STUDIES ON THE FORMATION OF OXALIC ACID BY ASPERGILLUS NIGER*

BY W. W. CLELANDt AND MARVIN J. JOHNSON

(From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wisconsin)

(Received for publication, October 7, 1955)

Although Aspergillus niger, strain 72-4, has been studied previously be- cause of the high yields of citric acid it gives when fermenting sugars, it also produces oxalic acid under the proper conditions. The present work is a study of the conditions under which oxalic acid is formed, the substrates that are converted to it, and the mechanism of its formation from these sub- strates.

Oxalic acid is never found in normal growth fermentations with strain 72-4 until after autolysis has begun. However, in replacement fermenta- tions in which preformed, washed mycelium is resuspended in nitrogen-free medium, oxalic acid is often formed together with citric acid. It was orig- inally suggested (1) that oxalic acid is made by hydrolytic splitting of ox- alacetate. Later work (2, 3), although consistent with this theory, gave data more in line with the results of the present work, which indicate that oxalacetate is oxidatively split to 2 molecules of oxalic acid. Direct oxida- tion of acetate to oxalate was ruled out by the work of Bomstein and John- son (3) and also by the present work.

The present study also shows that, at neutral pH, a great variety of sugars and related compounds can be fermented to oxalic acid by fluoride and fluoroacetate-insensitive pathways. Compounds part of or related to the Krebs cycle still appear to form oxalic acid by oxidative splitting of oxal- acetate.

iMethods and Materials

Culture and Fermentation-A. niger, strain 72-4, was used throughout this work. The methods and fermentation media for sporulation and growth of the mycelium were the same as those used previously in this laboratory (446). The replacement fermentations described here were carried out with preformed, washed mycelium resuspended in sterile nitro- gen-free media. Low pH fermentations contained substrate, 1.0 to 2.5 gm.

* Published with the approval of the Director of the Wisconsin Agricultural Ex- periment Station. Supported in part by a grant from the Atomic Energy Commis- sion .

t Predoctoral Research Fellow of the National Science Foundation, 1954-55.

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596 OXALIC ACID FORMAT105 BY A. NIGER

of KH&‘O+ 0.25 gm. of MgSO*.7H,O, 700 y of iron, and 240 y of zinc per liter, in addition to sufficient hydrochloric acid to lower the pH to 2. Keu- tral pH fermentations contained substrate and 0.2 to 0.4 M potassium phos- phate, pH 7.2, except when the substrates were acids, in which case the potassium or sodium salts were used, together with 0.005 to 0.01 M phos- phate. Fermentations of radioactive substrates were carried out in the closed system described below xvith 25 ml. of medium containing 5 ml. of packed mold suspension. Other fermentations were carried out in 500 ml. Erlenmeyer flasks with 50 ml. of medium containing 10 ml. of mold pellets. Aseptic conditions mere maintained throughout.

Closed System-When it was desired to obtain complete carbon, radio- activity, and oxidation-reduction balances, fermentations were run in a totally closed system with oxygen supplied on demand by an electrolysis cell. The apparatus is the same as that described in an earlier paper (7), except that a two level 300 ml. flask holding up to 20 ml. of alkali in the upper level is substituted for the fermentation flask and gas reservoir of the other system. The two level flask is similar in shape to that used by Dickens and Simer (S), but has no side arms except the one delivering oxygen from the electrolysis cell. As modified for carbon dioxide absorp- tion, the system may be used to study any aerobic fermentation producing carbon dioxide as the only gaseous product wit,hout sacrificing the high aeration rates available from a rotary shaker.

Analytical Techniques-Citric acid was determined by the calorimetric method by use of the pyridine-acetic anhydride complex (9). Oxalic acid was precipitated with calcium at pH 6 and titrated with permanganate after solution in 2 N sulfuric acid. In the closed system, carbon dioxide was absorbed in 4 N alkali and the amount was determined by titration of the carbonate with 1 N hydrochloric acid in the presence of excess BaC12. Glu- conic acid was determined by periodate oxidation at room temperature at pH 1 for 3 seconds (3 moles of periodate reduced per molecule) or 1 hour (4 moles of periodate reduced per molecule). Glycolic acid was determined calorimetrically \yith 2,7-naphthalenediol reagent (10). oc-Keto acids were determined calorimetrically as their 2,4-dinitrophenylhydrazones in basic solution. Tartaric acid was precipitated as potassium acid tartrate from 80 per cent alcohol and titrated to pH 9. Sugar alcohols were determined by periodate oxidation at room temperature. Sugars and ketogluconic acids were determined by the copper reduction method (11).

