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
culture, from various carbohydrates, such as cane and beet
molasses, cane sugar juice, starch hydrolysates or pure
sucrose-containing media (1, 2). Molasses is known to
affect citric acid fermentation negatively because o f its high
ash and trace metal content (mainly iron and manganese).
Also, this substrate suffers from the shortcoming of yield
variations that depend largely on the quality o f the beet or
cane. As a consequence, each batch requires standardisa-
tion o f the substrate and a d d i t i o n o f high and variable
a m o u n t s o f ferrocyanide (1-3).
Submerged citric acid fermentation is only feasible if the
yield is a b o u t 70-90%o o f citric acid (based on the sugar in-
itially added) within a fermentation period o f 5-10 d (1, 4,
and U.S. Patent 3290227, 1966). However, in the course of
scaling-up, the standard strains available from various col-
lections lose their productivities, even if yields beyond
70°//oo could be obtained in shaken flasks. This is why suffi-
cient oxygen supply is necessary to achieve high yields o f
citric acid. In this connection, when using fungi like A.
niger, the growth o f the culture in the form o f small pellets
is desirable to avoid high viscosity o f the fermentation
broth, which in turn leads to lower oxygen transfer coeffi-
cients (5). It was reported that at the l a b o r a t o r y scale,
pellet f o r m a t i o n and high citric acid yield could be obtained
by exposing A. niger to a high stirrer speed (1000 rpm)
(6). However, these results are not feasible under a scaled-
up process because this leads to a very high specific power
input in industrial scale reactors (7). F u r t h e r m o r e , they are
in contradiction with the results o f Ujcova et al. (8), who
reported a negative influence on A. niger at higher stirrer
speeds in that range.
In the present paper, relatively low revolution speeds
have been used to obtain c o m p a r a b l e high citric acid yields
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 ,
three substrates have been examined using the same
organism under otherwise constant conditions. This
enables a real feasibility test o f the process, which ordina-
rily would not be possible by comparing the results o f
different authors.
A strain o f Aspergillus niger was used as described
earlier (9) and grown in the following media: (i) Defined
medium: Sucrose 100g, N H 4 N O 3 1.5 g; KH2PO4 0.25 g;
* Corresponding authors.
72
M g S O 4. 7 H 2 0 0.25 g; K4Fe(CN)6 6 mg, deionized water to
1 I. The initial p H was adjusted to 5.0. (ii) Sugar cane juice:
Fresh sugar cane was crushed. The juice so obtained was
heated to 85°C after adjusting the p H to 6.5. The cooled
juice was then filtered and diluted with deionized water to
give a sucrose concentration of 100 g/l. The following salts
were added per liter of the diluted juice: NH4NO3 1 g;
KH2PO4 0.6 g; M g S O 4 . 7 H 2 0 0.16 g; K4Fe(CN)6 180 mg.
The initial p H was adjusted to 5.0. (iii) Cane molasses:
The cane molasses was diluted with deionized water to give
a sucrose concentration of 100g//. This solution was
heated to 85°C after adjusting the p H to 6.5. The cooled
medium was filtered and then 1.8 g/l of K4Fe(CN)6 was
a d d e d to the final medium.
After sterilization o f the media, K4Fe(CN)6 was added at
a temperature o f 90°C. Spores were inoculated to each
medium at a concentration or 1 × 108 per liter.
Fermentations were carried out in a jacketed stirred tank
reactor with a working volume of 500 l. The reactor has a
height to diameter ratio of H / D = 2 with a diameter of
D - - 0 . 7 m. It is provided with four baffles ( w i d t h = 0 . 1 × D)
and four 6-bladed turbine agitators ( d / D = 0 . 4 , height o f
blades 56 ram) three o f which are used for stirring and one
as a foam breaker. The fermentor is made entirely of
stainless steel (AISI 316). Agitation and aeration were car-
ried out at 155 r p m and 150l/min, respectively. Further-
more, fermentations o f A. niger in the chemically defined
medium at three different stirrer speeds were carried out in
a geometrically similar stirred tank reactor o f 2.6 m 3 work-
ing volume. The selected stirrer speeds were 68, 91 and
155 rpm. The temperature was controlled at 30°C. The p H
was kept at p H 3 by the addition of NazCO3 solution.
Substrate and p r o d u c t concentrations were determined as
described earlier (9). The dry weight o f the mycelium was
measured by filtering 2 0 m l o f the fermented broth
through balanced filter-paper and drying at 95°C till con-
stant weight. The presented results are average values of
two fermentations.
Figure 1 shows the production of citric acid with the
different media. It can be seen that the acid production
usually starts at the second day o f the fermentation. A f t e r
6 to 7 d, when the citric acid production rate had slowed
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
is evident that for both the sucrose medium and cane juice,
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.
(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
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
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
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 -
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 .
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
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
acid p r o d u c t i o n .
The authors are grateful to the Council of Scientific and Industrial
Research, New Delhi, India and International Bureau of Kern-
forschungsanlage, JiJlich, FRG for supporting the collaboration.
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