Production of Citric Acid in Continuous Culture

B. KRISTIANSEN* and C. G. SINCLAIR, Department of

Chemical Engineering, University of Manchester Institute of
Science and Technology, Manchrster M60 1 QD, England

Summary
Work has been carried out on the production of citric acid by Aspergillus foeridus
in single-stage continuous culture, operated under nitrogen-limiting conditions at
dilution rates between 0.04 to 0.21 hr-'. Citric acid concentration increased rapidly
as the dilution rate decreased and appears to be critically dependent on the pH in
the culture vessel and the nitrogen concentration in the feed. A mathematical model
based on a distinction between basic cells, which require nitrogen but do not produce
citric acid, and storage cells, which accumulate carbon and simultaneously produce
citric acid, is proposed.

INTRODUCTION

Citric acid is one of the few bulk chemicals produced by fermen-

tation. Current practice is to carry out such fermentation in batch
or fed-batch submerged fermentations or on a quasicontinuous basis
by passing media over a stationary surface culture of the appropriate
microorganism. Continuous culture in submerged fermentation is
an obvious step for consideration in the development of this process
and this study was carried out in order to try to identify and solve
some of the associated problems.
In batch fermentation' it was found that the dry weight increased
throughout the fermentation. The supply of nitrogen was exhausted
early and the subsequent increase in dry weight was due to an
accumulation of carbon by the cells. Citric acid was produced during
this phase of carbon storage and nitrogen limitation, apparently by
the carbon-storing cells.
Generally, it has been thought that the growth and acid production
phases must be separated into two stages in a continuous process.
The only published attempt to produce citric acid in a single-stage
continuous-stirred-tank reactor (CSTR) was unsuccessful.*
Present address: Dept. of Chemistry, University of Manchester, Manchester,
MI3 9PL, England.

Biotechnology and Bioengineering, Vol. XXI, 4. 297-315 (1979)

@ 1979 John Wiley & Sons, Inc. 00063592/79/OO21-0297$01.OO
2% KRISTIANSEN A N D SINCLAIR

MATERIALS AND METHODS

Organism
The organism used in the experiments was Aspergillus foetidus
IMI 15954 obtained from the Commonwealth Mycological Institute,
Kew, Surrey. It was maintained on PDA slopes stored at 4°C and
renewed every other month.

Medium
The medium composition and operating conditions are: 50 kg/m3
glucose; 0.5 kg/m3 NH,NO, ; 0.5 kg/m3 KH,PO,; 0.1 kg/m3 MgS04-7
HzO; 0.1 X lop3 kg/m3 Fe3+ [as (NH4),SO,Fe,(SO,),.24 H,O]; 0.1
X kg/m3 Zn2+ (as ZnSO4.7H,O); 0.06 x kg/m3 Cu2+ (as
CuSO4.5H,O); distilled water made up to 1 liter; 32°C; 350 rpm; 8.5
liter reactor volume; and 0.7 v/v/m air flow rate. The medium was
made up with distilled water in 20 liter aspirators and sterilized at
15 psig (2 bar absolute) for 35 min. Contamination of the medium
was never observed.

Inoculum
Spores were harvested from Petri dishes containing 12 ml of the
relevant medium and transferred to 500 ml conical flasks containing
100 ml fermentation medium. The flasks were incubated for 24 hr
at 32°C on an orbital shaker and the pellets formed were then
transferred to the main fermentor. This was batch operated until
the expected continuous-culture cell population was reached. At
this point continuous operation commenced.
Some of the runs were started from the end of batch runs. It was
found that after about three residence times the past history had no
influence on the dry weight and citric acid production.

Equipment
The fermentor used in these experiments has been designed es-
pecially to avoid the use of a constant overflow tube to control the
culture level, which is not satisfactory for fungal cultures. Details
of the design are available from the manufacturers (SK Fermenters,
121 Princess Road, Manchester MI 7AG, England). Basically it
consisted of a 10 liter glass vessel with stainless-steel end plates
that carried the normal inlet and outlet ports for air, steam, electrical
heating, mechanical agitation, medium, titrants, as well as the level
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 299

control transducers. The agitator was a six-bladed turbine of con-

ventional design.
The following variables were controlled: medium level; temper-
ature; pH; air flow rate; agitator speed, and foam level. Dissolved
oxygen concentration was continuously recorded.
The pH control titrants were NaOH (1N) and HCI (IN); SAG 470
(Union Carbide) was used as the antifoam agent.