Citric and oxalic acids were separated and isolated by partition chroma- tography with a 35 per cent butanol-65 per cent chloroform mixture as the flowing phase and 0.5 N sulfuric acid supported on Hyflo Super-Cel (1 ml. per gm.) as the stationary phase.

Degradation Procedures-Citric acid was degraded as described previously

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IV. TV. CLELAND AND M. J. JOHNSON 597

(7). Gluconic acid (about 0.02 M) was degraded by allowing a 20 per cent excess of 0.5 M periodic acid to oxidize it for 20 minutes at room tempera- ture at pH 1. Carbons 1 and 2 form glyoxylic acid, carbons 3 to 5, formic acid, and carbon 6, formaldehyde. The formaldehyde and glyoxylic acid were isolated as their 2,4-dinitrophenylhydrazones, which were separated by solution in ether and extraction of the glyoxylic 2,4-dinitrophenylhydra- zone into Xa,HPOd solution. The formic acid was isolated by distillation.

Radiocarbon Counting Techniques-The sample to be counted was diluted with a known amount of gelatin and adjusted to pH 12; 1 ml. aliquots were then placed on 5 sq. cm. copper disks and air-dried. Sample thicknesses were commonly between 1 and 10 mg. per sq. cm. Samples were counted

TABLE I

Effect of pH on Oxalic Acid Formation

Products Initial pH

Net citric acid Oxalic acid

srmole c ntmole c

2* 6.4 3.4 1.85 9.5 1.3 3.15 1.7 7.4 4.0 -0.1 13.3 5.1 -0.6 18.2

Substrate, 5 per cent glucose. 6 day fermentation. 0.1 M citrate buffer present (30 mmoles of carbon).

* Xo citrate buffer.

in a flowing gas counter, at least 1000 counts being totaled for all but the weakest samples.

Xubstrates-Potassium gluconate-l-C?* was obtained from Dr. Laurens Anderson. Glucose-2-C?*, glucose-6-C’*, xylose-l-C?4, n-arabinose-l-Cl*, and n-arabinose-5-0” were obtained from the n’ational Bureau of Stand- ards.

Results

Effect of pH and Bu$er on Oxalic Acid Formation from Glucose-Although in normal growth fermentations at low pH oxalic acid is not formed, it frequently is formed with citric acid in replacement fermentations. If one increases the pH of the medium by addition of buffer, oxalic acid formation is favored over citric acid production and completely replaces it at a pH above 5, as can be seen from Table I. It can also be seen that the presence of 0.1 M citric acid lowers the oxalic acid yield to one-third that of the

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598 OXALIC ACID FORMATIOK BY A. NIGER

control. This effect was found to bc reproducible and a function of the citric acid concentration. These data suggest that oxalic acid formation is due to a higher than normal pII inside t,he cell. In low pH replacement fermentations, t,his raised internal pII is probably generated by the wash- ing during replacement. At t,he low external pI1 of the growth medium, the cell buffers are likely to be citrate or phosphate anions and protein cations. With the cell wall impermeable to w-ionized acids, it is possible by a Donnan equilibrium to keep the internal pH above 3.3 Jvith a citrate concentration of 1 M inside the cell and up to 0.4 M outside. Washing re- moves the anions, together with hydrogen ions stripped from the proteins, thus raising the pH. Added citric acid would restore the internal cell buf- fer, thus lowering the internal pH and preventing oxalic acid formation. If a buffer of a higher pH is added, the internal pH will remain high, and oxalic acid will be formed.

