ARTICLE

:
THE CITRIC ACID FERMENTATION
OF ASPERGILLUS NIGER

Access the most updated version of this article at
http://www.jbc.org/content/31/1/15.citation
Find articles, minireviews, Reflections and
Classics on similar topics on the JBC Affinity
Sites .
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail
alerts
This article cites 0 references, 0 of which can be
accessed free at
http://www.jbc.org/content/31/1/15.citation.full.html
#ref-list-1

Downloaded from http://www.jbc.org/ at CAPES - UFC on November 6, 2014

James N. Currie
J. Biol. Chem. 1917, 31:15-37.

THE

CITRIC

ACID

FERMENTATION
NIGER.*
BY

the Research

Laboratories,

Dairy

of Agriculture,
PLATES

(Received

N. CURRIE.
Division,
Washington.)
1 AND

for publication,

United

States Departmeut

2.
April

20, 1917.)

INTRODUCTION.

The citric acid fermentation induced by certain fungi has been
elaborately studied, especially by Wehmer.l
His work on this
subject and also on the oxalic acid fermentation is familiar to all
students of fungi. Wehmer2 believed that the production of
citric acid in more than mere traces was characteristic of the
group of fungi to which he gave the generic name Citromyces
and that the oxalic acid fermentation was characteristic of Aspergillus niger. This seems to have been accepted by all the other
workers who have investigated either ‘the citric or oxalic acid
fermentation.
Martin,3 in a very recent study of the citric acid
with the assumpfermentation, discarded all cultures of Aspergilli
tion that their fermentative action was well known and that they
did not produce citric acid.
It has been noted4 that many cultures of Aspergillus niger
produced citric acid. Although the literature on the chemical
activity of Aspergillus niger is .voluminous, only one reference
has been found relative to citric acid production by this group
Published with the permission of the Secretary of Agriculture.
1 Wehmer, C., Beitr. zur Kenntnis einheimscher Pilse, Hannover, 1893,
No. 1.
2 Wehmer, in Lafar, F., Handb. technischen Mykologie,
Jena, 2nd
edition, 1905-07, iv, 242.
*Martin, J. A., Am. d. Pharm., 1916, lxxxviii, 337.
4 Thorn, C., and Currie, J. N., J. Agric. Research,
1916, vii, 1.
15

Downloaded from http://www.jbc.org/ at CAPES - UFC on November 6, 2014

(From

JAMES

OF ASPERGILLUS

For conducting the citric acid fermentation a well selected culture of Aspergillus wiger is far superior to any culture resembling Wehmer’s Citromyces with which the writer has ever worked. requirements.066. This impression was probably wrong. B. What is t. however.jbc. Downloaded from http://www. another set of conditions differing ever so little. July 1. In the beginning it was hoped that Aspergillus niger cultures could be divided into two general groups.4 Zahorski. supposed that Zahorski had worked with some very unusual culture of Aspergillus niger. In 1913 Zahorski5 was granted a patent in the United States on a method for producing citric acid by fermenting sugar solutions with Sterigmatocystis nigra. and (3) the reaction of the medium.. (2) the general equation of metabolism. Few concise statements can be made concerning the metab&= of an organism capable of producing such a variety of chemical transformations as Aspergillus niger. U.xperimenter will not be .UFC on November 6. Patent No. Much of the preliminary work which served no other useful end than to inform the e. states that Sterigmatocystis differs distinctly from Aspergihs. The writer at first. In upon concentrated fact almost any culture of Aspergillus niger sugar solutions will produce much more citric acid than oxalic acid. for any one of about twenty cultures studied under certain conditions produced citric acid in abundance. 1913. 2014 of fungi. 1. S.org/ at CAPES .rue for one set of conditions may not be true for.358. This is one of the many names that has been used to designate fungi of the black Aspergillus group. Many of the workers who have studied the citric acid fermentation performed only a few experiments without being guided by a fundamental knowledge of the metabolism of fungi or of the conditions favorable to the reaction with which they were concerned.uzzling group of black Aspergilli. In this paper three fundamental factors with regard to Aspergillus niger have been considered : (1) The inorganic salt. one of which produced citric acid and the other oxalic acid. This would lend some aid to the problem of classifying this p.i6 Citric Acid Fermentation 6 Zahorski. No cultures produced citric acid only under all conditions or oxalic acid only under all conditions. In this respect the data are disappointing. Experiments conducted in this way are not likely to make a very definite contribution to any problem.

2014 The cultures were selected from those used in the previous study and are designated by the same numbers. U.org/ at CAPES .7 While either method will give fairly satisfactory results if used with discretion and patience. S. 285. This peculiarity of growth enables the mold to exhaust a deep substrate much more rapidly than if it depended on diffusion alone.. Results were sometimes obtained which could not be duplicated.. N. 6 Pratt. Carbon dioxide was determined by drawing a gentle current of . 1914. Dept. Erlenmeyer flask was used in nearly all of the experiments here reported. This floats on the surface of the substrate but wrinkles in such a manner that it presents an enormous surface of contact. Currie 17 described in detail. D. S. the medium was drained off and the mycelium repeatedly washed. Nevertheless the ratio of the surface to the volume of the media must be uniform in order to obtain results that have comparative values. The cultures were grown at 28°C. them. Bureau of Chemistry. These wrinkled structures often project 5 to 6 cm. vii. 88. Downloaded from http://www.J. of media contained in a 200 cc. into the substrate. The citric acid was estimated either by the method of Pratt! or the method of Kunz. The chemical reactions must proceed in the mycelium. Agrie. and sugar. with the omission of the first two digits. 7 Kunz. Oxalic acid was in all cases estimated by double precipitation as calcium oxalate and titration with standard permanganate. To determine the quantitative relations between the products formed and the sugar consumed. citric acid. A volume of 50 cc. Methods Employed. Mikros. a really convenient and accurate method for estimating citric acid is still wanting. Such results have not been included without comment to this effect. although general conclusions from such preliminary work may be related.. Arch. Separate portions of this solution were then taken for the estimation of oxalic acid. The medium and washings were combined and made up to a definite volume.UFC on November 6. Circ.jbc. The sugar was estimated by reduction with Fehling’s solution and calculated from the reducing factor of invert sugar. R. 1912..

