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Production of Citric Acid in Continuous Culture Kristiansen - Sinclair 1979
Production of Citric Acid in Continuous Culture Kristiansen - Sinclair 1979
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
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
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.
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
kglrn3
60-
L.0.
2-0
50-
kg1m3 0
: O
30
2 0.
10
CONTINUOUS-CULTURE CITRIC ACID PRODUCTION 303
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.
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
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
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
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
TABLE 111
Storage Cell Growth
f X C F C
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 , .
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
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
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
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
CONCLUSIONS
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
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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