Eleven different buffers were tested for their ability to cause oxalic acid formation from glucose at neutral pH. (pH values ranged from 5.6 to 8.0, most values being between pH 6.9 and 7.5. All buffers had a so- dium or potassium concentration of 0.6 M.) Except for acetate and ben- zoate which were toxic, and bicarbonate which became too basic, all the buffers gave some oxalate. Citrate, oxalate, phosphate, succinate, and tar- trate gave the best yields; glycolate, phthalate, and pyruvat,e gave less. The yields from citrate, succinate, and tartrate may have been high because of utilizat)ion of the buffer itself; for this reason phosphate was used rou- tinely as a buffer to study the oxalic acid fermentation at neutral pH, ex- cept when acidic substrates n-hich could serve as their own buffers were employed.

Identijication of Gluconic Acid As Intermediate-Study of the fermenta- tion of glucose to oxalic acid at neutral pH showed that glucose disappeared completely in 24 hours, but that oxalic acid was formed more slowly over a period of a week or more. Since the u n k nown intermediate appeared to be an acid because of the large drop in pH accompanying sugar utilization before oxalic acid was formed, gluconic acid formation was suspected. The intermediate was proved to be gluconic acid by preparing the phenylhydra- side and benzimidazole derivatives from a 24 hour sample of a glucose fermentation at neutral pH and comparing them with authentic samples.

It appears that this strain of A. niger possesses a very active glucose dehydrogenase operative only at neutral pH. Glucose is thus rapidly con- verted to gluconic acid or to its lactone which is then slowly converted to oxalic acid and carbon dioxide. 50 other products or intermediates have been isolated. Furthermore, glucose is the only substrate yet observed that is converted t’o an aldonic acid before being oxidized to oxalic acid. All other substrates tried accumulate no observable intermediates.

Oxalic Acid Formation from Various Substrates and E$ect of Inhibitors-

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0.001 M fluoride + 1.001 x fluoroacetate

present iXo inhibitors

Potassium Substrate phosphate,

pH 7.2 -__ ubstrate

used 0 xalic acid ubstrate used C lxalic acid

I.- .I- -__ - M >Iw& c mmole c nmuze c mlilole c

4% glucose .................. 2% mannose. ................ 4% fruct.ose ..................

.) 0.4 67* 21.8 67* 7.11 0.2 28.0 16.2 0.4 23.3 19.8 21.7 14.0

2% galactose ............... 0.2 10.0 5.7 2% sorbose. ............... 0.2 8.0 4.9

4% gluconolactone. .......... 4% 2.ketogluconic acid ........ 4% 5-ketogluconic “ ......

0.4 41.3 25.8 10.9 7.3 0.3 31.3 12.8 0.3 17.4 3.8

4yo xylose .................... 4% n-arabinose .............. 4$$ n-arabinose ............... 2yo ribose .....................

0.4 35.0 17.7$ 31.6 13.3 0.4 11.7 20.4$ 6.7 15.8 0.4 0 10.11 0.2 3.5 3.9

2% mannitol. ................. 2% dulcitol. ......... 2% sorbitol. ................ 4% glycerol. .................

0.2 7.1 5.3 0.2 20.5 12.4 0.2 19.3 12.0 0.4 18.6 16.3 13.5 12.0

2% K glycolate. ........... 0 28.4 18.3 1.5yc K glyoxylate. ......... 0 6.4 7.5 3.5 3.4 4% Na tartrate. ............ 0 11.1 12.9

3% ‘( pyruvate .......... 0 40.0 6.1 2% K citrate ........... 0 13.3 5.1 1.7 0.2 2% “ (‘ ........ ... 0 31.2 25.4 31.2 23.2s

2% “ cis-aconitate ............ 0 23.4 13.9 23.6 13.18

2% “ d-isocitrate. .......... 0 9.9 8.45

2% “ succinate. ... ....... 0 9.3 4.8 4.13 - 4 to 6 day fermentations.

\I:\‘. IV. CLELAND AKD M. J. JOHNSOK 599

As can be seen from Table II, a wide variety of substrates has been shown to be fermented to oxalic acid at neutral pH. The yields shown are in most cases the best obtained; considerably lower yields are frequently ob- tained from glycolate and from citrate (as shown), for example. The inter-

TABLE II Oxalic Acid Form&on ,from Various Substrates and E#ect of Inhibitors

* All of the glucose is converted to g1uconat.e before being fermented further. t Only fluoride present as inhibitor. $ n-Arubinose (and to some extent n-arabinose) at neutral pH causes some autoly-

sis, with apparent formation of oxalic acid from the endogenous carbon released; see also the n-arabinose fermentation in Table III and the discussion in the text.

