Citric acid production by surface culture using Aspergillus niger: Kinetics and simulation

JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 72, No. 1, 15-19. 1991

Citric Acid Production by Surface Culture Using Aspergillus niger: Kinetics and Simulation A K I H I K O S A K U R A I , 1 H I R O S H I I M A I , 1. T E T S U O E J I R I , 1 K A Z U O E N D O H , I AND S H O J I U S A M I 2

Department of Chemical Process Engineering, Faculty of Engineering, Hokkaido University, Sapporo 060, t and Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 160,2 Japan Received 6 October 1990/Accepted 22 April 1991 The citric acid production rate for batch surface culture using Aspergillus niger Yang no. 2 was examined experimentally in terms of rate coefficients and the effect of various depths of medium. The citric acid production rate was affected by the biofilm mass expressed as mg-Celi/cm2-fllm. The biofilm mass increased in parallel with medium depth. The citric acid production rate divided by the biomass weight at the end of cultivation was constant when the biofilm mass was less than 40 m g / c m 2, but decreased above this critical value. The citric acid fermentation time course was expressed by the Luedeking-Piret type rate expression. The time course of the surface cultivation was simulated well with the expression when biofilm mass was less than 40 m g / c m 2. In this range, rate coefficients were obtained by the nonlinear least squares method. Comparison in regard to the citric acid productivity between surface and other types of cultures in the literatures was made.

Citric acid is p r o d u c e d using Aspergillus niger in an aerobic fermentation. Much oxygen has to be supplied for the fermentation. The usual submerged cultural m e t h o d which needs aeration o f the medium, is often accompanied by foaming due to substrate c o m p o n e n t or fermentation products such as organic acid in the medium. The surface culture needs no aeration into the medium and no agitation for the medium. Thus it needs no powers for agitation and aeration. A n d it is an economical m e t h o d from a view point o f energy. A n o t h e r advantage o f surface culture is that the separation o f product from a m e d i u m is easier than mixed submerged culture, because a fungus is not dispersing into a medium. Several investigators have reported the citric acid production using A. niger orfoetidus (1-10, 13-17). Most o f these reports (1-7, 13, 14, 17) were concerned with submerged culture and solid culture, and discussed the effects o f the nutrient contents. There have been a few reports (8-10, 15, 16) for the effect o f the operational conditions on citric acid p r o d u c t i o n and for kinetic analysis of the citric acid p r o d u c t i o n (8, 9, 17), but they were not concerned with surface culture. The purpose of the present research is to obtain kinetic d a t a o f citric acid p r o d u c t i o n by surface culture applying the kinetic equation and elucidate the effect o f the surface density o f the biomass on the p r o d u c t i o n rate.

mg; FeC13.6H20, 21 mg; deionized water. Cultivation method Two kinds of culture bottles were used. To obtain the time courses o f biomass, sugar, citric acid, and nitrogen concentrations simultaneously, 70 ml o f medium was prepared in 16-34 pieces of 100 mibottle. The bottle was 5 . 0 c m in diameter and 10cm in height, and the bottleneck was 3 . 0 c m in diameter and 3.0 cm in height. In most experimental runs, the time course o f the biomass was not pursued and a single 500 ml-bottle was used, in which 150-400 ml portion o f the medium was prepared. The 500 ml-bottle was 8 . 4 c m in diameter and 13.5 cm in height, and the bottleneck was 4.5 cm in diameter and 4.0 cm in height. The bottleneck was plugged by a cotton plug during the cultivation. Before inoculation, the bottle containing the medium was autoclaved at 120°C for 15 min. The spore suspension was prepared by adding a 10 ml p o r t i o n of the liquid medium on the preculture o f the agar slant in a test tube and vibrating it for 30 s. The cultivation was started by inoculating the spore suspension into the culture bottle. The resulting initial spore concentration in the medium was about 1 × 10 ~° spores//. The initial p H o f the medium was adjusted to 4.15, but the p H was not controlled thereafter during the cultivation. The prepared bottles were set in a constant temperature chamber kept at 30+_I°C. The medium beneath the surface biofilm was gently agitated for 2 min every 3 d by a magnetic stirrer. Analytical methods In the case of the 500 ml-bottle culture, a 2.5 ml portion of culture b r o t h was sampled at intervals and filtered by m e m b r a n e filter (pore size; 0.45/~m) in order to remove the filamentous suspended fungus. In the 100 ml-bottle culture, one or two bottles were removed from the constant temperature chamber. A l0 ml p o r t i o n of liquid medium was filtered in the same manner as mentioned above. The whole remaining content in the bottle was centrifuged to separate the biomass. The filtrate was analyzed for total acid, sugar, citric acid, a m m o n i u m - , and nitrate-nitrogens. Total acidity was

