BIOTECHNOLOGY AND BIOENGINEERING VOL.

XVIII (1976)

Mass and Energy Balance Analysis of Metabolic Pathways

Applied t o Citric Acid Production by Aspergillus niger

INTRODUCTION
For various reasons it is useful to know the metabolic pathways by which
organisms convert substrates to a given product. Often, metabolic schemes are
complicated and there are many routes to this product. Mass balance and energy
balance techniques can be employed to eliminate metabolic schemes from con-
sideration and to suggest which of the possible schemes are most probable. Also
these balances can indicate restrictions upon the directions of certain reactions
which can kinetically go in two directions.
The actual application of these principles will depend upon the given situation.
Herein they are applied to citric acid production by Aspergillus niger. Because
conversion of glucose to citric acid is high, the mass balances are pivotal in the
analysis with energy balances only providing qualitative information. However,
for different products, energy balances might be critical.
Aspergillus niger fermentation is the world's leading source of commercial
citric acid and metabolic pathway information is important to understand
enzyme control mechanisms and to build a mathematical model of the citric
acid production process. To obtain and comprehend information sufficient to
understand, biochemically, the process by which A . niger produces citric acid is
a significant undertaking. However, the important pathways of citric acid
production can be illuminated by applying the principles of conservation of
mass and energy along with other knowledge about the organism and its products.
The primary goal of this investigation is to illustrate the application of mass
balances, stoichiometric information, and energy balances to discover all possible
metabolic pathways and then to ascertain the most probable metabolic pathway
by which A. niger synthesizes critic acid. Further, some minor speculation
concerning the dynamics by which A niger is induced to produce citric acid is
included.

SOURCES OF 1NFORMATION
The following items of information were used in analyzing metabolic pathways
by which A . niger could produce citric acid.
It is presumed that citric acid is produced only by known metabolic pathways
and, in particular, by the citric acid cycle and known variations thereof. This
information is obtained from the metabolic charts.'
It is generally known that 70 to 90% of the glucose can be converted to citric
acid and that oxalic acid is often produced as an undesirable by-product. Oxalic
acid is removed during purification. Also, during the fermentation little carbon
dioxide is produced.2
Organisms such as AspergiUus have the capability of forming considerable
quantities of lipids for fat storage, to the extent that lipids can constitute a
fairly high percentage of the dry weight of the myce1ium.J
425
@ 1976 by John Wiley & Sons, Inc.
426 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976)

It is known from work a t Miles and by others,4e6 that the enzymes pyruvate
carboxylase, isocitrate lyase, and oxaloacetic hydrolase are more active in A .
niger during oxalic acid and citric acid production. Also, all enzymes of the cit-
ric acid cycle are active.6
Some pathway information was obtained from radiocative tracer studies
performed on A . niger citric acid production by Bomstein and Johnson7 in 1952,
Cleland and Johnsons in 1954, and Shu et al.9 in 1954.

APPLICATION O F MASS AND ENERGY BALANCES AND OTHER

INFORMATION
The major fact which allows a mass balance analysis of the metabolic pathways
is the high conversion of glucose to citric acid. Only 10% of the glucose is
available for the production of mycelia and carbon dioxide, both of which occur
primarily during the growth phase early in the fermentation.* Thus, practically
all the glucose which enters the cell during the citric acid production phase (after
the growth phase) must be converted to citric acid. Hence, in constructing
metabolic pathways for citric acid synthesis, one need only consider those which
result exclusively in citric acid.
Since oxalic acid is often generated by A . niger during citric acid production,
the metabolic pathways proposed for citric acid production must contain the
possibility for oxalic acid production. A pathway for oxalic acid could be
independent of citric acid, but the metabolic charts indicate that oxalic acid is
inextricably tied to the production of citric acid through acteate. The metabolic
schemes to be considered then should satisfy the conservation of mass and contain
the possibility of oxalic acid production.