3 Only fluoroacetate present as inhibitor.

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600 OXALIC ACID FORMATIOK BY A. NIGER

esting thing is that, for the most part, the fermentation appears unaffected by fluoride or fluoroacetate, indicating that the Krebs cycle and glycolysis beyond the stage of trioses do not participate as pathways for oxalic acid formation. (Roth inhibitors prevent germination of the mold and fluoride inhibits the formation of citric acid from glucose at low pH completely.) Sugars, sugar alcohols, and related acids are possibly converted to oxalate through glycolaldehyde, glycolate, and glyoxylate (see under “Discussion”). Compounds that are part of, or closely related to, the Krebs cycle probably form oxalate by oxidative splitting of oxalacetate to glyoxylate, which would be oxidized to oxalate. The quantitative conversion of tartrate to oxalate suggests that it might be an intermediate (formed by hydration of an enol form) in the splitting of oxalacetate. Since carbons 1 and 4 of optically active tartrate are indistinguishable, equilibration of oxalace- tate with tart.rate could be an alternative explanation to equilibration with fumarate for explaining the randomization between the carboxyl groups of oxalacetate that is frequently observed. Such a hypothesis is especially inviting for highly oxidative mold fermentations in which reduction of oxal- acetate to malate is most unlikely.

Of special interest here are the results with citrate. In the case in which over 80 per cent of the carbon was converted to oxalate, fluoroacetate had no effect, while in the other case shown in Table II, oxalic acid formation was completely inhibited. It appears that, in the first case, citrate has been broken down by reverse operation of the Krebs cycle (as normal opera- tion of the Krebs cycle could not convert more than 67 per cent of the citrate carbon to oxalate by any method), and in the second case by normal forward operation of the Krebs cyc1e.l Fluoroacetate inhibits aconitase only when condensed to form fluoroeitrate; this does not happen when the citrate condensation is being reversed. The data for cis-aconitate and d-isocitrate were obtained with the same lot of mold mycelium as was used in the first case above, and fluoroacetate again had no effect. The decrease in yield from citrate to aconitate to isocitrate supports the idea of reverse operation of the Krebs cycle under t.hese conditions.

Tracer Experiments on Oxalic Acid Formation-The results of tracer fer- mentations in Ivhich oxalic acid formation is studied are given in Table III. (The data for the first two fermentations have been published previously

1 The data here show that at neutral pH the Krebs cycle may be active in mycelium grown at low pH, while such mycelium at low pH appears to lack aconitase and causes accumulation of citric acid with no recycling through the Krebs cycle (7). Citric acid formation is evidently the result of inactivation of the aconitase by the low pH caused by the citric acid already present.. This process begins as soon as growth of the mold reaches the point at which the glycolytic enzymes can convert sufficient glucose to citric acid to overload the small amount of aconitase present and cause sufficient accumulation of citric acid to inactivate it.

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W. W. CLELAND AND M. J. JOHNSON 601

(7).) The fermentation of glucose-3,4-P at’ low p1-I produced oxalic acid of specific activity identical with t,hat of the glucose used. The oxalic acid

TABLE III

Tracer Experiments on Oxalic Acid Formation

I I Substrates 1 Citric acid

Glucose-3,4-C’*, pH 2...........21.213.8* 13,300 5.7

Glucose.. 30.724.3 C’40*. 0.5 5T8,0009.8~ K citrate, pH 4.5. 14.9 Gluconolactonc-

l-V, pH 7.2. 32.4 23.9 -L2,000 Glucose, pH 2.... 32.119.9 3.2 K gluconate-l-C14. 0.1 $ 20,000,OOO Xylose-1 -CY, pH

