pe-riod.The citric acid fermentation is very sensitive to the
components of the medium, especially to iron, manga-nese, and zinc. However, it has been shown by Moyer(1951 and 1953) that the addition of slightly toxic con-centrations of the low molecular weight alcohols to themedium increases the tolerance level of these traceelements in the fermentation. The purpose of the present
' One of the laboratories of the Bureau of Agricultural andIndustrial Chemistry, Agricultural Research Administration,U. S. Department of Agriculture.
investigation was to determine further the value ofalcohols, especially methanol, in stimulating the pro-duction of citric acid by Aspergillus niger from suchcrude carbohydrate sources as starch, starch slurries,crushed grain, and various kinds of molasses in bothsurface and submerged culture.
MATERIALS AND METHODSProcedures used in culture preparation, inoculation,
incubation, harvesting and analyses, unless otherwisespecified, have been described (Moyer, 1953). The cul-ture medium, incubation temperature, and culture ageare stated for each experiment. All citric acid yields arecalculated on the basis of the anhydrous acid. Fermen-tation efficiency is considered to be 100 times the ratioof the weight of anhydrous acid to weight of sugar con-sumed. The surface and shaker flask cultures contained50 and 100 ml of solution in 200 and 300 ml PyrexErlenmeyer flasks respectively.The three lots of non-Steffenized beet molasses em-
ployed were supplied by: (1) The Heyden ChemicalCorporation, which reported it as one of the bettergrades for the citric acid fermentation; (2) the GreatWestern Sugar Company; and (3) the Stauffer Chem-ical Company, this sample being from the western area.The lots of blackstrap molasses were untreated exceptthose from Godchaux Sugars, Inc., which had beensubjected to a treatment to remove part of the aconiticacid.