ii. 1159.. The conclusions of Meyer are in all likelihood correct. 2014 carbon-dioxide-free air through the flasks containing the growing mold and absorbing the carbon dioxide produced in caustic potash. J. There is some discussion in the literature on the isomerism of citric acid. Botanique). Ann.. On the other hand. His conclusion was based on chemical data. These were never found and in all probability are never formed at all. The total acidity is nearly exactly accounted for by the sum of the oxalic and citric acids regardless of the proportion in which they occur.acid was obtained by recrystallizing a sample of ordinary citric acid which had been dehydrated at 130°C. 9 Meyer. especially malic and tartaric.org/ at CAPES . Several pounds of calcium citrate have been prepared which by analysis corresponds to the formula Caa(CaHs0J24H~0 and in no wise differs from calcium citrate prepared from the citrus fruits. xxxvi. H. Meyer9 concluded from physicochemical data that the acids were identical. Both citric and oxalic acids have repeatedly been isolated and identified.UFC on November 6. The anhydrous acid was the more stable form at high temperatures and the hydrated acid the more stable form at low temperatures. nat. The oxalic acid can be recovered directly from the fermented liquors by evaporation and crystallization. 1869... J. SC. Ges. Downloaded from http://www. Raulin’slo fluid which is still much used by mycologists is a good illustration of this type of medium. them. 3599. Diligent search was made for other organic acids. them.. The composition as given by Raulin is as follows: 8 Witter. 224. 1903. Ges. Earlier workers with fungi used very complex culture media.18 Citric Acid Fermentation Mineral Requirements of Fungi. The citric acid prepared from this calcium citrate by decomposition with sulfuric acid has sometimes crystallized out anhydrous and sometimes with one molecule of water. Ber. (sir.jbc. 6. Ber. xxv. 1892. lo Raulin. Citric acid because of its very great solubility has been recovered only through the calcium salt. . Witter* claimed that an anhydrous isomeric citric .

6 0. .... .. .J. ..... In some cases when no iron salts were added to the media there was no sporification while in other cases there was an abundant development of spores.07 0... H. . Ammonium tartrate.. Indeed in some media iron salts do not even stimulate the development of mycelium or accelerate the rate of metabolism in any way. ... . . .. 1895.” Molisch.... Tartaric acid.. .... Sitzungsber. . Akad..... . After observing a very large number of cultures of Aspergillus niger upon media to which no iron salts had been added the writer is of the opinion that iron is not at all necessary for the development of spores.. four elements only were necessary for growth-potassium..... . ...07 0. . . .. . 1880.... ... Ferrous sulfate...12 and Benecke. .. Potassium carbonate... .. M&den...... . I2 Molisch. 2014 Although Raulin conducted a very elaborate research on the mineral requirements of fungi.. 554... .. Currie gm. Downloaded from http://www. . Benecke tried to settle this question but acknowledged that his results were inconclusive.. Akad. . .. Wien......UFC on November 6.6 0...... Sitzungsber. .. .jbc.. ..... Wissensch. . No one of these four elements can be replaced by any element of the same chemical group.... xxviii. . . .. of which the ammonium phosphates serve best. .... 487..org/ at CAPES .. . Cane sugar. Ammonium sulfate. .. Zinc sulfate.. .. Botanik.. Ammonium phosphate...... 340.... Wissensch. . W... . magnesium... The addition of iron salts to media containing nitrates always accelerates the rate of metabolism and increases the weight of mycelium... C...... . Distilled water...500 cc. . . The only practicable forms in which nitrogen can be offered in inorganic combination are ammonium salts and nitrates. 1894... .... 70 4 4 0. .. Potassium silicate. . . ciii. . . .... .. .” The combined work of these three investigators showed that after a mold had been supplied with available sources of carbon and nitrogen. ... This was shown by the later work of Nageli.... while if nitrogen be supplied by ammonium salts...4 0.. ... 13 Benecke. he made his formula much more complex than was really necessary. . wissench. . .25 0.. .... .. . Magnesium carbonate. phosphorus.. .... .07 I1 Nageli. 1. . Molisch thought iron was necessary for the complete development from spore to spore... Pringsheim’s Jahrb. . N.. . and sulfur. x.. ...