Measurements of Process Variables

Air flow rate was controlled using a flostat (G. A. Platow Ltd.)
and measured with a rotameter. Flow rates were accurate to CO.l
literimin.
Medium flow rate was set by means of a peristaltic pump (Wat-
son-Marlow Ltd.) and checked by timing the fall of the level in the
feed vessels. The outlet flow rate was also checked by collecting
the effluent in a graduated aspirator.
Culture volume was maintained constant by using a pressure
transducer at the base of the fermentor to control the outflow of
culture through a pinch valve in the bottom plate. The system
controlled the liquid level to within CO.1 kg of the set point.
Temperature was controlled to within kO.2"C of the set point
using an immersion heater and cooling water in a tube wrapped
around the outside of the glass vessel.
pH control was effected using a combined glass electrode (W. G.
Pye Co. Ltd.) and a conventional controller (Analytical Measure-
ments Ltd.) manipulating two small peristaltic pumps on the titrant
supplies.
Dissolved oxygen was measured with a galvanic electrode-type
probe similar to that described by Borowski and Johnson3 (L. H.
Engineering Ltd.). The output was recorded directly on a mV re-
corder. The probe, which had a linear response, was calibrated
against air and nitrogen saturated fermentor medium at the operating
temperature.

Analytical Techniques
Samples: Three 50 ml samples were taken from the base of the
fermentor, the first was discarded and the remaining two used as
duplicates for analysis. The samples were found to be true repre-
sentatives of the culture as a whole.
Dry weight: The sample was filtered on No. 1 filter paper that
had previously been dried for 24 hr at 85°C and cooled in a dessi-
300 KRISTIANSEN A N D SINCLAIR

cator before weighing to the nearest mg. The filter cake was washed
twice with 50 ml aliquots of distilled water, dried at 85°C for 10 hr,
and cooled in a dessicator before weighing. Incomplete washing
was indicated by charring of the mycelium or filter paper and these
samples were discarded.
Citric acid: The method of Marier and Boulet4 was used.
Sucrose was analyzed on a Technicon Autoanalyser.
Nitrogen was supplied as ammonium nitrate, which was com-
pletely metabolized. It was found that the concentration of total
nitrogen in the broth could be adequately described by the ammonia
concentration and a Technicon Autoanalyser was used for the anal-
ysis. Tests for total nitrogen in the fermentor liquor by the Kjeldahl
method were carried out intermittently.
Organic acids: Qualitative analysis for organic acids was carried
out with paper chromatography using a descending technique. Two
solvent systems were used: ethanol/20% ammonidwater (80:5 : 15)
and ~~-butanol/formic acidiwater (70: 30: 120). The chromatograms
were developed using the spray methods described by Ferraz and
R e l ~ a sStandards
.~ were developed for the following acids: citric;
isocitric; a-ketoglutaric; succinic; fumaric; malic; oxalacetic; py-
ruvic; oxalic; aspartic; glutamic; gluconic, and lactic.
Trace metals: The metals analyzed for were copper, zinc, iron,
and manganese. The analysis was carried out using an atomic ab-
sorption spectrophotometer (Corning EEL Scientific Instruments

Fig. 1. Dry weight and citric acid concentration vs. dilution rate. pH 2.60 (A)
dry weight; (A) citric acid; pH 3.00: (0)
dry weight; ( 0 ) citric acid; pH 3.40: (0)
dry
weight; (m) citric acid.
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 301

Ltd.) The standards for copper, zinc, and iron were prepared by
dissolving the metal in acid, and for manganese the sulfate was
dissolved in deionized water.

RESULTS AND DISCUSSION

Effect of Dilution Rate

Steady-state values were obtained throughout the dilution rate
range at pH 2.60, 3.00, and 3.40. The results are given in Figure 1.
The citric acid concentration increases rapidly as the dilution rate
decreases. It can be seen in Figure 2, however, that the productivity
has an optimum at a dilution rate of approximately 0.075 hr-l. The
dry weight, which is independent of pH in this range, does not
increase significantly until the dilution rate falls below 0.1 hr-l.

