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JOURNALOF FERMENTATIONAND BIOENGINEERING

Vol. 69, No. 1, 72-74. 1990

Pilot-Scale Citric Acid Production with Aspergillus niger


under Several Conditions
G H U L A M N. Q A Z I , I* C H A N D N. G A I N D , l S U R Y A K. C H A T U R V E D I , l C H A R A N J I T L. C H O P R A , I
M I C H A E L TR)kGER, 2 AND U L F E R T O N K E N 2.
Regional Research Laboratory Jammu, Canal Road, Jammu-Tawi, 180 001 India ~ and Universitiit
Dortmund Lehrstuhl Technische Chemic B, Postfach 50 05 00, 4600 Dortmund 50, F.R.G. 2
Received 3 February 1989/Accepted 25 October 1989

Citric acid fermentations with Aspergillus niger in stirred tank reactors (5001 and 2.6 m 3) w e r e carried out
using cane juice, cane molasses and a synthetic medium. Highest yields were obtained with the cane juice and
synthetic medium. The rate of stirring had a pronounced influence on the morphology of A. niger.

Citric acid is p r o d u c e d , either by surface or submerged M g S O 4. 7 H 2 0 0.25 g; K4Fe(CN)6 6 mg, deionized water to
culture, from various carbohydrates, such as cane and beet 1 I. The initial p H was adjusted to 5.0. (ii) Sugar cane juice:
molasses, cane sugar juice, starch hydrolysates or pure Fresh sugar cane was crushed. The juice so obtained was
sucrose-containing media (1, 2). Molasses is known to heated to 85°C after adjusting the p H to 6.5. The cooled
affect citric acid fermentation negatively because o f its high juice was then filtered and diluted with deionized water to
ash and trace metal content (mainly iron and manganese). give a sucrose concentration of 100 g/l. The following salts
Also, this substrate suffers from the shortcoming of yield were added per liter of the diluted juice: NH4NO3 1 g;
variations that depend largely on the quality o f the beet or KH2PO4 0.6 g; M g S O 4 . 7 H 2 0 0.16 g; K4Fe(CN)6 180 mg.
cane. As a consequence, each batch requires standardisa- The initial p H was adjusted to 5.0. (iii) Cane molasses:
tion o f the substrate and a d d i t i o n o f high and variable The cane molasses was diluted with deionized water to give
a m o u n t s o f ferrocyanide (1-3). a sucrose concentration of 100g//. This solution was
Submerged citric acid fermentation is only feasible if the heated to 85°C after adjusting the p H to 6.5. The cooled
yield is a b o u t 70-90%o o f citric acid (based on the sugar in- medium was filtered and then 1.8 g/l of K4Fe(CN)6 was
itially added) within a fermentation period o f 5-10 d (1, 4, a d d e d to the final medium.
and U.S. Patent 3290227, 1966). However, in the course of After sterilization o f the media, K4Fe(CN)6 was added at
scaling-up, the standard strains available from various col- a temperature o f 90°C. Spores were inoculated to each
lections lose their productivities, even if yields beyond medium at a concentration or 1 × 108 per liter.
70°//oo could be obtained in shaken flasks. This is why suffi- Fermentations were carried out in a jacketed stirred tank
cient oxygen supply is necessary to achieve high yields o f reactor with a working volume of 500 l. The reactor has a
citric acid. In this connection, when using fungi like A. height to diameter ratio of H / D = 2 with a diameter of
niger, the growth o f the culture in the form o f small pellets D - - 0 . 7 m. It is provided with four baffles ( w i d t h = 0 . 1 × D)
is desirable to avoid high viscosity o f the fermentation and four 6-bladed turbine agitators ( d / D = 0 . 4 , height o f
broth, which in turn leads to lower oxygen transfer coeffi- blades 56 ram) three o f which are used for stirring and one
cients (5). It was reported that at the l a b o r a t o r y scale, as a foam breaker. The fermentor is made entirely of
pellet f o r m a t i o n and high citric acid yield could be obtained stainless steel (AISI 316). Agitation and aeration were car-
by exposing A. niger to a high stirrer speed (1000 rpm) ried out at 155 r p m and 150l/min, respectively. Further-
(6). However, these results are not feasible under a scaled- more, fermentations o f A. niger in the chemically defined
up process because this leads to a very high specific power medium at three different stirrer speeds were carried out in
input in industrial scale reactors (7). F u r t h e r m o r e , they are a geometrically similar stirred tank reactor o f 2.6 m 3 work-
in contradiction with the results o f Ujcova et al. (8), who ing volume. The selected stirrer speeds were 68, 91 and
reported a negative influence on A. niger at higher stirrer 155 rpm. The temperature was controlled at 30°C. The p H
speeds in that range. was kept at p H 3 by the addition of NazCO3 solution.
In the present paper, relatively low revolution speeds Substrate and p r o d u c t concentrations were determined as
have been used to obtain c o m p a r a b l e high citric acid yields described earlier (9). The dry weight o f the mycelium was
and a p p r o p r i a t e mass transfer conditions. F u r t h e r m o r e , measured by filtering 2 0 m l o f the fermented broth
three substrates have been examined using the same through balanced filter-paper and drying at 95°C till con-
organism under otherwise constant conditions. This stant weight. The presented results are average values of
enables a real feasibility test o f the process, which ordina- two fermentations.
rily would not be possible by comparing the results o f Figure 1 shows the production of citric acid with the
different authors. different media. It can be seen that the acid production
A strain o f Aspergillus niger was used as described usually starts at the second day o f the fermentation. A f t e r
earlier (9) and grown in the following media: (i) Defined 6 to 7 d, when the citric acid production rate had slowed
medium: Sucrose 100g, N H 4 N O 3 1.5 g; KH2PO4 0.25 g; down, all fermentations were stopped. During all fermenta-
tions, no other b y p r o d u c t acid was detected. F r o m Fig. 1 it
* Corresponding authors. is evident that for both the sucrose medium and cane juice,
72
V o L 69, 1990 NOTES 73