When the solution has cooled.. In order to settle this point a medium was prepared of the following composition : . but failing in all but two cases to form more than a few scattered patches of spores..UFC on November 6......000 cc... Transfers to this iron-free medium from cultures already grown upon it likewise showed no impairment of ability to produce spores (Fig.. Distilled water. when cool. 1.” The contention is frequently made that this “trace” which is necessary can be obtained from the walls of the glass containers.2 The water used in making up the medium and for recrystallizing the salts was doubly distilled and received in flasks lined All the salts were recrystalwith a pure high melting paraffin... . This suggests that some definite chemical reaction involved in the utilization of nitrates is accelerated in the presence of iron. ...... . 2 grown upon a medium containing iron.. ... 30 2 0..000 cc.org/ at CAPES . The cane sugar was crystallized five times from redistilled alcohol and acetone.. Nearly all media used by mycologists contain at least a “trace of iron.... . ..... and finally twice from alcohol in a platinum dish. To what extent this method has been used to purify saccharose is not known to the writer. lized three times in porcelain dishes and finally twice in a platinum dish... Potassium chloride....... poured into sterile paraffined flasks............ acetone is added until an abundant precipitate of saccharose falls out. ... The medium was sterilized in a platinum dish covered with tin foil and. The same cultures are seen in Fig... Cane sugar... Aspergillus niger spored just as abundantly as on a medium of the same composition to which iron salts were added.. Downloaded from http://www. . ... Magnesium sulfate. ...........20 Citric Acid Fermentation the addition of iron is entirely without effect. This medium had the following composition per ‘1. . With all of these precautions to assure the absence of iron..jbc. . . Ethyl alcohol of 90 per cent strength is saturated at the boiling point with saccharose.. ... Ammonium dihydrogen phosphate.. 2014 gm. . 1)...... . Data in support of this are offered in another section of this paper.2 0. ..

.. .. .... . .. . . Torrey Bot. . 0. It supplies the required nutrients in about the correct proportion. .. . MgSOa..5 per cent of agar has been used for some time to carry about 60 stock cultures of Aspergilli and Penicillia.. .... Club. . ......... . Although their proportion may vary widely with the culture employed and the conditions of growth. KH2POb... ..... FeSOd.. .. ...... .. Currie Saccharose. . . ..... a further word might be included concerning the action of stimulants. .. . ..... 0. . . . and remains clear when sterilized... ... .jbc. .... .2 . .. ...5 .UFC on November 6... 150 . One might suppose that the addition of some of these stimulants would accelerate the rate of the citric acid fermentation and perhaps even increase the yield of acid. .. has a reaction favorable to most fungi.... .... Nearly every culture grows more luxuriantly on this simple three salt medium than on many of the more complex media proposed in the botanical literature.. .. . .. ..7HzO. . Many other substances will also stimulate growth... 19 CC. . . A.. . .. 1.... . Downloaded from http://www. . ... .. HCl. . ..org/ at CAPES . Bull.. ... .... .. . ... ..7H20.. niger may dioxide-tmycelium. . ....J. In accord with this principle it is clear that the highest yield of fermentation by-products will be had when the development of mycelium is restricted and not when stimulated.. . . ... . 291. 21 . .. . . . .. .. . .. .... their sum will account for approximately 95 per cent of the consumed carbohydrate... . . . ... 2014 It might be added here that the iron-free medium described above plus 1. .. . .01 . These four products are nearly always present... ...a little zinc sulfate to a medium stimulated growth.. Raulini” noted that the addition of .. . ..... NHaNOa. 1904. . ... . .. While considering the subject of the mineral requirements of fungi.0 .. . . On this point the writer’s experience agrees in general with the conclusion of Watterson14 that the action of such stimulants enables the fungus to transform more of the food material into its own body substance and less into waste products... . xxxi... . . 2... .. N. .. 14 Watterson.. . N/5 The General Equation of Metabolism The general equation of metabolism be written in the following form: Carbohydrat@citric acid+oxalic of Aspergillus of Aspergillus acid+carbon niger..

0 1.5 0. The measure of control is the measure of success in conducting the oxalic acid Generally speaking. NaNOa ‘I ‘I “ 100 150 150 50 50 50 50 50 50 50 50 50 50 50 50 50 50 NH&O3 ‘I NHnH*POd “ “ ‘I “ “ ‘I “ Asparagine.01 0.UFC on November 6.25 0. and carbon dioxide.2 0.01 0.2 0.5 2.000 KC1 0.0 2. This reaction can be controlled to a very considerable extent. per T Nitrogen.0 3.jbc.01 - these acids have been anticipated as products of the action of fungi but only oxalic and citric have ever been definitely identified.0 1. -- 50 50 50 in KHaPOa -- 1.0 0.0 2. tions most favorable for a high yield of the end-products. tartar& oxalic.0 2.0 2.01 0.0 2.4 0.2 0.5 1.2 1.01 0.2 0.01 0. citric and oxalic acids.0 2.2 0.0 2. All of TABLE Composition oj Media - NO.0 2.5 2.0 2.0 1.2 0. “f$. and results in the formation of the common organic acids. are least favorable for the formation of the intermediate products.2 2 -- 2%3 0.0 1.0 1. ‘I NaN03 ‘I 1NH.NOs “ - Gm.5 0.0 2.01 0.5 0:2 0.5 2.2 1.0 1. oxalic acid.2 0.0 1.5 0.0 Cc.0 1.25 0.0 3.2 0. 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sacchal-X0. carbon dioxide and mycelium.0 2.2 0.2 0. malic.4 0. the condior the citric acid fermentation. The fermentation of a sugar by Aspergillus niger may be considered as an oxidation proceeding in three stages.0 2.2 1.org/ at CAPES .2 1.2 0.2 0.Citric Acid Fermentation 22 We commonly think of respiration as an oxidation which proceeds with the consumption of ‘oxygen and the elimination of carbon dioxide. and producing citric acid.2 0.0 1. 0. citric.2 0.0 2. I. .0 1.2 0. Throughout the plant kingdom there are numerous instances where this respiratory process stops short of carbon dioxide.2 0. and succinic. Downloaded from http://www.5 2.

