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T H E MECHANISM OF FORMATION OF CITRATE AND
OXALATE BY ASPERGILLUS NIGER*
Bf R. A. BOMSTEIN AND MARVIN J. JOHNSON
(From the Department of Biochemistry, College of Agriculture, University of
Wisconsin, Madison, Wisconsin)
(Received for publication, March 14, 1952)
From the distribution of radioactivity in the citrate they obtained, using
labeled acetate and C0 2 , Lewis and Weinhouse (1,2) have concluded that
the citric acid cycle is a major pathway for the formation of citrate by
Aspergillus niger under the conditions they employed. It is obvious that
in glucose fermentations in which citrate accumulates (60 to 75 per cent
molar yield) reactions of the citric acid cycle, but not the complete cycle,
could form the major pathway for citrate formation. Similarly, while a
condensation of three 2-carbon fragments could account for the formation
of some citrate, as suggested by Foster and Carson (3), this reaction (assuming the C 2 compound is formed via the Embden-Meyerhof scheme)
permits maximum citrate yields of 67 per cent and, by itself, could not
account for yields above this value. In our experiments the conditions
found by Shu and Johnson (4) to be optimum for obtaining citrate yields
of about 60 to 70 per cent from hexoses were employed to determine to
what extent the conclusions reported above for fermentations giving low
citrate yields (10 to 25 per cent) are valid for fermentations of high citrate
yield. The results obtained, in general, support the C 2 -C 4 condensation
and the pyruvate carboxylation reactions proposed by Lewis and Weinhouse (1) and indicate that the citric acid cycle is not operative to any
significant extent when glucose is the chief substrate.
According to Lewis and Weinhouse (2) oxalate may be formed from splitting of oxalacetate, but probably not from splitting of oxalosuccinate, nor
by direct oxidation of acetate. Their interpretation of their results was
hampered by the fact that A. niger in their experiments had a high endogenous metabolism relative to its exogenous metabolism. In the experiments to be detailed here this obstacle was not encountered. The
results obtained indicate that oxalate might be formed by splitting of
oxalacetate or oxalosuccinate, but not by direct oxidation of acetate.
Methods
Culture and Fermentation—In order to obtain high yields of citrate A.
niger 72A (5) was used throughout this work. Shu and Johnson (5, 6),
* Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Atomic Energy Commission.
143
144
CITRATE FORMATION BY A. NIGER
among others (7-9), have shown that certain concentrations of iron and
zinc are optimum for obtaining high yields of citrate, and that traces of
manganese may adversely affect the yield. Therefore, chemically cleaned
Pyrex glassware, doubly distilled water, and chemicals of analytical reagent grade were used throughout this work. The sporulation and fermentation media found by Shu and Johnson (5, 6) to give high yields of
citrate were used. The mold was permitted to grow 5 to 7 days on a
rotary shaker at 25° in the fermentation medium. The mycelial pellets
from six flasks were then harvested, pooled, and washed six times with
100 ml. portions of distilled water. Aliquots of a suspension of the mycelium in the replacement medium (same as the fermentation medium
above, but with no ammonium nitrate, and half the iron concentration)
were pipetted into the reaction vessels. All operations were performed
under aseptic conditions.
Analytical Techniques—Citric, oxalic, succinic, and acetic acids were
isolated chromatographically essentially by the methods of Zbinovsky (10).
Succinic, malic, oxalic, and acetic acids were determined by titration of
the effluent. Citric acid was determined colorimetrically by the method
of Saffran and Denstedt (11) after extraction from the effluent with a
calculated (from titration of an aliquot of the effluent) amount of alkali.
The RF values of the above acids were very constant when care was taken
to maintain constant conditions for chromatography. This enabled identification of the acids by comparison with known acids chromatographed
under the same conditions. Succinic acid was also identified by comparison of the rate of decolorization of methylene blue with that of known
samples of succinic acid when a pig heart preparation (12) of succinic dehydrogenase was added to the unknown and known samples. Oxalic
acid was also identified by titration of the silver salt (prepared from the
column effluent after extraction with alkali) with thiocyanate and comparison of the amount of oxalate calculated from this determination with
that calculated from direct titration of the effluent. The procedure of
Feigl (13) was employed for more positive identification of acetate.