MATERIALS AND METHODS

A. niger Yang no. 2 (5), a g o o d p r o d u c e r o f citric acid, was used t h r o u g h o u t this research. It has been stored on the agar slant medium at 10°C. Before the experiment, the spores were precultured on an agar slant m e d i u m in a test tube at 28°C for 715d. The liquid m e d i u m used for this experiment contained (per liter of solution): sucrose, 140g; KHzPO4, 10g; NH4NO3, 2.0 g; MgSO4.7H20, 250 mg; M n S O 4 . 5 H 2 0 , 14 Microorganism and m e d i u m

*Corresponding author. 15

16

S A K U R A I ET AL.

J. FERMENT.

determined by a titration m e t h o d using 0.1 N N a O H solution. Citric acid concentration was calculated from the total acidity minus that o f the blank by assuming the acid was composed o f only citric acid. The citric acid concentration was occasionally analyzed by liquid c h r o m a t o g r a p h y with a Y a n a p a k column (SAX-801) to confirm the validity o f the above assumption. A l m o s t 100% o f total acid was citric acid, while the a m o u n t o f oxalic acid was less than 3%o of total acid. Sucrose and reducing sugars in the b r o t h were converted to trimethylsilyl (TMS) ethers by trimethylsilylation reagent (TMSI-C) supplied by G a s u k u r o Kogyo, T o k y o . The procedure for the p r e p a r a t i o n o f T M S derivatives was as follows: 0.5 ml p o r t i o n o f the T M S I - C reagent was placed into a 1 ml test tube, then 5 pl o f the sample was added. The tube was kept at 60°C for 15 rain to complete the reaction. T M S derivatives were analyzed by a flame induced gas c h r o m a t o g r a p h y (Shimadzu G C - 6 A P F ) with OV-17 3% column. Inositol was used as an internal standard to analyze quantitatively. The a m m o n i u m - , and nitrate-nitrogens in the broth were analyzed by a gas c h r o m a t o g r a p h y nitrogen analyzer (Shimadzu Sumigraph GCT-13N). The biomass concentration was measured as dry cell weight as follows. In the 500 ml-bottle experimental run, the whole biomass in the bottle was sampled at the end o f the cultivation, whereas in the 100 ml-bottle run, samples were taken at intervals. The whole culture broth inside the bottle was centrifuged for 5 m i n at 10,000rpm and washed with 4 0 m l p o r t i o n o f deionized water 3 times. The precipitate was dried at 105°C for 24 h to weigh the biomass. The oxygen uptake rate (OUR) was measured by an automatic BOD meter (11) in the 500 ml-bottle run.

RESULTSANDDISCUSSION Cultivation time course The upper surface o f the medium was covered with a biofilm 3 d after the inoculation. Sucrose was hydrolyzed to glucose and fructose completely after 9 d. In this research, the sum of the a m o u n t o f detected glucose, fructose, and sucrose is termed the sugar concentration. Time courses o f the concentrations o f citric acid, sugar, biomass, and total dissolved nitrogen ( N H 4 - N + N O 3 - N ) for a 100ml-bottle run are shown in Fig. 1.