353 1. - h3lic o.520 0. 2014 - of Metabolism of - .3139 0. The stimulating effect of various substances has been repeatedly studied by determining only the weight of the mycelium. Currie 23 The equation has been written in this order because Aspergilluo niger will readily form oxalic acid when given only citric acid as a source of carbon.2719 0.7 28.6048 0. 0.7 28.0945 0.4475 0. -_ II.4914 0.410 * * 8 9 8 8 8 8 8 8 7 7 7 7 7 7 7 7 8 8 7 7 2.629.3169 1.3106 0.372 0.530 1.126 2.254.7905 0.7835 0. There is a common tendency among investigators to study TABLE Products No.org/ at CAPES .1220 0.597.4 69.5000 3.2907 0.2167 0.4 69.969 0.926 1.707 1.3638 1. I‘ “ ‘I 0.3038 0.0158 0.6401 0.6785 1.4 69.481 0. Carbon dioxide always accompanies the development of mycelium.4 28.9605 0.380 0.7028 * * 0.477 * * - 0. 4 Carbon dioxide.5353 0.1310 Trace.5801 0.475 0.6743 0. in niaer upon !l%ble i.7 142 69.053 2.632 5.417 0.636 0.4133 0.3342 0.7288 0.6345 0. N.f Asvergillus thg Composition-G&en - - a Age.J.2041 0. ABycelium.558 1.7 28. determined.5012 0.2828 - 0.5514 1.jbc.1083 0.UFC on November 6. Many of the students of the chemical activities of Aspergillus niger have taken account of only one or at most two of the products of metabolism.0439 0.507 0. In.268 3.0756 * * 0.094 2.6348 0.0000 0.7 69.4356 0.0869 0. I.7 28.5812 3. -- culture. -_ -_ dagys 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 - * Not 142 142 142 142 28.797 2. In fact it is given off in greatest quantities between the 2nd and 5th days when the growth of mycelium is most rapid.1739 0. in 6%~ o. only the major or more obvious reactions with which they are concerned.7 28.679 0.9757 1.2061 1.7715 1.582 1.7 28. this study an effort has been made Downloaded from http://www.482 1.686 1.2906 0.5254 0.f Media - ah 1.7 28.029 - 1.4 - 1.7368 0.4116 0.985 0.7809 0. ach 1.498 0.7 28. t3tric -- -- 60 Cc.378 2.775 0.525 0.4221 0.468 6.641 0.

iron does not stimulate the metabolic processes in any way. It is now clearly recognized by most workers who are concerned with the reaction of biological fluids that their hydrogen ion concentration is a much more important factor than their titratable acidity. The introduction of calcium carbonate. This gives a much more adequate picture of what has occurred and affords data capable of specific interpretation. 3. . Cultures which cannot be distinguished on morphological grounds give quite different results under the same conditions. The proportion in which the products of the metabolism of Aspergillus niger appear can be varied at will. 2. iron has a marked stimulating effect. Table I shows the composition of the various media and Table II the products of metabolism on the medium bearing the same number. 4. When nitrogen is supplied as nitrates. By a judicious selection of cultures and conditions citric acid can be varied from none at all to over 50 per cent of the sugar consumed. high concentration of sugar.org/ at CAPES . 2014 Some of the more important conclusions to be drawn from an analysis of these data are: 1. When nitrogen is supplied as ammonium salts or as asparagine. Wehme? and also others who have studied the citric acid fermentation worked on the supposition that the acid should be neutralized as formed or the rise in acidity would interfere with the growth of the mold. and nitrogen supplied as ammonium salts rather than as nitrates. Reaction of the Medium. especially noticeable in the increased production of carbon dioxide and weight of mycelium. it is believed the facts brought out here will show.jbc.UFC on November 6. causes many diEculties and. 6.Citric Acid Fermentation to estimate all the products of metabolism in the same culture. It has long been a custom among mycologists to add some organic acid to media intended for the cultivation of Downloaded from http://www. 5. The conditions especially favorable to the citric acid fermentation are low nitrogen supply. Tables I and II are self-explanatory. the only practicable neutralizing substance. is wholly unnecessary.

2014 Lime juice Lime juice Lemonjuice Lemon juice . ‘There seems to be a general impression that the mineral acids and also oxalic acid were too toxic to be used for ’ ’ *HCl AskiTer AskiTer 2 yeasts yeasts Vinegar 3 Wine.coli Streptococci 5 6 7 Water 8 TEXT-Fb. this purpose.J. Currie 25 fungi. The basis of this impression lies in the relatively low dissociation constants of the citric and tartaric acids in comparison with the mineral acids and oxalic acid.org/ at CAPES .s BbuLgaricus 4 Beers B. results generally show that the mineral acids are less toxic than the organic acids. N.jbc. In fact if these acids be used at the same hydrogen ion concentration. It was recognized that these acids would restrain the growth of many types of bacteria without interfering with the growth of molds. 1. The acids most commonly used were citric and tartaric. pH of biological fluids.UFC on November 6. Downloaded from http://www.