T A B L E 1. C o m p a r i s o n of final yields and selectivities of


different fermentation media"
Yield Selectivity
(%) (-)
1 .o'-°-- 1 Synthetic m e d i u m 72 0.71
Cane juice 72 0.72
Cane molasses 58 0.50
a Yield and selectivity are defined according to Lockwood and
'-' O/L~ I Schweiger (10).
l. A
Y=~o • 100 (%)

.,OOo---_. CA. Ms
S= (Cso-Cs)'MA ~
1
( )
CA=concentration of citric acid m o n o h y d r a t e g/l
Cs = sucrose concentration g/l
Cso = starting sucrose concentration g/l
Ms, MA = molecular weight of sucrose and citric acid monohydrate,
~-~ 40 o respectively
r3 ~ uL_
growth, is unfavourable for submerged production of citric
[] cone molosses LO
acid despite its low costs. Based on our results, cane juice
100 O cone juice 0 can be considered as an alternative favourable raw material,
A synthetic medium which gives good yields with tolerable higher growth.
~- 80 Moreover, no problem arises due to fluctuating cane juice
E~ VR= 500 I composition. The results with this substrate were re-
producible, whereas they varied from 300/00 to 50%0 in the
case of using molasses as substrate.
During the course of further scale-up of citric acid
0 40 ~]
fermentation, various criteria were taken into considera-
tion. Since the stirrer speed has direct influence on other
scale-up parameters, such as power input or mass transfer,
o fermentations at different stirrer speeds were carried out at
0 2.6 m 3 working volume. Selected stirrer speeds of 68, 91
0 2 4 6 and 155 rpm are based on calculations for scale-up using
different scale-up criteria. Figure 2 clearly demonstrates
Cultivation time [d] that the highest citric acid productivity could be obtained
FIG. 1. Citric acid production, sucrose c o n s u m p t i o n and at 91 rpm. The fungus formed small pellets (diameter ca.
mycelial dry weight in stirred reactor with different media. Working 2 mm), whereas at 155 rpm a filamentous type of growth
volume: 500/. was observed. This is in contradiction with Gomez etal.