150

I

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I

I

T

[

1.0

I

_

&o.6

I

0

~ O

O O

~ %

~0.4 d (..p ~0.2

_

0

-

~ 1 0

0

0

°° 0.01

_

o

I

I

0.02 0.03 0.04 Consumed-N [g]

0.05

FIG. 2. Correlation between consumed nitrogen and biomass for 100 ml-bottle culture. Solid line shows Eq. 1.

In the 500 ml-bottle run, time course o f the biomass concentration (X) was not measured directly but estimated from the consumed nitrogen time course d a t a by use of the correlation Eq. 1,

DCW=18.67×N

( r = 0.9527, n = 3 3 )

(1)

where D C W is dry cell weight [g] and N is total nitrogen consumed [g] measured by the 100ml-bottle runs. The data for DCW, N and Eq. 1 were shown in Fig. 2. A n example o f time courses o f citric acid, sugar, and estimated biomass for a 500 ml-bottle run were shown in Fig. 3. The course o f citric acid concentration was divided into 3 phases. The first phase was defined as the period from inoculation to the time when the citric acid p r o d u c t i o n rate became a p p r o x i m a t e l y constant, for example, before less than 10 d o f cultivation time in the case o f Fig. 3. The second phase was defined as the period when the citric acid p r o d u c t i o n rate was constant: the period from 10d through 27 d o f cultivation time in the case of Fig. 3. The third phase was defined as the period when the citric acid p r o d u c t i o n rate decreased or the citric acid concentration in the medium decreased: this period was after more than 27 d o f cultivation time in the case o f Fig. 3. In all the runs in this research, sugar was consumed at the rate of 1 6 - 3 6 ~ in the first phase, 41-74% in the second phase, and 4-26%o in the third phase.

150

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5"

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50

0

6 v

u 15 20 25 30 Time [d] FIG. 3. Courses of (o) citric acid (C), (~)sugar (S), and ( [] ) biomass (X) for 500 ml-bottle culture. The solid lines are the simulation results calculated from Eqs. 2-4 using the parametric values of run no. 9 shown in Table 2. 0

0

I

~0.8

1

LolOC

I

BIOENG.,

0

5

10

15 20 Time [d]

25

30

0 35

FIG. 1. Courses of ( O ) citric acid (C), (±) sugar (S), ( [] ) biomass (X), and ( 0 ) nitrogen for 100 ml-bottle culture.

glo 5

~ 10

CITRIC ACID PRODUCTION BY A. NIGER

VoL 72, 1991

15O

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TABLE 1. Experimental data used for simulation

2~\c) 1 0 0 ~ 5 0

0 0

5 10 15 20 25 Time [d]

30

:_:55

FIG. 4. Comparison of the culture bottle shape in the parallel experiments: (A) 100 ml-bottle culture, (©) 500 ml-bottle culture. The difference in the time courses between the 100 mlbottle run (Fig. 1) and the 500 ml-bottle run (Fig. 3) was not clear. But, in the other parallel experiment using the 100 ml-bottles and a 500 ml-bottle, there was no difference in the citric acid time course as shown in Fig. 4. Hereafter, the discussion is only concerned with the 500 ml-bottle runs. Kinetics and simulation of surface culture The solid lines in Fig. 3 are the simulation results using the following Luedeking-Piret type (12) rate expressions. In modeling the surface culture, a complete mixing in the bottle was assumed, although biomass was separated as a thin layer film on the liquid. The mathematical expression is as follows:

d X / d T = kt. S. X / ( K x + S)

(2)

dC/dT=k2.(dX/dT/)+k3.S.X/(Ks+S)

(3)

dS/d T=

(4)

Ysx" ( d X / d 13

Ysc- ( d C / d T)

where C [g/0, S [ g / 0 , X [g//] and T [d] denote the concentrations o f citric acid, sugar, and dry weight of biomass in a culture bottle, and cultivation time, respectively; k, [ l / d ] , k2 [ - ] and k3 [ l / d ] denote the m a x i m u m specific growth rate, citric acid p r o d u c t i o n coefficient based on growth, and citric acid p r o d u c t i o n p r o p o r t i o n a l coefficient based on biomass concentration, respectively; K s [g//] and Kx [g//] denote the half velocity coefficients of citric acid p r o d u c t i o n and growth, respectively; Ysx [ - ] and Ysc [ - ] denote the reciprocal o f the yield coefficients o f the biomass and the citric acid based on the sugar consumed for TABLE 2.