This result together with the fact that the cultures readily produce a solution containing 10 per cent of citric acid shows that the limiting hydrogen ion concentration is very high in comparison with most organisms. A........ .. 17 This determination was made by Dr. . .... Bact..7)....” It is interesting to note that three or four of the cultures showed a decidedly greater resistance than the others..7. J. and hydrochloric.. 6.... J.... . .org/ at CAPES .. citric... ... It is of the same order of magnitude as that of lime and lemon juice (Text-Fig... This table shows that the critical pH for most of these cultures lies between 1. ........ The hydrogen ion concentration was determined by the calorimetric scheme of Clark and Lubs.......‘. 2 KHaPO ~..6 and 1. Observations were made at 3 days on the oxalic and hydrochloric acid media and at 5 days on the citric acid medium..... Cultures upon the medium containing citric acid grew slower than the others because citric acid is less readily consumed than saccharose. 33. 16 Clark and Lubs.... ..... ...OOOcc... H........ ..... Clark of this laboratory. ....... ....jbc.. ..... ... ... Water. 1 MgS04.. Saccharose was omitted in the medium to which citric acid was added because this acid provides a good source of carbon. .. 1917.. Downloaded from http://www. ..... .. ... ...46.... M.. .26 Citric Acid Fermentation m...... .. .. 0. . 1917.. .. . ... . 50 NaNO *... and Lubs... Ten cultures of Aspergillus niger and one culture resembling Wehmer’s Citromyces p. 1). .. Saccharose.... W. ii. ....... ......... oxalic......... ... and also that the culture showing the greatest resistance was the culture that 15 Clark.... . . W.... 1. This is in agreement wit’h results obtained by determining the hydrogen ion concentration of liquids fermented by Culture 28.. ...UFC on November 6. 2014 Clark and Lubsl5 give the hydrogen ion concentration of a culture of Aspergillus niger they examined as 2 X 10-Z N (pH 1.fefferianus were inoculated into media made up to definite hydrogen ion concentrations with three acids.7HzO ..2 Acid to designated pH....... .... ii.. . ... . . ... Results are shown in Table III.... .... . Bact...... M... . . . They did not attempt to determine a limit..16 The media employed had the following composition : .4..... The highest result determined by the hydrogen electrode was 1. ...

4 + + ? ? ? ? ? 0 ? 0 + -.5 1.7 1. . 0.8 1.11: 1. ++ + + + +t + t+++ + + ++ + + ‘I + ? 49 .writer cannot resist the temptation to point out the possible bearing of these facts on the problem of preserving the juice of the citrus fruits.7 . . 0 indicates no development. Chem. ). 6: ). ++ + + + + ++ +t + + t+++ “ 0 ? 0 t+ 50 . ..jbc. .4 1. After a concentration of 20 per cent of citric acid is reached the increase in hydrogen ion concentration is very slight in comparison to the added acid. In this connection the . .75 7._----pII:.3 I. ++ ++ 0 0 + + + + + C‘ ? ? ? 0 142 .5 1. Culture 28.25 0.4 is 62 times that of the medium containing the hydrochloric acid and having the same pH and the same inhibiting effect. I Concenlralion acid. + + + + ++ + ? I‘ ? ? 0 ++y ? + + ? 69.0980. ++ + + 0 + + ? + + “ ? 0 + + ? 0 57 .4. acid.7..0870. .E 1.lf3 1. T. - - Citric -- acid.18 I Effect ofHydrogen Hydrochlcric Ion III.org/ at CAPES .7 will make considerable growth on a medium containing 40 per cent of citric acid. The titratable acidity of the medium containing the citric acid at pH 1. R. J.7 1. 78. In& and Eng. viii. --A. 0~. 2014 TABLE Inhibiting .f 1. f-t + + ‘+ f-t f-l ++ +t + + t+++ + “ 96 . 1.0 - 1. ++ ++? 0 + + 0 + ? ‘I 0 0 0 111 .UFC on November 6. .. _ - It is hardly practicable to consider producing a liquid containing more than 10 per cent of citric acid for the concentration of sugar required would exceed the point where the fermentation proceeds most rapidly._ Downloaded from http://www.37 0.6 1. ++? ? + 0 ++ + ? + Penicillium 45 + ++ tt ++ ++ ++ + + t+ ++ ++ + .8 1.4 1. This concentration of citric acid does not interfere with the growth of the mold. I* Will. .t 10.+ indicates positive growth.13. ++ ++ + “ 0 0 0 47 . Per cent of acid. niger 28.. on Aspcrgillus Odie niger.6 1. Currie 27 had previously been selected as the best one for the citric acid fermentation. A glance at the columns representing the pII and the per cent of acid present shows clearly the importance of considering the hydrogen ion concentration... ? indicates germination. ----. N. 15. 1916.J. + + ? +t fS + + t+++ + ++ ++ +t “ 74 .

jbc. This is probably partly due to the reaction and partly to the disturbance of the equilibrium of salts in solution in the medium.. both of which are essential to the growth of the mold. W. The element of time should also be taken into consideration.Citric Acid Fermentation 28 I9 Clark and 2o Hallerbach. Hallerbach20 states that Wehmer’s process failed because it required too long a time on a technical scale. 219. would be at least partially precipitated. Even if calcium carbonate were present the substrate in immediate contact with the mold mycelium would contain free citric acid or calcium citrate in solution and the chance for the consumption of the products of fermentation would not be materially lessened.5. Downloaded from http://www. 2014 Wehmer2 states that one of the chief reasons for the failure of his process was the difliculty encountered in preventing his media from becoming contaminated with organisms which interfered with the citric acid fermentation. This is the best argument against combining the acid in the form of calcium citrate as the fermentation proceeds..UFC on November 6. It is therefore clear that the fermentation can be started off at a hydrogen ion concentration that will greatly reduce the chances of infection. &act. Die Citronenstiure and ihre Derivate. This . Wehme? states that the yield of citric acid was increased by adding calcium carbonate for some of the citric acid was thereby removed from the field of action and could not be consumed by the growing mold. There is also a possibility of recovering the citric acid directly from the fermented liquor. This observation is not in accord with the experience of the writer. Berlin. The fermentation proceeds much more rapidly in an acid medium than in one to which calcium carbonate has been added. 1917. When the fermentation proceeds properly there is little consumption of citric acid as long as sugar is present. without going through the expensive process of separating and decomposing the calcium citrate. Lubs. Higher yields have been obtained and in a shorter time in the absence of calcium carbonate.org/ at CAPES . It would require at least 10 per cent of citric acid to lower the pH from this point to the region where the hydrogen ion concentration would begin to interfere with growth. 25. J. 1911. ii. The magnesium and phosphate radicles. A glance at the tables given by Clark and Lubslg will show that few bacteria will grow below a pH of about 3.