Degradation Procedures—For chemical degradation of citrate essentially
the method of Lewis and Weinhouse (1) was used upon the chromatographically isolated acid. Essentially the procedures of Potter and Recknagel1 were employed for the enzymatic degradation of citrate. The reaction was stopped with 0.2 ml. of 50 per cent sulfuric acid and the reaction
mixture deproteinized by addition of 0.2 ml. of 20 per cent tungstic acid,
followed by centrifugation. 1.0 ml. of 10 per cent 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid was added to the centrifugate or an
aliquot thereof and allowed to react at room temperature for an hour.
The hydrazones formed were extracted by shaking the solution twice with
1
Potter, V. R., and Recknagel, R. O., private communication.
R. A. BOMSTEIN AND M. J. JOHNSON
145
0.2 volume of ether. The ether extract was evaporated to dryness. The
residue was taken up in 5 ml. of the upper phase, resulting from shaking
400 ml. of a solution of 35 per cent butanol and 65 per cent Skellysolve C
with 50 ml. of 0.5 M potassium citrate buffer at pH 5.0. The sample was
then chromatographed on a silica gel or Hyflo Super-Cel column 18 mm.
in diameter. 4.0 ml. of the citrate buffer were mixed with 4.0 gm. of the
Super-Cel for this column. The Super-Cel was then mixed with 40 ml. of
the butanol-Skellysolve-citrate solution and the slurry poured into the
column. To separate the hydrazones placed on this column the upper
phase described above was run through the column at the rate of 0.5 ml.
per minute. Excess hydrazine reagent was eluted first, the hydrazones
of pyruvic, a-ketoglutaric, and oxalacetic acids following in that order.
The fraction containing the a-ketoglutarate derivative was concentrated
in vacuo and taken to dryness in a vacuum desiccator. 20 mg. of carrier
were added and the hydrazone was degraded by the method of Krebs (14).
The carbon dioxide was absorbed in sodium hydroxide, while the reaction
mixture was extracted with ether continuously, the ether extract evaporated to dryness, and the succinic acid isolated chromatographically. For
quantitative determinations of the amount of keto acids the method of
Friedemann and Haugen (15) was used.
Counting Techniques—All radioactive substances were counted as such
by pipetting an aliquot of known amount into a gelatin solution. 1.0 ml.
aliquots of this solution were pipetted onto copper planchets 5 sq. cm. in
area and allowed to dry in air. The gelatin solution contained enough
gelatin to give a thickness of 4 to 7 mg. per sq. cm. and enough sodium
hydroxide to give a final concentration of 0.01 N.
Results
Carbon Recovery—Washed mycelium of A. niger was suspended in the replacement medium to which traces of C14-carboxyl-labeled acetate (about
0.0005 mole per mole of glucose) were added. The fermentation was carried out in Pyrex tubes (1 inch X 6 inches) placed on a rotary shaker.
Compressed air was freed from carbon dioxide, rehumidified, filtered
through cotton, and bubbled through the reaction vessels, and the carbon
dioxide evolved in the fermentation absorbed in sodium hydroxide solution. The results obtained in four of the runs with the above procedure
are given in Table I. The low yield obtained in Experiment 1 may have
resulted from accidental introduction of traces of manganese ions. It
may be seen that the only products formed were carbon dioxide, oxalate,
and citrate. No other products were found, and the total acidity produced
did not exceed the acidity due to oxalic and citric acids. The unaccounted
for sugar carbon was, therefore, presumably assimilated by the mycelium.
Degradation of Citrate—As no chemical method lacking optical specificity
146
CITRATE FORMATION BY A. NIGER
is satisfactory for differentiating 1 primary carboxyl of citrate from the
other, it was necessary to turn to the enzymatic degradation procedure of
Potter and Recknagel1 for information concerning the relative specific
activities of these carboxyls. Diagram 1 illustrates the presumable mode
of degradation of citrate initially labeled by incorporation of carboxyllabeled acetate. Carboxyl 1 is the carboxyl derived from that of acetate.
Carboxyl 2 is that derived from the /3-carboxyl of oxalacetate via carboxylation of pyruvate. Carboxyl 3 is that which would be derived from the
a-carboxyl of oxalacetate. Data to illustrate the validity of the degradation procedure and a comparison of the results of the enzymatic method
with those obtained by chemical degradation (Lewis and Weinhouse (1))
TABLE I
Submerged Fermentation of Glucose and Carboxyl-Labeled Acetate
!