Run no.

a (-)

fl ( )

Ysx (-)

P (l/d)

M (g/cm2)

1 2 3 4 5 6 7 8 9

0.796 0.790 0.706 0.794 0.925 0.865 0.856 0.572 0.583

0.051 0.037 0.049 0.071 0.030 0.036 0.033 0.088 0.077

4.933 6.987 6.874 3.626 4.502 5.219 6.001 5.271 5.896

0.2604 0.2290 0.2383 0.1902 0.2999 0.3028 0.3005 0.2253 0.3232

0.0153 0.0161 0.0223 0.0304 0.0362 0.0376 0.0396 0.0398 0.0361

Averagea

0.770

0.051

5.529

0.2652

0.0312

a The average values were calculated excluding the minimum and maximum values in each column. Ysx, Obtained by the yield coefficients a and ft. Y~p, Stoichiometrical coefficient, 0.9376 ( ). The data of the run no. 9 was used for Fig. 3.

the sake o f each production, respectively. Ysc was determined to be 0.9376 g - s u g a r / g - C A (CA is used hereafter for citric acid) stoichiometrically by assuming that 1 tool citric acid was produced from 1 mol glucose. Ysx was calculated from Eq. 5 which was obtained by integrating Eq. 4, AS =

YscAC - Ysx&¥

(5)

where A means the increase in an arbitrary time interval. In obtaining Ysx, the time interval in Eq. 5 was f r o m the start o f the second phase o f cultivation to T = Tm,~ at which point the citric acid concentration was at its m a x i m u m . The values o f Ysx for the data o f Fig. 3 as run no. 9 and also the values for the other runs are shown in Table 1. The biofilm mass of these runs used for the simulation was in the range o f M=<40 m g / c m 2, where M [ m g / c m 2] was the biofilm mass at the end o f the cultivation divided by the biofilm area. The average value o f Ysx which was calculated excluding the m a x i m u m and the m i n i m u m values for the 500ml-bottle run was 5.529 g-sugar/g-cell. The other parameters kl-k3, Ks, and Kx were determined by the nonlinear least squares method with regard to three variables C, S, and X simultaneously for the second phase o f the cultivation. Numerical solution to Eqs. 2-4 was obtained by the Runge-Kutta-Gill method. For the run shown in Fig. 3, the data at 10 d o f cultivation was chosen as the initial condition o f Eqs. 2-4. The values of the initial condition for simulation were summarized in Table 2. Also the deter-

Kinetic parameters obtained by simulation and initial condition for simulation

Kinetic parameters obtained by simulation k2 k3 Ks ( ) (I/d) (g//)

Run no.

kl (l/d)

l 2 3 4 5 6 7 8 9

0.045 0.037 0.070 0.033 0.056 0.100 0.087 0.070 0.125

21.0 21.0 10.0 18.0 22.0 19.0 17.0 10.0 9.5

0.0140 0.0100 0.0600 0.0340 0.0075 0.0300 0.0006 0.0010 0.0400

Averagea

0.066

16.6

0.0195

C

Initial condition for simulation S X (g//) (g//)

Kx (g//)

(g/l)

3.90 1.00 1.50 0.46 2.40 5.00 3.60 4.40 2.40

90.0 89.0 88.0 99.0 60.0 70.0 44.0 58.0 81.0

14.48 11.95 14.56 16.21 35.89 31. l 1 19.84 19.04 13.89

111.3 116.0 112.0 109.7 95.9 92.0 102.6 95.0 109.8

4.81 1.69 3.57 3.44 3.73 4.20 4.27 6.49 3.49

11 11 9 13 11 11 11 9 10

2.74

76.6

18.45

105.2

3.93

11

The average values were calculated excluding the minimum and maximum values in each column. T, The cultivation time when the second phase was started. The date of the run no. 9 was used for Fig. 3. a

17

T (d)

18

SAKURAI ET AL.