Effect of p H
pH had a marked effect on the citric acid production rate both by
its action on the concentration at any given dilution rate and by its
effect on the maximum dilution rate at which citric acid is produced.
The former effect is shown in Figure 3 where a series of readings
at a dilution rate of 0.06 hr-I and over a pH range of 1.60 to 3.90
are shown. There is a sharp maximum at pH around 3.40. The latter
effect is evident from the curves shown in Figure 1.
Although the CO, production rate was not measured directly,
indirect calculations can be made using a carbon balance based on
the information in Figure 3 (constant dry weight), Figure 4 (slightly
decreasing residual sugar as pH increases), and the fact that citric
acid was the only organic acid formed. These show that the CO,

0.5
kg/m3 h 1

Fig. 2 . Productivity (citric acid) vs. dilution rate.

 302 KRISTIANSEN A N D SINCLAIR

kglrn3
60-

L.0.

2-0

' 1:8 ' 22 ' 26 ' 30 . 34 ' 38 p'H

Fig. 3. Effect of pH at dilution rate 0.06 h r - ' . ( 3 )Dry weight: (0)
citric acid.

production rate is substantially independent of the pH. This suggests

that the pH does not affect citric acid production through any direct
influence on the Krebs cycle. I t is possible that pH affects the
enzymes that are active in degrading the substrate and/or the perme-
ability of the cell membrane to sucrose and citric acid.

.5fpc , t (? f I1is s o11s c d O.rygc I I

'

Sparging a batch culture with oxygen instead of air will lead to

a reduction in the fermentation time without affecting the final
yields. Thus the growth and production rates increase. In continu-
ous culture the fermentation time is fixed, increasing the dissolved

50-
kg1m3 0

: O
30

2 0.

10
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 303

oxygen concentration may therefore lead to a greater yield of citric

acid.
The effect of dissolved oxygen was investigated in the 0.07-0.30
atm range, using nitrogen and oxygen enrichment of air as required.
The gas flow rate was kept constant so that the aeration pattern was
not altered. The results, obtained at a dilution rate of 0.06 hr-' and
pH 3.00, are given in Figure 5.
The dry weight stayed relatively constant, indicating that the
growth rate was not affected by the dissolved oxygen in the oper-
ating range. The citric acid concentration increased significantly,
which indicates that the role of acid production increased with the
dissolved oxygen concentration.

Ejfect o j Nitr-ogoii
The effect of the nitrogen concentration on the steady-state be-
havior of the culture was investigated at a dilution rate of 0.06 hr-'
and pH of 2.60. The results are given in Figure 6. The basic cell
concentration was calculated using the yield coefficient on ammo-
nium nitrate evaluated below.
The dry weight increased with the nitrogen concentration as ex-
pected. There was, however, an optimal concentration for the pro-
duction of citric acid. Increasing the ammonium nitrate concentra-
tion above 0.8 kg/m3 resulted in a drastic reduction in the citric
acid.
We have postulated earlier' that citric acid is produced by cells
that are accumulating carbon under nitrogen limitation. In Figure
6 it is clear that the concentration of storage cells increases with

I 010
020 D o tbtm) 030
Fig. S. Effect of dissolved oxygen tension at dilution rate 0.06 hr-' and pH 3.00.
(a)Dry weight; (0)
citric acid.
304 KRISTIANSEN AND SINCLAIR

9.0-
kgh3

70-

50-

Fig. 6. Effect of NH,NO, concentration at dilution rate 0.06 hr-’ and pH 2.60.
Dry weight; (0)
(0) citric acid; (---) basic cells.

the nitrogen concentrations. If the formation of storage cells is the

only prerequisite for the production of citric acid, a similar increase
should be expected for the citric acid concentration. The reason
why this did not happen is not clear. A possible explanation may
be obtained from Zalokar’s6observations on fungal cell growth. He
noted that the cytoplasm in the hyphae was streaming forward into
the tip where the new cells were formed. The rest of the filament
contained storage material, except for the posterior part of the
hyphae, which was devoid of functioning cytoplasm.
If we assume that streaming occurs and Zalokar’s observations
are valid in our case, we may use the following hypothesis as a
possible explanation of the effect of the nitrogen concentration. The
nitrogen in the feed is accounted for by the new cells growing at
the tip of hyphae. The older cells will suffer nitrogen limitation and
consequently store carbon and produce citric acid. The transfor-
mation of basic into storage cells may be brought about by the
streaming of cytoplasm.
The effect of nitrogen concentration on the production of citric
acid may then be described as follows. The number of cells formed
will increase with the nitrogen concentration and the streaming will
increase with the number of cells formed. Thus, at low nitrogen
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 305