Experiment
No.
;
Citrate
!
1
2
3
4
Products formed
Glucose used
90.0
56.1
65.0
90.0
f«
32.3
36.2
46.3
48.5
Oxalate
Carbon
dioxide
Molar yield Sugar carbon
of citrate
"lost"*
Titratable
acidity
M»
llM
per cent
IM
ml. 0.1 x
NaOH
11.2
16.2
15.7
24.5
170
37.5
60.3
88.0
35.9
64.5
71.3
53.9
25.8
8.2
3.5
18.6
1.20
1.45
1.65
2.00
All data based on 1 ml. reaction mixture. Fermentation conducted with washed
mycelium in replacement medium (traces, about 0.0005 mole per mole of glucose, of
carboxyl-labeled acetate added) under aeration with C02-free air.
* Lost sugar (probably assimilated by mycelium) is sugar not accounted for as
citrate, oxalate, or carbon dioxide.
of the same samples are given in Table II. The values calculated for the
number of moles of a-ketoglutaric acid formed from the oxidation of
citrate to a-ketoglutarate are lower than those obtained or calculated from
the manometric data. This may be explained by endogenous activity
of the enzyme preparation. The activities of carboxyls 1 and 3 (Diagram
1) as determined by the two methods checked well. The activities of
carboxyl 2 checked less well. The reason for this is unknown.
Validity of C2-C4 Condensation—In Table III the results of the enzymatic degradations are given in more detail, as well as the specific
activities of the carbon dioxide and oxalate formed during the conversion
of glucose to citrate. It may be seen that citrate has by far the highest
specific activity of any product and that the greatest activity is in carboxyl
1. This is the result to be expected if the present, most widely accepted
mechanism for citrate formation is valid. According to this mechanism,
R. A. BOMSTEIN AND M. J. JOHNSON
H.CCOOH (1)
I
I
HOCCOOH (2)
H»CCOOH (3)
Citric acid
k.
cia-Aconitic acid
I
Isocitric acid
i
i
(1)
Oxalosuccinic acid
HjCCOOH
I
I
H,C
+ COs (2)
0=CCOOH (3)
a-Ketoglutaric acid
i
H,CCOOH (1)
I
H.CCOOH
Succinic acid
+
CO, (3)
Diagram 1. Enzymatic degradation of citric acid
TABLE
II
Enzymatic and Chemical Degradations of Citrate
a-Ketoglutaric acid
Counts per /ill
Calculated
Experiment
No.
Citrate carboxyl*
Counts
recovered
Found
From From
added
0,
citrate uptake
Citrate chain
1+ 3
2
Enzy- Chemi- Enzymatic
cal
matic
Chemi- Enzycal
matic
Chemical
per cent
1
2
3
4
14.4
15.3
15.5
13.7
14.1
14.4
15.5
15.3
16.4
75
79
83
447
214
182
177
418
211
176
1611
62
41
34
37
47
29
34
22f
(0)
(0)
(0)
(0)
Citrate samples are from corresponding experiments of Table I.
* The carboxyl designations are from Diagram 1.
t Only 94 per cent recovery was obtained in this chemical degradation.
7
6
20
6
148
CITRATE FORMATION BY A. NIGER
glucose is split into 2 molecules of pyruvate, 1 of which is oxidized to
"active acetate" and carbon dioxide, the other combining with this carbon
dioxide to form a C4 acid. The C4 acid then condenses with "active acetate" to form a molecule of citrate. If the assumptions are made that this
mechanism is correct, and that radioactive acetate equilibrates with active
acetate from glucose, the specific activity of acetate, and, thus, of carboxyl
1, may be calculated. These values are given in the 6th column of Table
III. As may be calculated from the data of Table III, the observed counts
in carboxyl 1 represent 72 to 120 per cent equilibration of added acetate
TABLE
III
Radioactivity of Fermentation Products
Counts per /xu
Experiment
No
Counts
recovered
Citrate
Carbon
Oxalate dioxide
Found Calculated!