2...... . ...34.822.3 57,800 2.7 Xylose-l-P, pH

7.2.. 16.716.6 38,800 Glucose-2-C”, pH

7.2.. 16.810.611 172,500 Glucose-B-W, pH

7.2.. _. 16.810.9[/ 209,000 n-Arabinose-

I-CP, pH 7.2. 16.8 0.1 D-Arabinose-

212,000

5-W, pH 7.2. 16.8 0 136,000

jpecilic ctivity

7,SSOt

2,700t

6,5001

‘5,SOOl

Oxalic acid

Specific activity

Carbon dioxide

My-

Specific c&urn

activity

1.61 12,650i 3.11 7,860i 23,500

7.1 5,500 1.538,400

4.1 20,300 9.458,900 15,100 1.7 21,400 6.475,400 48,000

3.1 27,200 10.441,000342,000

9.2 25,000 6.432,300 188,000

5.4198,OOO 5.130,900503,000

5.3246,OOO 5.240,500685,000

1.8 28,000 2.226,200 81,900

1.5 72,800 1.9 7,000 21,600

The data for the first two fermentations have been published previously (7). The fermentations at pH 7.2 contained phosphate buffer.

* 3.4 mmoles of carbon converted to gluconic acid. t Distribution of labeling given in the text. $ Net citrate formed. $ Final gluconate concentration, 0.5 mmole of C; specific activity, 1,230,OOO c.p.m.

per mmole of C. [I The remainder of the glucose was converted to gluconic acid.

could have come from oxalacetate (the citric acid formed contained a full label in the tertiary carboxyl group and one-seventh of a label in the pri- mary carboxyl that came from oxalacetate), or from the gluconic acid in the fermentation with slight] dilution. In any case, t,he 3 and 4 carbons of glucose appear to be good oxalic acid precursors.

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602 OXALIC ACID FORMATION BY A. NIGER

Oxalic acid became labeled during the fermentation of glucose at pH 4.5 in the presence of labeled carbon dioxide. As noted in the earlier paper that described this experiment (7), an analysis of the labeling pattern pro- duced has been made. The analysis (which is analogous to the one made in that paper for a low pH fermentation) yields three equations for the specific activities of citrate, oxalate, and carbon dioxide, which, if citric acid is assumed to be formed from oxalacetate made from pyruvate and carbon dioxide, can be simultaneously solved in pairs to give a value for the fraction of theoretical incorporation of carbon dioxide into oxalic acid. The average of the three values obtained is 0.07. If all the oxalate were formed from oxalacetate, the expected value would be 0.25 (since 1 of the 4 carbons of oxalacetate comes from carbon dioxide); thus 28 per cent of the oxalate appears to have come from oxalacetate. The rest must have been formed from glucose by another pathway.

A trace amount of acetate-2-C4 was added to a fermentation of glucono- lactone at neutral pH to see whether acetate could be directly oxidized to oxalate. Two-thirds of the label appeared in the carbon dioxide, which had a specific activity (in counts per minute per millimole of carbon) 1.3 times that of the oxalate. It appears that acetate is not oxidized directly to oxalate, but probably enters the Krebs cycle and is oxidized to carbon dioxide. The oxalate label probably came from splitting of some oxalace- tate, which would be highly labeled.

To test whether oxalic acid could be oxidized to carbon dioxide, xylose was used as a substrate at neutral pH with tracer amounts of labeled oxalic acid present. Although sufficient oxalic acid was produced to dilute the specific activity of that added by a factor of 8, no label at all appeared in the carbon dioxide produced. Oxalic acid is evidently an inert end-product of metabolism under these conditions.

Fermentation of gluconolactone-l-Cl4 at neutral pH (Table III) gave carbon dioxide containing 50 per cent more label, per molecule, than the oxalic acid, suggesting that conversion to pentose is a major pathway for met,abolism of gluconate. The label in the oxalate is too high, however, to be accounted for by carbon dioxide fixation into oxalacetate, alone, and must represent either another carbon dioxide fixation reaction (which seems unlikely) or a pathway for metabolism of gluconate which does not involve decarboxylation of carbon 1.

Similar results were obtained by fermentation of glucose at low pH in the presence of tracer amounts of gluconate-l-Cl4 (Table III). The carbon dioxide has twice the label, per molecule, of the oxa1at.e and contains over 10 times the total number of counts, indicating that most of the gluconate was decarboxylated to pentose. A considerable amount of the oxalate could have come from oxalacetate, although it has 15 per cent more label

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W. W. CLELAND AND M. J. JOHNSON 603

than the corresponding carbons of the citric acid which come from oxal- acetate. (The tertiary carboxyl has 30 per cent, the primary carboxyl com- ing from oxalacetate 60 per cent, and the other 4 carbons 10 per cent of the label.) The labeling in the citric acid formed indicates that some of the gluconate label has found its way into pyruvate, suggesting that here, too, some of the gluconate may be metabolized by a pathway other than decarboxylation.