J. FERMENT•BIOENG.,

mined values of the parameter were shown in Table 2. Kristiansen and Sinclair (7) reported the specific growth rate of 0.696-1.584d ~ of A. foetidus in a submerged batch culture. The average value of k~ (which is equal to the m a x i m u m specific growth rate) shown in Table 2 is much smaller than that in the literature (7). With regard to citric acid production, the growth associated term ( d C / d T ) o may be expressed as (17) (d C/d 7")G: k 2 ( d g / d T)

= (dC/dS)(dS/dX)(dX/d 7)

(6)

k2 = (dC/dS)(dS/dX) = a/i~

hence

of

medium

depth

on

citric

acid

production

Figure 5 shows the correlation between biofilm mass (M [mg-cell/cm2]) and the initial medium depth. Here, the biofilm mass M was defined by the dry cell weight at the end of cultivation divided by biofilm area, which was assumed to be equal to the cross-sectional area of the cultivation bottle. The biofilm mass increased in parallel with the medium depth. Assuming that the biofilm mass represented the biofilm thickness, the biofilm thickness increased in parallel with the medium depth. There was a period when the time course of citric acid concentration was approximately linear with time in each run. This period coincided with the second phase as shown in Fig. 3. The average slope of the time course was decided

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~:0.2

-

~0.1

-

(D I

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0

0

(900 0 ~ 0 o o 0 o-

I

0

I

I

1

20 40 60 80 B i o f i [ m mGss [ m g / c m 2]

1O0

FIG. 6. Correlation between citric acid productivity (P-R/Xe) and biofilm mass M for 500 ml-bottle culture. The solid line is the simulation result using the average parametric values shown in Table 2. CA, Citric acid. by the least squares method. This slope is defined as the citric acid production rate R [g/(l. d)]. A simulation curve of citric acid production was not linear, as shown in Fig. 3, but the average of the slope over the simulated period was chosen as the average production rate obtained by the simulation. The citric acid productivity (P) that is defined by R/Xe, where Xe [g//] is the biomass concentration at the end of cultivation, was correlated with the biofilm mass (M) at the end of cultivation as shown in Fig. 6. The productivity was constant in the range of M < 4 0 m g / c m 2 and decreased with increasing of biofilm mass in the range above this value. The solid line in Fig. 6 shows the average slope of the simulation curve which was calculated from the Eqs. 2-4 by use of the average parametric values shown in Table 1 and Table 2. In solving the differential equations, the initial values of the variables were set to the average concentrations of sugar, biomass, and citric acid at the time when the second phase started in the range of M=<40 mg/cm2; 105.2 g/l, 3.93 g/l, and 18.45 g/l. These values were shown in Table 2. Average slope was calculated in the range of T = 0 - 3 6 d of the simulation. The simulated production rate agreed well with the average experimental data in the range of M=<_40 m g / c m 2 as shown in Fig. 6. The decrease in the productivity in the range of M > 4 0 m g / c m 2 in Fig. 6 was considered due to the oxygen limitation. The oxygen in the air diffuses into the biofilm

I

I

.L

o0,6-

E 8O (_1

I

O E

I

0.4-

-~.0.8

1O0

1

0 (D

(7)

where a and ,~ are yield coefficients of citric acid and biomass based on total sugar consumed in the second phase, respectively, a and ~f were defined by the a m o u n t of citric acid produced divided by the a m o u n t of sugar consumed, and the a m o u n t of biomass divided by the sugar consumed from the time when the citric acid production rate became constant to the time when the citric acid concentration was maximum, respectively. Using the value of a and ~ in Table 1, the average of ~/~f is t7.1 which is comparable with the average of k 2 in Table 2. Roehr et al. (17) reported that k 2 = 1 . 9 - 2 . 0 g-CA/gglucose and k3=0.912-2.16 g-CA/(g-cell.d) using a submerged culture. These values disagreed with the present ones. The strain of A. niger and the cultivation method were different from those of the present research, so a direct comparison could not be made. Half velocity coefficient Ks [g-sugar//] with regard to citric acid production was small enough to be neglected compared with the substrate concentration. The other half velocity coefficient Kx [g-sugar//] was comparable to the substrate concentration and could not be neglected. Effect