Citrate carboxyl*
1
2
3
382
(100)
173
(100)
156
(100)
139
(100)
62
(16)
41
(24)
34
(21)
37
(17)
65
(17)
29
(17)
26
(17)
38
(28)
2 or 3 calculated
from total counts
inCOj
per cent
1
88.0
38
18
508
320
2
86.5
29
17
243
232
3
87.5
4
100
3.1
6.6
208
203
6.7
5.0
202
147
(8.3)
(5.1)
(5.3)
(3.4)
The products are from corresponding experiments of Table I.
The figures in parentheses are values of carboxyls on the basis of carboxyl 1 = 100.
* Carboxyl designations are from Diagram 1.
t From the assumption that acetate from glucose is equilibrated with radioacetate.
with acetate from glucose. Probably, then, the C2-C4 condensation reaction is valid for the formation of citrate.
Origin of Activity of Carboxyls 2 and 8—The counts in carboxyls 2 and 3
might be ascribable to recycling of part of the citrate formed via the citric
acid cycle, resulting in formation of oxalacetate having a "half label"
in each of its carboxyls. It may be seen, however, that, in Experiments
2 and 3 (Table III), carboxyls 2 and 3 are unequally labeled. The lower
activity of carboxyl 3 might be ascribed to partial equilibration of the
"carbon dioxide carboxyl" of the oxalacetate with carbon dioxide. There
is, however, a serious objection to the assumption that the activity of
carboxyls 2 and 3 results from recycling of citrate. The maximum amount
of recycling that could have occurred may be calculated from the total
R. A. BOMSTEIN AND M. J. JOHNSON
149
count in the carbon dioxide. For every mole of citrate recycled, 3 moles
of carbon dioxide are evolved, 2 moles of which have half the activity of
carboxyl 1, the 3rd mole being unlabeled. The last column of Table III
shows the labeling of carboxyls 2 and 3 that would result from the amount
of recycling that could have occurred. It will be seen that the values
fall far short of those observed. Moreover, if any incorporation of activity into carbon dioxide occurred by mechanisms other than recycling,
even less recycling could have taken place.
It therefore appears impossible that the radioactivity of carboxyls 2
and 3 is due solely to recycling. A similar conclusion can be reached by
calculation of the maximum amount of recycling from the molar yield of
citrate or from the total carbon dioxide production.
As has been pointed out, the formation of oxalacetate from two 2-carbon
fragments is excluded as a main pathway, because observed yields of citric
acid are higher than those possible by this reaction. However, the formation of about 20 per cent of the total citrate from oxalacetate formed
in this way would adequately explain the labeling of carboxyls 2 and 3.
C2-C2 Condensation—Supporting evidence for the occurrence of a C2
to C2 condensation was obtained from the use of carboxyl-labeled acetate
as the sole substrate for washed mycelium in submerged fermentation.
As may be seen from the data of Table IV, the succinate isolated at 40, 66,
and 92 hours had an almost constant specific activity, about 24.5 c.p.m.
per ixu, and the malate at 40 hours had 24.6 c.p.m. per >IM. If these acids
were derived from citrate by the scheme of Diagram 1, they would have the
same activity as carboxyl 1 of the citrate. However, enzymatic degradation of the citrate at 40 hours showed that carboxyl 1 had 19.5 c.p.m. per
JUM, making it improbable that the succinate and malate were derived from
citrate alone. In support of this conclusion it will be noted from Table IV
that the quantity of succinate present in the medium increased with the
amount of citrate, but practically disappeared as the citrate was utilized.
On the other hand, the sum of the activities of carboxyls 2 and 3 was 24.6
c.p.m. per JUM, about the same as that of the succinate and malate. As
carboxyls 2 and 3 are the carboxyl groups of the 4-carbon precursor of
citrate, the succinate or malate could be identical, or in equilibrium, with
this precursor and would have been synthesized before formation of citrate. The only apparent substrate from which they could have been
synthesized was acetate. However, acetate had a specific activity of 17.1
c.p.m. per HM, and a C2 to C2 condensation from this substrate would
yield 4-carbon acids with an activity of 34.2 c p.m. per JUM. The observed
activities of the C4 acids could be accounted for by the assumption that
they were formed from acetate, but were in equilibrium with oxalacetate
(oxalacetate was isolated at 40 hours from the medium) that exchanged
its carboxyl initially with carbon dioxide of lower activity. Subsequent
150
CITRATE FORMATION BY A. NIGER
equilibration of the carboxyls of oxalacetate through a 4-carbon acid cycle
might yield the observed activities. An alternative explanation is that
about 50 per cent of the C4 acids was formed by direct C2 to d condensation, the other 50 per cent by degradation of citrate. This would give C4
acids with a specific activity of 26.8 c.p.m. per IXM.