The data also allow a calculation of how much carbon passed through the gluconate pool during this fermentation. The maximal figure obtain- able (which assumes that all of the gluconate was formed at the start of the fermentation and later partly metabolized) is only 0.56 mmole of carbon, showing that most of the oxalic acid did not come from gluconate.

Comparison of the results of fermentations of xylose-l-C* at low and neutral pH (Table III) shows that in both cases the carbon dioxide has a specific activity higher than that of the oxalate by nearly the same ratio, suggesting that similar mechanisms may be operating at both pH values. The labeling in the citric acid formed at low pH is not of too much help (15 per cent in the tertiary carboxyl, 18 per cent in the primary carboxyl coming from oxalacetate, and 67 per cent in the other 4 carbons). A more complete degradation is needed to tell whether the label is spread evenly throughout the molecule or concentrated in the chain carbons coming from the methyl of pyruvate (which one might expect if xylose were converted to xylulose and split to non-labeled oxalate and triose which would give pyruvate-3-C’* or hexose-1 , 6-C14).

Fermentations of glucose-2-C’* and glucose-6-U* at neutral pH provide nearly similar results (Table III). The oxalate is very highly labeled and appears to have been formed by complete conversion of carbons 2 to 6 of the hexose to oxalate after decarboxylation of carbon 1. The residual glu- conate in both fermentations, when degraded, showed no activity in posi- tions other than the one originally labeled in the glucose. The carbon dioxide is partly formed from carbon 1 by decarboxylation, but appears to have come mostly from low activity carbon exchanged from the mycelium. The large amount of activity recovered from the mycelium in the fermenta- tions of labeled glucose and xylose suggests that there is a sizable reserve of carbon in preformed, washed mold mycelium which may exchange with substrates under proper conditions, but is not readily removable from the cell. (Shaking of preformed, washed mycelium in distilled lvater for 3 days causes no autolysis or release of titratable acid, but the presence at neutral pH of a compound such as D-arabinose causes extensive visible autolysis and formation of oxalic acid from endogenous carbon.)

The fermentations with labeled n-arabinose (Table III) support the idea of a reserve pool of carbon in the mycelium. n-Arabinose (and to some

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604 OXALIC ACID FORMATION BY A. NIGER

extent n-arabinose and ribose; see Table II) appears to be a poor substrate for the mold, very little of it being utilized. Either the breakdown of the n-arabinose produces sufficient reducing substances to falsify the analysis, or D-arabinose causes the carbon reserve of the mycelium to be mobilized and converted to oxalate and carbon dioxide. The latter possibility seems more probable, as there appears to be considerable autolysis with D-arabi- nose as substrat,e.

DISCUSSION

The results above leave little doubt that oxalic acid is formed as the result of raised internal pH in mycelium grown at low pH for citric acid production. The mechanism of its formation is not so clear, but the follow- ing general picture is indicated.

At low pH, a large proportion of the oxalic acid formed is derived by oxidative splitting of oxalacetate. Oxalacetate is probably present in the mold in larger amounts than acetyl coenzyme A, since, with the overloaded carbohydrate metabolism supplying high energy bonds faster than they can be used, phosphoenolpyruvate mill tend to be carboxylated rather than transfer its phosphate to adenosine diphosphate. Thus, when the rise of the internal pH of the mold due to the washing permits oxalacetate to be split, sufficient substrate is available. Also, if tartrate is postulated as an intermediate in this splitting, the relatively large amount of oxalacetate will be sufficient to overcome the unfavorable equilibrium which probably exists for the conversion of oxalacetate to tartrate.