....... .. FeSOa. . ...org/ at CAPES ...25 0. ......01 quantity.. pectins.: Saccharose.. KH2P0. ... ... . .. . ... .. ... 50 2 1 0... . ... Varying 1. If only a liquid is to be removed this can very readily be drained off and a new batch run into the fermentation pans. . ...... . ... .. .... .. ...... . .. ... ..... . . ...... .....OKL 50 1 0. . .. .. .. .. .... KC1 ... ...... .... ......... .. .. .. ........ ... ..01 In order to study the effect of nitrogen on the acid fermentation of Aspergillus niger.. .... .. . FeSOa.... ...5 0..... and show clearly that the restriction of the supply of Downloaded from http://www. . . .. . The cultures were examined on the 7th day...t ....... ... .0. Currie cannot be done with lime juice because of the high percentage of sugar... ........... which has the following composition: Pm. The results in Table IV are in accord with all the other data on this particular question. Water.UFC on November 6....OOOcc. .... ...... . ... KHgPO+. . ............. ..... .iHzO............ It is possible to ferment a second and perhaps even a third or fourth batch of medium with the same mycelial felt with a considerable saving of time. 2014 Selection of a Medium for the Citric Acid Fermentation... 1. Saccharose.. . .... .. ... . .7HzO. .... . .. an effort was made to determine not only the simplest salt mixture favorable for the acid fermentation of Aspergillus niger but also the minimum quantity of each salt necessary.. ..... ...25 .. .. ..........jbc. . .. . .. . . . . ... .J... .. .. and proteins....... . The removal of the products of fermentation would be a much more difficult problem if it were necessary to remove solids such as calcium carbonate or citrate. . MgSOc7HzO. ........... . ... ......5 0. .. NaNO a.. In the work recorded in the pages that immediately follow......7HzO..... . MgSOa. .... ... ... .. . ... . . .. ...... .. . .. ... ... .. NaNO a. . Culture 142 was grown upon a medium of the following composition : Water...... .. . .... KC1 . N.. .. In the beginning a medium was employed after the formula of Czapek’s solution.....

6 137..o 143...jbc.org/ at CAPES .........548 0.... on the Acid Fermentation in ~/lo Cc.. W&high Citric acid. TABLE Effect gillus of Va@ng Quantitiies 1..6 1.5 141 ....695 In order to determine the quantity of potassium the medium should contain. .3 39... 0..42.9 153..30 Citric Acid Fermentation nitrogen tends to increase the amount of citric acid at the expense of the end-products of metabolism..........2 gm.8 3...2 161 ..5 154...... The results indicate that the potassium requirements are very low...Varying gm.... 50 .8 060 gm.2 124...3 162......UFC on November 6. KC1 ......... ...2 quantity...5 150..0 152..... of medium of the following composition: Water ..1 - 58..606 0.. per 60 Cc...2 1......4 2..644 0. Oxalic IV...9 152...500 0..5 14. In order to determine the effect of varying quantities of magnesium sulfate on the acid fermentation of Aspergillus kger.....9 169.2 24....... 2014 1.........0 .....8 2........ MgSOa . four cultures were grown upon 50 cc.1 130.......637 0...000 cc.. 1.......0 ...616 0.4 1......9 149...... of Asperof Medium.. Saccharose. The cultures were examined when 8 days old. 2..8 - 108.7 27... in most cases..........7 6.....2 114... For tabulation of results see Table V......6 2..........4 14.. yy- _- 166.... .634 0.. per liter being sufficient..........0 2....5 7..O 152...2 158... 0... Results niger I kidity of Nitrogen Expressed by titration....4 158......643 0......: NHdHzPO..2 2.1 5...398 0.. acid. Culture 142 was grown upon a medium of the following composition : Downloaded from http://www... 0.

..8 83.. .460 0. ... Acidit:z titra- VI.5 31. .8 133. Results of M&O4 per 1.3 86.... .5 31 Trace. of Potassium Quantities Results Expressed - Acidity by titration. . . ..1 98.. ..0 75.. . FeS04.6 55.... . Wm.6 55. . per liter (Table VI). Citric gm. .0 0..2 0.4 0.543 0.8 97.2 45.20 0. 0.382 69.. . .25 0.. Citric 1 acid. ... ......From the results it appears that the most favorable quantity of MgS04.40 acid.3 94. .2 17. . .30 0. ..2 0. 31.6 0.05 0.1 and 0.. ..535 0..4 0.6 0..498 0. .10 0..7 52. on the Acid E”&mentation in ~/lo Cc...123 0..1 10..520 0. .... . of MgSO4.7 0.. .25 0. ..5 63.7H20 Expressed Medium..0 0.. . . .547 0.551 0.. . ... TABLE Effect of Varying pergillus niger.4 124. MgS01. KC1 per l.jbc... . . . Saccharose.6 110... . between 0.8.. -- -- gm.. ... . . . ...2 93. ..6 lO9. . ‘KC1 . .. of Midium.. ..394 0.. .494 28. .631 0. ...5 8. .4 0.8 151. . .572 0. . .. . ...8 9.8 0.3 89..OOOcc. ...oo( 1 cc. . .8 103. . culture..2 136. 50 2 1 0. .335 0. .. 2014 0..4 16. .8 117.UFC on November 6.2 120..0 0. Oxalic acid.. . ... .. . .2 0.4 0.. .492 The cultures were examined when 5 days old.. . ..3 72.7Hz0. .8 8.org/ at CAPES ...8 78... .522 74 0..0 6.....4 118. ..2 10.. . per - Oxalic -- - acid.521 0...gm.6 159...6 142 .. .0 69.. .. ... ..... .. . “ 2... Weid&. . .4 0.0 82.. .Varying quantity.2 0..4 94.01 0.01 - Fermentation of As60 Cc. - TABLE Effect of Varying Aspergillus Quantities niger 14. .. Water. .8 64...0 103. ... . . .. 115.. . . -- V.8 81.436 of Downloaded from http://www..6 22. .6 143.4 127. NaNOa.0 109.7Hz0 for a medium of the above composition lies. .3 101.8 23.p1y- am.0 14............. .8 93.. on ths Acid in ~/lo Cc. .2 gm.. .. . . . ....7HzO .. 1. KHePOh.. _. -- .. per 60 Cc. . . . .4 52..2 0...4 119.0 6....457 0.5 112.. .... . .15 0.456 0. .OCOcc.. ..