concentration few cells are formed and with little or no streaming

the citric acid production will be low.
When the nitrogen concentration increases, the rate of formation
of storage cells will increase and more citric acid will be produced.
The acid is produced by the mitochondria, which may, if the
streaming becomes too pronounced, be carried along toward the
nonproducing tip of hyphae, which is not suffering nitrogen limita-
tion, and the citric acid production will drop.
This offers a possible explanation for the drastic reduction in the
citric acid concentration at high nitrogen levels. It is clear that the
nitrogen concentration is of great importance and an optimum value
must be determined to maximize the citric acid production.

Trace Metals
The following solutions were analyzed: A, distilled water (used
to make up the feed); B, fermentor liquor prior to inoculation: C,
fermentor broth at steady-state operation. The results are given in
Table I . The broth appears to be free from impurities, and of the
supplied trace metals only zinc and copper were taken up by the
cells.
When the Krebs cycle is operating, citrate is formed from con-
densation of acetyl-CoA and oxalacetate, the latter being a product
of the cycle. In high-yielding citric acid fermentations, an anapler-
otic production sequence for oxalacetate must be established.
Woronick and Johnson7 discovered two CO, fixation reactions in
citric acid-producing strains of Aspergillus niger. One originated
with pyruvate and the other with phosphoenolpyruvate. both lead-
ing to oxalacetate. The latter reaction was inhibited by zinc and
copper. Assuming that the pathways of A . /rigor-and A . Jiwridrrs are
the same, this suggests that the main anzplerotic sequence for the
supply of oxalacetate is the reaction leading from pyruvate, involv-
ing pyruvate carboxylase.
The role of iron has been much discussed in relation to the

TABLE I
Trace-Metal Analysis

A 0 0 0 0
B 0.1 0.1 0.07 0
C 0.1 0.02 0.02 0
306 KRISTIANSEN A N D SINCLAIR

deactivation of the Krebs cycle in citric acid production. However,

both La Nauze* and Ahmed et al.9 found all the Krebs enzymes to
be active during the accumulation of citric acid. Seichert et al.l0
have suggested that the decisive factor in the production of citric
acid is the regulation of CO,. It has been reported that high con-
centrations of citric acid will inhibit isocitric dehydrogenase. If
oxalacetate is supplied by an anaplerotic sequence, rapid excretion
of citric acid should follow.

Production ($OthLjr Organic Acids

Analysis showed that citric was the only organic acid produced.
The mycelium may therefore be used as a raw material for animal
protein supplement without having to be washed for the removal of
undesirable compounds such a s oxalic acids.

THEORY

The production of citric acid in batch culture a s reported earlier'

suggested that normal growth of cells without citric acid accumu-
lation occurs while nitrogen is present in the medium. When the
nitrogen is exhausted, the dry weight continues to increase but at
a lower rate, and during this phase citric acid accumulates in the
medium. The increase in dry weight is due to carbon storage in the
older hyphal cells.
In formulating a mathematical model, it is reasonable to postulate
initially two types of active cells. These are a) basic cells produced
during nitrogen-unlimited growth and containing the expected
amount of cell nitrogen and b) storage cells produced during the
nitrogen-limited phase by streaming of the genetic material from
basic cells into the growing tips. Storage cells can be expected to
eventually lose all mitochondria1 enzymes becoming deactivated
and losing their citric acid-producing activity.
Continuous-culture conditions are somewhat different from batch
in that nitrogen levels in the medium within the fermentor are
always low, providing nitrogen is the limiting substrate. However,
we can still envisage growth of basic cells in the normal fashion
with transformation through citric acid-producing cells to the deac-
tivated form.
The equations describing the concentration of the three cell types
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 307