Existence of Initial Pool of Citrate Precursor—It was suggested by Dr.
Harland G. Wood that in the experiments in which only traces of acetate
were added initially to the medium the data obtained from analyses at the
conclusion of the experiments might not represent truly the conditions
obtaining during the fermentation. Therefore, an experiment was perTABLE
IV
Submerged Fermentation of Carboxyl-Labeled Acetate As Sole Substrate
Time
Acetate
utilized
c.p.m.
per ftn
hrs.
0
40
66
92
117
Residual
acetate
0
63
110
193
201
16.6
17.1
17.1
18.0
Oxalate formed
"
pap*
28 | 11.5
30 j 11.8
93 ! 9.54
141
8.61
Succinate found
**
1 per UM
0.33
1.05
1.95
0.04
23.8
24.8
24.5
Malate found
Citrate formed
c.p.m.
per nil
0.33
24.6
c.p.m.
per pM
0.32
2.0
3.8
6.4
1.5
44.0
44.0
44.0
44.0
The enzymatic degradation of citrate at 40 hours gave for carboxyl 1,19.5 c.p.m.
per JIM, carboxyl 2, 12.3 c.p.m. per JUM, and carboxyl 3, 12.3 c.p.m. per J»M. Fermentation conducted in 500 ml. Erlenmeyer flasks on a rotary shaker with
washed mycelium in a replacement medium: 20 gm. of acetic acid as sodium acetate, 2.5 gm. of KH 2 P04, 0.25 gm. of MgS0 4 -7H 2 0, 0.7 mg. of Fe, 0.25 mg. of
Zn, about 53 yXL of C14-carboxyl-labeled acetate, all dissolved in 1 liter of water
and adjusted to pH 6.8. At least three flasks were pooled for analysis at the
times given in the table. All data based on 1.0 ml. of medium.
formed in which trace quantities of carboxyl-labeled acetate were continuously fed to a glucose replacement medium undergoing submerged
fermentation. The results of this experiment are given in Table V. The
yields of citrate obtained were somewhat lower than those in previous
experiments with glucose as the substrate. This probably resulted from
introduction of trace impurities of metal with the acetate, probably from
the rubber tubing used to introduce the acetate.
It may be seen that the specific activity of the citrate increased during
the experiment. As the added acetate was fed at an almost constant rate
through 27 hours, and the glucose was used at an almost constant rate, the
specific activity of the citrate formed should have been almost constant.
If, however, a pool of acetate or citrate precursor existed initially in the
mycelium, the specific activity of citrate, once the pool was exhausted,
would increase above the initial activity as radioactive acetate was added.
151
11. A. BOMSTEIN AND M. J. JOHNSON
The following calculations support this hypothesis. If the specific activity of the acetate for any interval between the times given in Table V
is calculated by the methods previously described, the values given in
parentheses are obtained. Comparison of these values with those actually obtained by enzymatic degradation shows that the calculated values
are much higher. However, if a pool of citrate precursor, about twice the
size of that formed from glucose in the first 15 hours, existed initially
and was utilized in that period, the calculated specific activity of the
TABLE V
Submerged Fermentation of Glucose and Carboxyl-Labeled Acetate*
Citrate carboxyl
Time
Glucose used
Molar yield
Oxalate formed
Citrate formed
liu
c.p.m.
per tiH
1
krs.
txM
per cent
HM
c.p.m.
per ixu
0
15
0
46.0
52.5
5.9
20
23.8
106
23
63.0
51.3
6.8
30
32.0
170
27
73.3
47.6
8.1
36
34.0
183
31
80.0
47.5
7.8
21
38.0
686
c.p.m.
per pM
68
(206)
111
(288)
131
(254)
435f
(5900)
2
3
c.p.m. c.p.m.
per IAM per pM
20
18
33
26
39
13
148t
102t
* Acetate was fed in a trace quantity continuously into replacement medium at
the rate of about 3.65 X 102 c.p.m. per ml. of medium per hour. At the end of 27
hours about 3.67 X 104 c.p.m. per ml. of medium were added at one time; then continuous feeding was resumed.
t Chemical degradation of this sample gave for carboxyls 1 + 3 , 539 c.p.m., for
carboxyl 2,136 c.p.m., for chain carbons, 6.1 c.p.m. All data based on 1.0 ml. reaction mixture. The carboxyl designations are those of Diagram 1. The figures in
parentheses are calculated from the assumption that radioacetate is equilibrated
with acetate from glucose.
acetate would be only 68 c.p.m. per MM, the same as that of carboxyl 1.