Water. .. . .......000~~... ... ..... 2014 The cultures were examined on the 7th day.. 1.... . ...... Water.. .. ..... 1. of medium were employed). FeS0.25 .. . .. .. 50 1 3 0.7Hz0.. . ..... were not more complex than really necessary five cultures of Aspergillus niger were grown upon media of the composition shown below: om.000 cc.. .....5 NO.. . ..... 0.... ....... .. ...32 Citric Acid Fermentation In order to determine the effect of iron on the acid fermentation of Aspergillus niger. .....5 “ + 0..UFC on November 6.. 1.. ...... .. MgS04.... .. .... . If the fermentation be continued for periods longer than 6 or 7 days. ...... .. “ “ “ + 0. .. KHzPO4... .jbc. ...... KC1 .... ... ........ .. .5 NaCl. ....... . .. . contained in Czapek’s media. .. 50 ................. Downloaded from http://www.. . . ....7HzO.. The addition of about 0.... ......5 KCl........ ........ .org/ at CAPES ..... The above “ 2 ‘L ‘< ‘I ... .. . ... ...... No determinations were made other than total acidity. The results are shown below (50 cc.... .... + t-o. .. ...... . .. ...25 0.... ..... .. .. .. . ... . 2 KH2PO. In general the results were in agreement with those shown above. In order to determine whether the mixture of inorganic salts.. . .. .. . .rJ “ ... . Culture 142 was grown upon a medium of the following composition : gm... ..... ... .. ... especially in the earlier days of the fermentation... . ... . . Varying quantity.7Hz0. ....$ “ I‘ ‘I “ salts. ... ..... NaNOa........... Saccharose........ . .. ............ . . _...... ..... .. . .......01 gm... Saccharose..... 0... .... ..01 FeS04.. ............. 1 MgSOa......... . Other studies have been made on the effect of iron. of ferrous sulfate per liter to media containing nitrates stimulates the growth of mycelium and increases the rate of metabolism. NaNOs... ...7HzO. the unstimulated cultures tend to overtake and even to surpass the stimulated ones in total acidity.

469 0.5 136.617 0.4 152.493 0.521 0. the reaction of media.0 32.472 0. 2014 148.0 42.4 39. and from many experiments not reported in this paper it was concluded that the most suitable medium for conducting the citric acid fermentation with Aspergillus niger should have about the following composition per 1.3 42.7 47.6 61.9 75. 0. Citric -Of acid - -- Medium.0 27.4 41.000 cc.506 0.1 35.6 142 . -- in - ( hlic Acidity.0 94. of medium. Currie 33 The results in Table VII show that the duplication of the potassium radicle and the introduction of chlorides and iron are not only unnecessary but in most cases even unfavorable to the acid fermentation of Aspergillus niger.8 60.7 .570 0.0 om.4 119.1 43.1 78.8 0.0 82.8 67.4 i 28.6 111.4 44.2 32.486 135. TABLE - Results Expressed - No.8 47.0 137.438 96 91.6 88. N.4 38. -- VII.6 157.0 90.0 37.6 118.3 10.8 83.4 19.4 42.8 71.591 69.2 81. Downloaded from http://www. _ _ _ 91. citric.4 34.6 92.597 0.0 43.4 79.511 0.8 142. and the general equation of metabolism of Aspergillus niger.462 0.0 41.9 133.461 140.5 0.458 0.8 78.3 37.9 44.9 80.0 34.5 88.0 12.3 117.2 78.2 0.8 44.0 40. Culture.0 34.J. Cc.6 37.2 121.4 36.9 51.8 45.4 72.4 149.8 147.532 74 115.UFC on November 6.3 76.549 0.5 42.495 0.2 3 4 - - - - - From the experience gained in all of the foregoing studies on the mineral nutrition.8 152. - Weight of Oxalic and I mycelium.0 103.5 85.6 103.2 45:4 44.438 0. ~/lo acid ?r 60 Cc.0 66.1 78.9 0.5 79.jbc.org/ at CAPES .454 0.