in a single-stage ideally mixed CSTR are

where D is the dilution rate and the subscripts b , c , and d refer to

basic, citric acid-producing (storage), and deactivated cells, respec-
tively. The nonlinear rate constants are assumed to take the follow-
ing forms:
pb = pmb"/(kN + N)l
pc = pmc[S/(ksxc + S)l
kt = ktm[ki/(ki + N)1
The first is the Monod expression for growth on a limiting substrate.
The second is the rate constant for the increase in mass of storage
cells and was chosen for the following reason. Burrows et al.,"
growing Gibberc2ffafujikori in a nitrogen-limited batch culture, ob-
served that the growth changed from exponential (nonlimiting con-
ditions) to linear and finally cube root. In our own batch studies we
noted that the specific growth rate of the storage cells decreased
with an increasing cell population. To model the effect of the cell
concentration on the growth rate the expression suggested by Con-
toisl2 was used. The third, the rate of transformation of basic to
storage cells is written in this form to accommodate the known
batch information and can be thought of as a nitrogen-inhibited rate.
Citric acid formation is assumed to follow a Ludeking and Piret13
type of expression to give
dP
- -- rp - DP
dt
where r, = apex, + px,. The remaining equations then fall out
naturally
dN - -pbxb + D(N, - N)
dt YN
308 KRISTIANSEN A N D SINCLAIR

and since we can only measure the total cell concentration:

x = x* + x, + X(,

The steady-state solution of these equations is found either by

setting the derivatives to zero and solving the resulting set of non-
linear algebraic equations or by integrating the differential equations
forward in time to steady state. We found the latter technique to be
the most satisfactory.

DETERMINATION OF KINETIC AND STOICHIOMETRIC

COEFFICIENTS
Brrsic Cc1l.s
mb

The maximum specific growth rate was obtained from a standard

washout procedure and batch data at a pH of 2.60 and 3.40.14
Nitrogen limitation was avoided so it can be assumed that only
basic cells were present in the culture. The results are given in
Table 11. The average value for P m b is 0.26 hr-l. It is independent
of pH in the operating range.

kN
According to Pirt,I5the saturation constant for growth in nitrogen-
limiting conditions is of the order of lo-* kg/m3, i.e., 3 X kg/m3
based on ammonium nitrate, and this value was used.
k tm
It is assumed that the maximum transformation rate constant is
equal to the highest dilution rate at which citric acid will appear.
This was obtained from Figure 1 by extrapolating the P vs. D curve
at pH 3.40 to P = 0 giving a value of kt, = 0.23 hr-l.
ki
This is obtained from the expression given for the transformation

T A B L E I1
Maximum Specific Growth Rate, Basic
Cells
Washout Batch
PH (hr-') (hr-')
2.60 0.263 0.271
3.40 0.248
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 309

rate coefficient. Estimates for k , were obtained from the experi-

ments used to give p m b since direct measurements of the rate of
cell transformation were beyond the scope of this investigation. It
was found that the concentration of ammonium nitrate when the
culture reached the end of the exponential growth phase was 5 X
kg/m3. Since the change from basic to storage growth was not
instantaneous, we estimated that the rate of cell transformation was
of the order of 10% of its maximum value at this point. This gives
a value of ki = 5 x kg/m3.
It must be emphasized that this is only an estimate of k i . The
rate of transformation must be measured experimentally to obtain
actual values of ki .

Storage Cr 11s
The kinetic coefficients for the increase in mass of storage cells
were obtained from a run that was started as a continuous run and
switched to batch when steady state was reached. Only the batch
part of the run was used. It can be assumed that the basic cells
were transforming at the maximum rate and that their concentration
was low enough to be ignored. By using only the first half of the
run the deactivated cell concentration will be low and the measured
dry weight will be equal to the storage cell concentration.
The specific growth rate was calculated from the slope of the x
vs. t curve and the data are given in Table 111. The expression for
p , can be rearranged to give

1 / ~ c= (ks/S~mc)xc + 1/pmc

A plot of Up, vs. x, is shown in Figure 7. The slope and intercept

of the straight line produced were 10.2 and 11. I , respectively, giving
values for p m cand k , / S of
p m c = 0.09hr-l, k , / S = 0.90m3/kg

TABLE 111
Storage Cell Growth

f X C F C

(hr) (kglm'l) (hr-')

0 6.1 0.013
10 7.0 0.012
20 8.0 0.01 1
30 8.9 0.010
310 KRISTIANSEN A N D SINCLAIR

1 3 5 7+((kg;m3) 9
Fig. 7. Graphical determination of p m cand k , .