In the next 8 hours 8.2 MM of citrate were formed from acetate having 288
c.p.m. per MM. If the sum of the number of moles of citrate formed in the
first 15 hours multiplied by the specific activity of the citrate, plus the
number of moles of citrate formed in the next 8 hours multiplied by the
calculated specific activity, is divided by the total number of moles of
citrate formed, the specific activity obtained, 124 c.p.m. per MM, agrees
fairly well with the observed value at 23 hours. The same calculations
for the citrate specific activities at 27 and 31 hours give values of 120 and
744 c.p.m. per MM. The observed value for 27 hours agrees with that
calculated, but the observed value for the 31 hour sample is only 60 per
152
CITRATE FORMATION BY A. NIGER
cent of that calculated. The discrepancy at 31 hours may result from the
formation of some citrate from non-radioactive substances. As will be
shown, some oxalate in this experiment was formed from non-radioactive
substances from 27 to 31 hours. The validity of the enzymatic degradation at 31 hours was checked by chemical degradation of the citrate formed.
As may be seen from Table V, the two methods gave results that are in
good agreement.
DISCUSSION
Origin of C4 Acids—As has been pointed out, the major pathway for
formation of the C4 precursor of citrate cannot be via the C2 to C2 condensation reaction, but more probably by fixation of carbon dioxide. It is
improbable that this occurs by a simple reversal of oxalacetate decarboxylation for two reasons. First, the condensation of oxalacetate with "active
acetate" is not sufficiently exergonic to operate at a reasonable reaction
rate at the low oxalacetate concentration which would be present if the
fixation reaction were a simple reversal of oxalacetate decarboxylation.
Second, the authors have observed in some fermentations oxalacetate
concentrations (about 10~4 M) about 100-fold those which could occur as a
result of simple pyruvate carboxylation. Whatever the actual mechanism of fixation may be, that it does occur has been shown by Lewis and
Weinhouse (1), Martin el al. (16), and Foster and Carson (3). However,
that it is the sole mechanism of C4 formation is not likely from the data
presented here, the Thunberg condensation probably occurring simultaneously, but to a lesser extent. Recycling of citrate as a source of the C<
precursor of citrate, when the conditions of fermentation are such as to
give 50 to 70 per cent yields of citrate, probably occurs, if at all, to a very
minor extent.
Origin of Oxalate—The data of Table III show that the oxalate formed
had too low an activity to have been formed from acetate or from any of
the citrate carboxyls. However, inspection of Table V shows that, between 27 and 31 hours, the amount of oxalate decreased, and at the same
time its specific activity decreased, showing that formation, as well as
utilization, took place. It will further be noted that, in the early stages
of the fermentation, the activity of the oxalate was such that it might
have been formed by splitting of oxalacetate or oxalosuccinate. In the
later stages of the fermentation, however, it could have been formed only
from an essentially non-radioactive compound. At no stage of the fermentation could it have been formed from acetate or a 2-carbon compound
in equilibrium with acetate. When carboxyl-labeled acetate was used as
the sole substrate, data in essential agreement with the above conclusions
were obtained, as may be seen from Table IV.
R. A. BOMSTEIN AND M. J. JOHNSON
153
SUMMARY
Citrate is probably formed by Aspergillus niger by condensation of
"active acetate" and a C«-dicarboxylic acid.
In fermentations yielding high citrate the Cj-dicarboxylic acid is probably formed by condensation of 2 acetate molecules to a small extent, but
mainly by condensation of carbon dioxide with a 3-carbon compound.
The mechanism of carbon dioxide fixation is probably not by a simple
reversal of oxalacetate decarboxylation.
Oxalate may be formed by splitting of oxalacetate or oxalosuccinate, but
not by direct oxidation of acetate.
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