. .UFC on November 6.. 25 21 Wehmer. .. KH. N/5). 2. MgS04. about 2 per cent in 24 hours. .4 .___.... 5) containing 1 liter of a I5 per cent cane sugar solution.....” Zahorski5 used “small*amounts of nutrient salts such as ammonium nitrate.75-1. until the 7th or 8th day.0 -2. 2014 The medium proposed by Wehme+ contained “a small quantity of mineral salts (NHdNOs.. Curve II shows the development of acid in a 15 per cent cane sugar solution.. 0..033. It raises the hydrogen ion concentration to a point that makes complete sterilization possible at a single heating in steam at atmospheric pressure for 30 minutes. 3 and 4. were removed at intervals and the acidity was determined by titration. 0. HCI to pH 3. After remaining nearly constant for 2 or 3 days the acidity begins to decline... 5 cc... ...5 ~ .... .__. ..... 1894... potassium phosphate. . . ... . As previously pointed out.....POI. . 2.7H10. .._. The course of the fermentation under varying conditions is shown by the curves in Text-fig.5 per cent cane sugar solution._.jbc.34 Citric Acid Fermentation Saccharose.. KHzEOa _. fermentation proceeds properly the mold does not spore but remains white as shown in Figs. .3. Patent No.20-O. The general course of the fermentation is quite similar... ... 515... . .5 (5 .. U.4 cc.. There is little development of acid during the first 2 or 3 days. 20. . Feb. . .. Erlenmeyer flask.. and magnesium sulfate. 100 cc.. of the medium were contained in a 300 cc. . C.....org/ at CAPES ........ it greatly reduces the dangers of infection of the liquors with organisms which might interfere with the citric acid fermentation without inhibiting the growth of the mold with which the liquors are inoculated.” The addition of hydrochloric acid to the mixture of inorganic salts proposed above has several advantages. 125 -150 . _ . Curve I shows the course of the development of acid in a 12. NHdNOs. When the. Curve III shows the course of the fermentation in a shallow pan (Fig. When a vigorous mycelial felt has developed the rise in acidity is very rapid...0 .. Downloaded from http://www.. S.... .. and MgS04) . The mycelium had been developed in a previous fermentation and the rise in acidity began at once. Many fermentations have been conducted in Erlenmeyer flasks on this medium. ... ..

All of the coriditions that majr influence the course of the fermetitation are not within the control of the experimenter. The variability of the fermentation under what appears to be identical conditions is a difficulty that has not been entirely overcome.J.7 the acidity generally reaches about 10 per cent on the 8th day. N.org/ at CAPES .jbc. was the varying fermentative power of his cultures (Variabilitdt des Garverm6gens). Downloaded from http://www.2 TEXT-FIG. Good results can be had only by the adoption of conditions that prove successful and can be duplicated with a high degree of uniformity. representing the oxidation of a sugar to citric acid. The course of the cit. Along with the citric acid there are generally traces of oxalic acid and sometimes this latter acid may account for 3 or 4 per cent of the total acidity. Not infrequently the acidity is as high as 12 per cent although sometimes it fails to rise above 8 per cent. 2014 With Culture 28. 2. Wehmer2 stated that the greatest difficulty he had to . cdntend with. The removal of this oxalic acid by partial neutralization of the. fermented liquors with calcium carbonate would probably present no serious difficulties. Throughout this study it has been emphasized that a complex biological reaction is involved which results in a number of products and which cannot be expressed by a simple equation. Currie 35 14 12 246 8 16 18 20 22 .ric acid fermentation.UFC on November 6. aside from the infection of his liquors with undesirable organisms.

51 ll.95 4. Many such substances are known to occur as metabolic products of microorganisms.07 10. and bacteria. the fermentation can be conducted with equal success in shallow pans.8789 3. The painstaking investigation of all the conditions favoring the production of such substances will lay the only sure foundations for the development of a chemical fermentation industry It is the hope of the writer that the work here recorded may prove a definite contribution to this much neglected but promising field of scientific endeavor. Ten strains of Aspergillus niger. FIG. acetic acid. butyric acid.05 per cent 3.54 11. xxvii. and lactic acid are prepared by biochemical processes. 2014 per cent 1 2 3 . 1 liter of a 15 per cent saccharose solution was fermented in each of three pans (Fig... 1914. Showing failure to spore on a medium containing iron. 4).Citric Acid Fermentation While most of the experiments have been conducted in Erlenmeyer flasks of various sizes. of liquor could be recovered from each pan by pressing out the mycelium in a hand filter press. About 800 cc. Downloaded from http://www.8209 3. angew. 3. chemical fermentation industry in which many substances would be prepared which are now manufactured by expensive synthetic methods. Chem. Showing sporification on a medium coritaining no iron.35 10. Ten strains of Aspergillus niger. 2.52 15.org/ at CAPES . Analytical data on the liquid from each pan are shown in the table below: 11.jbc. Z. ff Ehrlich. EXPLANATION PLATE OF PLATES. F. molds.ocl Elm. 1. 48.UFC on November 6.93 Already many substances of great technical value such as ethyl alcohol.19 10. 1. FIG. In a recent paper on the chemical activities of yeasts.28 15.7673 per cent per cent 15.69 5. Ehrlichz2 concluded with the prophecy that in time we would have a great. The fermentation was continued for 8 days.

J.7 fermenting 1 liter of liquid in’s shallow pan. 5. N.org/ at CAPES . 2014 . FIG. Downloaded from http://www. 3. I and III show the appearance of the culture when the citric acid fermentation proceeds properly.UFC on November 6. FIG. Showing the appearance of the mycelium at the beginning of the third fermentation. Culture 28. Currie PLATE 37 2. Note the increased thickness and the deeply wrinkled structure of the mycelium.7. II shows the same culture on a medium which favors sporification.7.jbc. FIG. Culture 28. 4. Culture 23.

org/ at CAPES .THE JOURNAL OF BIOLOGICAL CHEMISTRY. (Currie: Citric Acid Fermentation.jbc. Downloaded from http://www.) . 2014 FIG.UFC on November 6. PLATE 1. 1. VOL XXXI.

4. Khnrie: Citric Acid Fermentation.org/ at CAPES .) . 5. 2014 FIQ. Downloaded from http://www. XXXI. VOL. 3.jbc.UFC on November 6. FIG. PLATE 2.THE JOURNAL OF BIOLOGICAL CHEMISTRY. FIG.