MASS BALANCES
The metabolic pathways considered for possible citric acid production are
shown in Figure 1. Also indicated are the notations for the various molar bio-
chemical reaction rates. The positive direction is indicated for those reactions
which can go in both directions. This scheme contains all the known active
enzymes and all the major products which result from this fermentation. Since
almost all the sugar utilized after the completion of mycelial growth is converted
to citric acid, oxalic acid, or carbon dioxide, these are the only products which
need to be considered and their molar production rates are labeled as y, z, and
w, respectively, in Figure 1. The input t o the metabolic scheme is pyruvate,
whose molar consumption rate is z in the figure. These variables are presumed
known and are related by the following equation on carbon.
32 = 6y + 22 + w
Mass balances of carbon on the chemical species yield ten equations. Since
there are 11 unknowns, this system cannot be solved directly. This should be
the case, since some of the reactions cannot be reversed, and hence there are
inequality restrictions on some of the fluxes. The steady-state mass balances are
listed in Table I along with the inequality restrictions.
From Table I, several things are evident. Mass balances preclude the citric
acid cycle from operating in reverse, i.e., if any of the known products are produced
then all the steps of the citric acid cycle operate in their normal fashion, i.e., e , k ,
 COMMUNICATIONS TO T H E EDITOR 427

NETABOLIC SCHENES

Ix
PYRUVATE

OMLOACETATE f

OXALATE * CITRATE ’-
GLYOXYLATE

2 cog

Fig. 1. Metabolic pathways for citric acid production.

and j must always be greater than zero whereas e and f must always be less than
zero. Also, it is noticed that an indeterminateness exists in the equations. The
cycle from oxaloacetate to acetate, oxalate, glyoxylate, malate, and back to
oxaloacetate can operate at any molar rate. However, its rate of operation
would probably be zero or small, since there would be nothing to gain by rapid
operation.
Table I1 contains the mass balances for the production of citric acid, o d i c
acid, and carbon dioxide. For citric acid there are two metabolic schemes for
its production. The classical scheme results when a = 1, and an alternative is a
consequence when a = 2. For a having a value between 1and 2, both metabolic
pathways are active. It should be noted that for pure citric acid production by
either scheme there is no net conversion between citric acid and isocitric acid.
The cycling through malate does not affect citric acid mass balances. From
mass balances it is concluded that there are two and only two metabolic pathways
for citric acid production.
The classical citric acid scheme, a = 1, involves the formation of acetyle CoA
(acetate) and oxaloacetate from two pyruvates and their combination into citric
acid. Although this scheme is plausible, it does not involve many of the enzymes
known to be active during citric acid production and oxalic acid production is not
428 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976)

TABLE I
r = 2y +
i z + f w

Mass balance equations

b=Zy+$z++w-a
c=a-y+g
d =y+fz++w
e = -iz-+w-g
f = - $ z - + w
9'8
h=a--y-z+g
i = y + z - a
j = a + + w - + z - y
k = i z + t w
Restrictions
a >O
b>O
c > o
g > O
j > O

a simple variant of this pathway. The alternative pathway, with a = 2, (Fig. 2),
utilizes most of the enzymes known t o be active during citric acid production.
The production of oxalic acid and carbon dioxide are simple variations of this
metabolic scheme. Finally, the oxalic acid produced is known to block the citric
acid cycle a t succinate, which is important for citric acid production according to
the second scheme.
For oxalic acid production, there are two oxalic acid production schemes for
g = 0. With a = 1, two-thirds of the oxalic acid comes from the glyoxylate and

TABLE I1
Mass Balance for the Production of Citric Acid, Oxalic Acid, and Carbon Dioxide
~~

Citric acid Oxalic acid Carbon dioxide

z = O w=O y = l ~ = w3= O y = O y=O z = O w = 6

b = 2 - a b = 2 - a b = 2 - a
c = a - 1 +g c = a + g c = a + g
d = l d = 2 d = 2
e = -g e = -2 - 9 e =-2-g
f = o f = - 2 f = -2
h = a - l + g h=a-2+g h=a+g
i = l - a i = 3 - a i = -a
j = a - 1 j = a - 1 j = 2 + a
k=O k = 2 k = 2
l < a < 2 l < a < 2 O<a<2
g = 0 probably 0 < g g = 3 - a probably 0 < g
 COMMUNICATIONS TO T H E EDITOR 429