0.5

60 --

0

o

O

O

\0.404 o

if)

6~ ~

Jo

o j ~

I

~ 40 E

E

o o

~ 2o% I

m 0

o

x

I

2 4 Medium d e p t h

w D

I

6 [cm]

8

FIG. 5. Correlation between biofilm mass at the end of cultivation and the initial medium depth for 500 ml-bottle culture.

o

J

0.2 ~

o

GD

o

_

o

1

0 R/X

1

0.5 1.0 [mg-CA/(mg-CeL[.d)]

1.5

FIG. 7. Correlation between specific citric acid production rate

(R/X) and specific oxygen uptake rate (OUR~X) in the second phase for 500 ml-bottle culture. CA, Citric acid.

CITRIC ACID PRODUCTION BY A. NIGER

VOL. 72, 1991 TABLE 3.

Comparison of citric acid productivity among various types of bioreactor

Method Submerged culture (batch) (continuous) (fed-batch) (continuous) Airlift fermentor (batch with Ca alginate) Tower fermentor (continuous with polyacrylamide) Dual hollow-fiber bioreactor (continuous) Shake-flask (repeated batch) Surface culture

Productivity (g/(l. d))

Reference

6.5 9.6 13.0 9.6 1.8

8 8 8 13 14

3.8

15

22.8

16

11.0

10

1.2-3.2

this work

f r o m the u p p e r surface, and the substrate in the m e d i u m diffuses into the biofilm f r o m the lower surface. T h e fungus c o n s u m e s the o x y g e n and the substrate, and p r o d u c e s the citric acid. A s the o r g a n i c substrate existed in excess inside the biofilm, the decrease in the p r o d u c t i v i t y in the range o f M > 40 m g / c m 2 was a t t r i b u t e d to the o x y g e n deficiency inside the biofilm. T h e discrepancy b e t w e e n the solid line and the d a t a in the range o f M > 40 m g / c m 2 in Fig. 6 was due to the increase o f the biofilm thickness leading to the o x y g e n deficiency, whereas the present m a t h e m a t i c a l m o d e l c o n s i d e r e d no o x y g e n l i m i t a t i o n . T h e range in which the rate expression using the average p a r a m e t r i c values c o u l d be applied to the present surface culture was M < 4 0 m g / c m 2. O x y g e n u p t a k e rate (OUR) O x y g e n u p t a k e is an imp o r t a n t f a c t o r for design o f a f e r m e n t a t i o n process. F i g u r e 7 shows the r e l a t i o n s h i p b e t w e e n the specific o x y g e n upt a k e rate (OUR~X) [ m g - O J ( m g - c e l l . d ) ] and the specific citric acid p r o d u c t i o n rate ( R / X ) [ m g - C A / ( m g - c e l l - d ) ] m e a s u r e d in the second p h a s e o f culture. O U R / X was correlated with R / X as: (OUR~X) = 0.2005 + 0.1805 × ( R / X ) [ r = 0 . 4 8 2 4 , n = 14, r(12, 0 . 0 8 5 ) = 0 . 4 7 6 6 ] . H e r e , r, n, and r(12, 0.085) represented the c o r r e l a t i o n coefficient, d a t a points, and p r o b a b i l i t y f u n c t i o n o f r test, respectively. T h e first t e r m o f the right expression is t h o u g h t to represent the specific o x y g e n u p t a k e rate for m a i n t e n a n c e . D a w s o n et al. (8) r e p o r t e d there was n o c o r r e l a t i o n b e t w e e n the citric acid p r o d u c t i o n rate and the o x y g e n u p t a k e rate. But the d a t a in the r a n g e o f 3-12 d in Fig. 2 o f the original p a p e r (8) was c o r r e l a t e d by us as: ( O U R / X ) = O . 1 4 4 5 + 0 . 9 6 0 8