the acid yield and process selectivity are the same. In the 100
case of molasses, the acid yield is apparently lower. Final
acid yields and selectivities for all substrates are given in
Table 1.
For sucrose and cane juice, the final yield amounted to
70%0, with a high selectivity of almost 0.75. However, for I J ~ , ~ ~ t ~ 40 o
molasses, the fermentation yielded 20%0 less citric acid synthetic medium
A~ I
with 30% lower selectivity. Since no acids other than citric vR~2 6 ~----; xA~ A 20 m
acid were produced, more carbon source must have been 100 ° revolution [rain-1 ] 0
used for growth and maintenance in the case of the & 68
molasses medium. This is further supported by the growth A gl ~A
of A. niger, which is also plotted in Fig. 1; cell growth was A 155 &
"o 6O
lowest in the synthetic medium. However, the higher cell u
growth in the cane juice medium was rather unexpected o 40
because of its similarity to synthetic medium with respect u
I
to yield and selectivity. One explanation for this behaviour ~- 20 J--a~._...._A~ • •
is that the microorganism in the cane juice medium re- 0
0
quires less carbon source for its energy generation as com- "~ 3 5 7
pared to the requirement within the defined medium. As a
Cultivation time [d]
consequence, the specific acid production rate is highest in
the defined medium. FIG 2. Citric acid production and sucrose consumption at
These aspects lead to the conclusion that, taken by and different stirrer speeds (68, 91 and 155 rpm) in synthetic medium.
large, molasses, with its low citric acid yield and high Working volume: 2.6 mL
74 QAZI ET AL. J. FERMENT. BIOENG.,

(6), w h o a c h i e v e d b e t t e r pellet f o r m a t i o n b y i n c r e a s i n g t h e tweinwirtschaft, 113, 373 (1972).


s t i r r e r s p e e d f r o m 450 t o 1000 r p m . A t s t i r r e r s p e e d s less 4. Nowakowska-Waszczuk, A. and Sokolowski, A.: Application of
t h a n 91 r p m , insufficient o x y g e n t r a n s f e r t o t h e b r o t h was carbon balance to submerged citric acid production by
c o n s i d e r e d t o l o w e r citric acid p r o d u c t i o n . T h e s e c o n d i - Aspergillus niger. Appl. Microbiol. Biotechnol., 26, 363-364
(1987).
t i o n s also led to m y c e l i a l g r o w t h a n d a m o r e v i s c o u s b r o t h .
5. Kubicek, C. P. and R6hr, M.: Citric acid fermentation, p. 331-
F r o m t h e d a t a p r e s e n t e d it c a n be c o n c l u d e d t h a t s t i r r e r 373. In CRC critical reviews in biotechnology, vol. 3. CRC Press,
s p e e d i n a v e r y n a r r o w r a n g e is i m p o r t a n t f o r h i g h e r citric Boca Raton (1986).
acid p r o d u c t i o n . 6. Gomez, R., Schnable, I., and Garrido, J.: Pellet growth and citric
acid yield of Aspergillus niger 110. Enzyme Microb. Technol.,
The authors are grateful to the Council of Scientific and Industrial 10, 188-191 (1988).
Research, New Delhi, India and International Bureau of Kern- 7. Aiba, S., Humphrey, A.E., and Millis, N.F.: Biochemical
forschungsanlage, JiJlich, FRG for supporting the collaboration. Engineering, p. 195-216. Academic Press, New York (1973).
8. Ujcova, E., Fend, Z., Musilkova, M., and Seichert, L.:
REFERENCES Dependence of release of nucleotides from fungi on fermentor
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473. In Rehm, H. J. and Reed, G. (ed.), Biotechnology, vol. 3. and Atal, C. K.: Production of citric acid by submerged fermenta-
Verlag Chemic, Weinheim (1983). tion; effect of medium sterilization at pilot-plant level. J. Chem.
2. Clark, D. S., Ito, K., and Horitsu, H.: Effect of manganese and Tech. Biotechnol., 31, 122-126 (1981).
other heavy metals on submerged citric acid fermentation of 10. Lockwood, L. B. and Schweiger, L. B.: Citric and itaconic acid
molasses. Biotechnol Bioeng., 8, 465-471 (1966). fermentations, p. 183-199. In Peppler, H . J . (ed.), Microbial
3. Kovats, J. and Niestrawsaki, Z.: Zuckerrohrmelassen. Die Brann- technology, Reinhold Company, New York (1967).

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