GLUCOSE
1
PYRUVATE

OMLOACETATE

OMLlC
b
GLYOXILATE CITRATE

NET REACTION
GLUCOSE- CITRATE

Fig. 2. Citric acid synthesis.

one-third comes from the oxaloacetate, and with a = 2, one-third of the oxalic acid
is generated from glyosylate and two-thirds is produced from oxaloacetate. How-
ever, since concentration of NADH is expected to be high, it is thought that the
transformation from glyoxylate to oxalic acid ( h in Fig. 1 ) might not be favored.
Hence, probably g = 3 - a, and thus, h = 0. This is shown in Figure 3.
Carbon dioxide is essentially produced by two pathways. In the first scheme
(a = 0) one glucose molecule generates two carbon dioxide molecules from
pyruvate and four from isocitrate; whereas the alternate pathway (a = 2) yields
eight carbon dioxide molecules via isocitrate and consumes two in the production
of oxaloacetate from pyruvate. Again, the production of malate from glyoxylate
and acetate does not affect the mass balance result concerning carbon dioxide.
Since the mass balances are linear, the sum of any combination of mass balances
will again yield a valid mass balance. For example, choosing y = 1, z = 3, and
w = 6, one could sum the three mass balances in Table 11. If a = 2, then the
citric acid, oxalic acid, and carbon dioxide would be produced according to the
schemes and should be the same. The results of Cleland and Johnsons agree
with these predictions. This is based upon the assumption that there exists a
pool of unlabeled acetate as was suggested by the previous radiotracer studies
(Bomstein and Johnson?) and that during citric acid production it is the only
product formed.
The oxalic acid radiotracer measurements provide some differentiation between
the two schemes. If, for the data of Cleland and Johnson,* the oxalic acid were
formed directly from pyruvate via oxaloacetate, the expected specific activity
would be 19,900 cpm per mM of carbon; however the measured specific activity
was 12,600. If oxalic acid were formed according to the second scheme, then
the expected specific activity of the oxalic acid would be less. Thus, approximate
430 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976)

PYRUVATE

OXALOACETATE

A GLYOXYUTE CITRATE

NET REACTION
GLUCOSE - 3 OXALIC

Fig. 3. Oxalic acid synthesis.

mass balance calculations indicate that the expected specific activity corresponds
reasonably with the measured value for the second metabolic scheme.
Mass balances were performed on both labeled acetate and 'carbon dioxide
but the results are difficult to interpret, since both enter and exit from a pool of
unknown and changing size during the fermentation.
The tracer studies are in agreement with the second schemes for the production
of citric acid and oxalic aicd. They also indicate that these two acids must be
produced at separate times and, probably, that the oxalic acid is produced first
(ashas been found a t Miles). It should be noted that the experiments of Cleland,
Bomstein, and Johnson7~8were performed with sugar concentrations considerably
less than those presently employed for commercial production of citric acid.'

ENERGY BALANCES
Energy balance techniques aan be applied to citric acid production ; however,
the information thus obtained would be minimal since the overall chemical
reaction for citric acid or oxalic acid from glucose (shown below) is very exother-
mic. In other words, almost any metabolic pathway chosen between glucose
and citric acid will satisfy the conservation of energy and the assooiated increase
in free energy.