In fermentations run at high pH values, less oxalate is made from oxal- acetate, as the carbon is diverted before reaching the Krebs cycle, unless the substrate is a compound such as pyruvate or citrate which has no choice other than to enter the Krebs cycle. The fermentation of citrate which gave over 80 per cent of the carbon in oxalate adds to the evidence that oxalacetate is normally present in larger amounts than acetyl coenzyme A. Here the rapid splitting of oxalacetate has drawn the equilibrium in favor of acetyl coenzyme A and oxalacetate formation, and at least some of the acetyl coenzyme A appears to have condensed to form succinate and oxi- dized to oxalacetate. This C&-C& condensation does not normally appear in fermentations with glucose as a substrate (7) ; it is logical to assume that it does not because the excess of oxalacetate removes the acetyl coenzyme A to form citrate before it can condense with itself to form succinate.

At neutral pH, glucose is oxidized rapidly to gluconic acid, which is mostly decarboxylated to pentoses; however, a part of it appears to be converted to oxalate by a pathway which preserves the label of carbon I in the oxalate. It is possible that the gluconate is converted to 2-keto-3- deoxygluconate and split to pyruvate and triose. The pyruvate-l-Cl4 that

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W. W. CLELAND AND M. J. JOIlNSON 605

would result, if carboxylated to oxalacetate, would give labeled oxalate when the oxalacetate was split.

Hexoses other than glucose, pentoses (including those derived from glu- conate), trioses, and compounds easily converted to these appear to be con- verted to oxalate by a pathway not involving the Krebs cycle or glycolysis beyond the stage of trioses. The results from fermentations of glucose-2- Cl4 and glucose-6-C14 (Table III) suggest strongly that all the carbon enter- ing this pathway is converted to oxa1at.e. It therefore seems reasonable to postulate a mechanism similar to the accompanying one (which would, no doubt, actually involve the phosphate esters of the compounds). This pen- tose cycle requires only transketolase, transaldolase, and pentose and tetrose isomerases in addition to the glycolytic enzymes converting hexoses to tri- oses. Hexoses and trioses could enter the cycle by the reaction between fructose and glyceraldehyde which produces ribulose and erythrose.

3 ribose + 3 ribulose + 3 glyceraldehyde + 3 sedoheptulose 4 glyceraldehyde + 4 sedoheptulose + 4 fructose + 4 erythrose 3 fructose + 6 glyceraldehyde Fructose + ribose + sedoheptulose + erythrose 5 erythrose + 5 erythrulose 5 erythrulose + 5 glyceraldehyde + 5 ribulose + 5 glycolaldehyde 2 ribulose --f 2 ribose

Net, 2 ribose --f 5 glycolaldehyde Glycolaldehyde + glycolate --) glyoxylate + oxalate

This system must be inactive at low pH, since citric acid formation takes place quantitatively under ideal conditions. As the pH rises, this system drains off more and more carbon, becoming at neutral pH the major path- way for metabolism of sugars.

This theory calls for complete conversion of sugar to oxalic acid with no carbon lost as carbon dioxide (except in the case of glucose and gluconate). Such yields are sometimes approached (see Table II, also the isotopic re- sults of fermentations of glucose-2-C14 and glucose-6-Cl4 in Table III), but more commonly considerable carbon dioxide appears to be produced. This may be the result of operation of the Krebs cycle (with only small amounts of pyruvate available, oxalacetat,e mill not be present in large quantities and mill not be readily split to oxalate), or of the hexose-monophosphate shunt in competition with the production of glycolaldehyde by the pentose cycle.

SUMMARY

Oxalic acid is formed by Aspergillus niger, strain 72-4, as the result of increased internal pH. This increased pH may be the result of loss of

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606 OXALIC ACID FORMATION BY A. NIGER

anions from the mycelium due to washing or of the addition to the medium of a buffer with a neutral pH.

At low pH values, oxalic acid is mostly formed by oxidative splitting of oxalacetate; this is postulated to involve tartrate and glyoxylate as inter- mediates. At neutral pH values, oxalic acid is formed from a wide va- riety of substrates. Compounds part of, or closely related to, the Krebs cycle form oxalate by splitting oxalacetate. Sugars and sugar alcohols are converted to hexoses and pentoses. These are rearranged by the enzymes of the pentose cycle to glycolaldehyde, which is then oxidized to oxalate. Glucose is an exception, being converted rapidly to gluconic acid, which is then more slowly metabolized to oxalate (mainly via pentoses).

BIBLIOGRAPHY

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