The concentration of sugar was kept at a high level in the contin-

uous runs and did not vary significantly with the dilution rate. If
the sugar had any other effect besides supplying carbon for growth
and citric acid formation, this would be constant for all D. The
sugar concentration would, therefore, not be a factor in determining
the fit of the model, which is achieved by using k, = 0.90s as a
value for the Contois coefficient.

Deactivated Cells
In some of the batch runs reported earlier,' it was noted that the
run came to a halt for no apparent reason. It was assumed, there-
fore, that this was because all the cells had become deactivated.
The rate of production of the deactivated cells can be described
by
dxd l d t = kdx,
kd was found by trial-and-error procedure, searching for the value
that resulted in the calculated deactivated cell concentration being
equal to the actual at the end of the run. It was assumed that only
storage cells were present at the beginning of acid production stage.
The above equation was integrated, using step lengths of 20 hr.
Runs B1 and B8,' were used, being at the two extremes of the pH
operating range (1.80 and 3.00) giving values for kd of 0.02 and
0.009 hr-l, respectively. Assuming a linear relationship between kd
and pH, the expression for k d will be
kd = 0.02 - 0.009 (pH - I .80)
= 0.036 - 0.009 pH 1.8 < pH < 3.4
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 311

The expression is assumed valid for pH values up to 3.4, which

will allow its use in the continuous-culture model.

PRODUCTION OF CITRIC ACID

The steady-state form of the product formation model is

D P = a p , ~ ,+ px,

A plot of D P / x , vs. p , should give a straight line with intercept ,6

and slope a . The results are given in Figure 8. It should be noted
that the points on the graph represent experimental values of D and
P but predicted x, and p,.
The straight lines produced go through the origin, indicating that
the production of citric acid was growth associated with respect to
the storage cells and p = 0. Clearly, the value of a depends upon
pH. The following empirical expression can be evaluated
a = exp[l.56(pH - 2.10)] 1.60 < pH < 3.40
It can be seen in Figure 8 that a growth-associated product for-
mation model will predict values for the citric acid at low dilution
rates that are too high. The decline in productivity may have been
due to a decay in the enzyme system that was responsible for the
production and an enzyme decay term - y P was included in the
model. The value for y was found by curve fitting to be y = 0.01
hr-'.

Fig. 8. Graphical determination of a and p. pH: ( 3 )3.40; (0)

3.00; (A) 2.60
312 KRISTIANSEN AND SINCLAIR

The product formation model is therefore

r , = apex, - yP

YIELD FACTORS

yb

The yield factor on sucrose for basic cells was obtained as fol-
lows. When no citric acid was present x c , k t , and r p could all be
taken to be zero. The model for cell growth and sugar consumption
reduces to
dxb
- - - pbxb - Dxb
dt

Hence at steady state

yb = Xb/(SR - S>
The values obtained for the yield factor are given in Table IV. The
average value was Y b = 0.64 kg cells/kg sucrose.

The run from which the kinetic coefficients for the storage cell
growth were obtained were used to calculate the yield factor on
sucrose for these cells, giving a value of Y , = 0.40 kg cellsikg
sucrose.
y,
This was evaluated in the same way as Yb , giving a value for Y ,
of 4.9 kg celldkg NH,NO,.

TABLE IV
Yield Factor on Sucrose

yb
pH (kg cells/kg sucrose)
2.6 0.67
3.00 0.61
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 313

O.'OL 0'08 m 016 020 Dc'h')

PH 3.00; (0)
Fig. 9. Dry weight vs. dilution rate. (A) pH 2.60; (0) PH 3.40; ( - - - )
experimental; (-1 predicted.

Y P

The yield factor on sucrose for citric acid was calculated from
the theoretical formation of 2 mol citric acid from 1 mol sucrose.
The citric acid was measured as the monohydrate. Thus Y , = 1.23
kg citric acidikg sucrose.
The fit obtained with the model for dry weight and citric acid
curves is shown in Figures 9 and 10 and it can be seen that a
reasonable agreement was obtained. It is still necessary, however,
to extend the model beyond 3.40 and to carry out further experi-

Fig. 10. Citric acid concentration v s . dilution rate. (---) Experimental; (-4
predicted. pH: (0) 3.00; (A) 2.60.
3.40; (0)
3 14 KRISTIANSEN AND SINCLAIR

ments to obtain better estimates of the model parameters and to

check the various assumptions made.