CeHlaOs + 32- Op -+ +
CoHaO~ 2 H a 0 -AH = 200 kcal/mol - A F
= 175 kcal/mol
9
CsHlnOs + - O2 -+ 3CaH204-t 3H20-AH = 490 kcal/mol - A F
2
- 450 kcal/mol
 COMMUNICATIONS TO T H E EDITOR 431

Possibly, further information concerning the relative merit,s of the second meta-
bolic pathway over the classical one could be obtained from scrutinizing the
overall equations which include the cofactors for citric acid production. Accord-
ing to the classifical scheme (a = l), the following stoichiometry holds.
H2O + CsH12Oe + ADP + Pi + 3NAI)+ -+ C&07 + 3NADH + 3H+ + ATP
For citric acid production via the second scheme (a = 2), the following balanced
chemical equation is applicable.
C,3Hr2Oe + 3NAD+ + HzO -+ C&07 + 3NAIIH + 3H’
From these reactions, it is evident that high energy compounds are generated as
would be expected from the excess energy associated with citric acid production.
I n fact, the process which is least efficient at coverting chemical energy into high
energy compounds may be favored, i.e., the second scheme. With the expected
excess of ATP and deficiency of ADP in the cells, the process which consumes
the least ADP might be favored; again, this would be the second scheme. In
either case, oxygen will be consumed during the conversion of NADH to NAD
in the cytochrome electron transport system with little generation of ATP accord-
ing to the following relationship.

NADH + H + + 51 0 2 -+ NAII+ + Hz0

BIOCHEMICAL DYNAMIC SPECULATION
The following sequence of events represents a guess as to how A . lziger bio-
chemically becomes induced to produce citric acid. Initially, consider cells
growing normally during the exponential growth phase. As they enter the
declining phase of growth, the media carbon to nitrogen ratio becomes high and
the cells produce lipids. As the lipid concentration gets high, the glyoxylate
cycle enzymes are induced in an attempt to reconvert the lipids to sugars. How-
ever, there is plenty of sugar and the net result is an increase in oxaloacetate
which induces the oxaloacetate hydrolase to produce oxalic acid and acetate.
Oxalic acid is an inhibitor in the citric acid cycle from succinate to fumarate
generating an excess of succinate. With the accumulation of both succinate and
oxalic acid which is converted to glyoxylate, the isocitrate lyase enzyme reaction
is reversed from that normally associated with the glyoxylate cycle. Before
the citric acid cycle is stopped and this reaction is completely reversed a certain
amount of oxalic acid production is possible. If the fermentation conditions
from this point forward are correctly maintained, A . niger can continue to pro-
duce citric acid according to the metabolic scheme shown in Figure 2.

CONCLUSIONS
The analysis herein indicates that mass and energy balances can be applied to
complicated metabolic pathway networks to quantitatively specify all possible
metabolic schemes for a given product and help select among these possibilities.
In particular it is concluded that only two metabolic pathways exist for citric
acid production and that the pathway with a = 2 is the most probable scheme
by which A . niger produces citric acid. The production of oxalic acid, carbon
432 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976)

dioxide, and growth intermediates are simple variations of this pathway. This
pathway information leads to speculation concerning the dynamics of induced
cit,ric acid production in A . nzger.
References
1. M. Ishimoto, S. Minakami, S. Mizushima, T. Oshima, and H. Wada,
Metabolic Maps, 3rd ed., Kyoritsu, Tokyo, 1971.
2. R. Noyes, Citric Acid Production Processes, Noyes I)evelopment, Corp.,
Park Ridge, New Jersey, 1969.
3. C. F. Schmidt, Jr., J . Riol. Chem., 110, 511 (1935).
4. V. Wonchai and W. E. Jefferson, Jr., Fed. Proe., 33, 1378 (1974).
5. A. P. Joshi and C. V. Ramakrishnan, Enzymologia 21, 43 (1959).
6. S. Usami, N. Saegusa, and T. Tateishi, Hakko Hyokaishi, 29. 399 (1971).
7. R. A. Bomstein and M. J. Johnson, J . Riol. Chem., 198, 143 (1952).
8. W. W. Cleland and M. J. Johnson, J . Biol. Chem., 208,679 (1954).
9. P. Shu, A. Funk, and A. C. Neish, Can. J . Riochem. Phys., 32, 68 (1954).
F. H. VERHOFF*
J. E. SPRADLIN

Miles Laboratories, Inc.

Elkhart, Indiana 46514

Accepted for Publication October 14, 1975

*Present address: Dept. of Chemical Engineering, West Virginia University,

Morgantown, West Virginia 26506.