CONCLUSIONS

The production of citric acid in a single-stage continuous culture

has been studied. It was found that under nitrogen-limiting condi-
tions A . foetidus produced citric acid rapidly. A process was estab-
lished and studied in the dilution rate range 0.04 to 0.21 hr-l. The
maximum productivity was found to be 0.43 kg/m3 hr (10.3 kg/m3
day) at a dilution rate of 0.075 hr-l. Small adjustments of the culture
conditions may increase the productivity substantially.
Both the dry weight and citric acid concentration increased with
decreasing dilution rate. The increase in the dry weight did not,
however, become significant until the dilution rate fell below 0.1
hr-l. The citric acid concentration increased more rapidly.
The production of citric acid may be explained by considering
streaming of the cytoplasms, a phenomenon well documented in
fungi. Assuming streaming occurs, the resulting differentiation along
the fungal filament seems to be necessary for the production of the
acid. New cells are formed at the hyphal tip and the cytoplasm
streams along the filament toward the tip. The streaming may lead
to rearrangement of the cell material and the transformation into a
storage or citric acid-producing cell. If the streaming is too pro-
nounced, it appears that the mitochondria will also move toward
the nonproducing tip of the hyphae. The production of citric acid
will then be reduced considerably. The cells will still store carbon,
however. Thus, storage of carbon is not synonymous with produc-
tion of citric acid, although this is produced by the carbon-storing
cells.
The production of citric acid is strongly dependent on the culture
conditions, especially pH. It appears that pH affects the enzymes
that are active in degrading the substrate and/or the permeability of
the cell membrane to substrate and product. The pH does not
appear to directly influence the mechanism of the citric acid for-
mation. Citric acid was the only organic acid produced.

Nomenclature
D dilution rate (hr-I)
kd deactivation rate coefficient (hr-I)
ki inhibition constant (kg/m3)
k, saturation constant for NH,NO, (kg/m3)
 CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 315

Contois coefficient (kg/kg)

transformation rate coefficient (hr-l)
maximum transformation rate constant (hr-l)
residual NH,NO, concentration (kg/m?
inlet NH,NO, concentration (kg/mq
product concentration (kg/m3)
rate of product formation (kg/m3 hr)
residual sucrose concentration (kg/m3)
inlet sucrose concentration (kg/m3)
total cell concentration (dry weight) (kg/m3)
basic cell concentration (kg/m3)
storage cell concentration (citric acid-producing cell concentration) (kgi
m7
deactivated cell concentration (kg/m3)
yield factor for basic cells on sucrose (kg/kg)
yield factor for storage cells on sucrose (kg/kg)
yield factor for basic cells on NH,N03 (kgikg)
potential yield factor for product on sucrose (kgikg)
specific growth rate, basic cells (hr-l)
maximum specific growth rate, basic cells (hr-l)
specific growth rate, storage cells (hr-*)
maximum specific growth rate, storage cells (hr-l)
product formation constant (kg/kg)
enzyme decay constant (hr-l)

References
1. B. Kristiansen and C. G . Sinclair, U.S. Patent No. 77-220.
2. Q. A. Choudhary and S. J. Pirt, J . Gen. Microbiol.. 43, 71 (1966).
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5 . P. G. P. Ferraz and M. E . A. Relvas, J . Chromatograph., 6 , 505 (1961).
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Suseman, Eds. (Academic, London, 1965), Vol. 1, Chap. 14.
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51 (1972).
10. L. Seichert, E. Ujcova, and 2. Fencl, Folia Microbiol.. 15, 212 (1970).
1 1 . A. Burrows, S. Brown, E. G . Jefferys, R. H . J. Kessell, E. C. Lloyd, P. B.
Lloyd, A. Rothwell, B. Rothwell, and J . C. Strait, C a n . J . Microbiol.. 10,407 (1964).
12. D. E. Contois, J . Gen. Microbiol., 21, 40 (1959).
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( 1959).
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1975).

Accepted for Publication June 3 , 1978