×(R/X). C o m p a r i s o n o f p r o d u c t i v i t y a m o n g v a r i o u s types o f bioreactor T h e citric acid p r o d u c t i v i t y o f the present w o r k and o t h e r investigators is c o m p a r e d in T a b l e 3. T h e values for b a t c h c u l t i v a t i o n were calculated f r o m the maxim u m citric acid c o n c e n t r a t i o n s and the c u l t i v a t i o n period. Because o f the difference in the culture c o n d i t i o n , a direct c o m p a r i s o n is difficult, but s o m e r e m a r k s can be m a d e .

19

T h e present surface culture has low p r o d u c t i v i t y c o m p a r e d with a s u b m e r g e d c o n t i n u o u s culture, but it has p r o d u c t i v ity c o m p a r a b l e to the airlift f e r m e n t o r and the t o w e r fermentor. T h e overall yields o f citric acid and b i o m a s s based on total c o n s u m e d sugar in this research were 30.5-90.4 (average 52.0) w / w % and 3.03-12.3 (average 6.72) w / w % , respectively. REFERENCES 1. Nowakowska-Waszczuk, A., Rubaj, E., Matsusiak, B., and Kosiek, E.: The effect of acetate on the production of citric acid by Aspergillus niger in submerged fermentation. Appl. Microbiol. Biotechnol., 20, 416-418 (1984). 2. Purohit, H. J. and Daginawala, H. F.: The relationship of some metal ions with citric acid production by Aspergillus niger using tamarind seed powder as raw material. J. Ferment. Technol., 64, 561-565 (1986). 3. Hossain, M., Brooks, J. D., and Maddox, I. S.: The effect of the sugar source on citric acid production by Aspergillus niger. Appl. Microbiol. Biotechnol., 19, 393-397 (1984). 4. Hossain, M., Brooks, J. D., and Maddox, I. S.: Production of citric acid from whey permeate by fermentation using Aspergillus niger. New Zealand J. Dairy Sci. Technol., 18, 161-168 (1983). 5. Usami, S. and Fukutomi, N.: Citric acid production by solid fermentation method using sugar cane bagasse and concentrated liquor of pineapple waste. Hakkokogaku, 55, 44-50 (1977). 6. Kumagai, K., Usami, S., and Hattori, S.: Citric acid production from mandarin orange waste by solid culture of Aspergillus niger. Hakkokogaku, 59, 461-464 (1981). 7. Kristiansen, B. and Sinclair, C. G.: Production of citric acid in batch culture. Biotechnol. Bioeng., 20, 1711-1722 (1978). 8. Dawson, M. W., Maddox, I. S., Boag, I. F., and Brooks, J. D.: Application of fed-batch culture to citric acid production by AspergUlus niger: the effect of dilution rate and dissolved oxygen tension. Biotechnol. Bioeng., 32, 220-226 (1988). 9. Kristiansen, B. and Chaley, R.: Continuous process for production of citric acid. Adv. Biotechnol., 1,221-227 (1981). 10. Tsay, S. S. and To, K.Y.: Citric acid production using immobilized conidia of Aspergillus niger TMB2022. Biotechnol. Bioeng., 29, 297-304 (1987). 11. Sonoda, Y., Kumagai, H., and Nakata, T.: Automatic determination of microbial respiration. J. Ferment. Technol., 50, 313-320 (1972). 12. Luedeking, R. and Piret, E. L.: A kinetic study of the lactic acid fermentation. Batch Process at Controlled pH. J. Biochem. Microbiol. Technol. Eng., 1, 393-412 (1959). 13. Kristiansen, B. and Sinclair, C.G.: Production of citric acid in continuous culture. Biotechnol. Bioeng., 21, 297-315 (1979). 14. Eikmeier, H. and Rehm, H.J.: Production of citric acid by Aspergillus niger immobilized in polyacrylamide gels. Appl. Microbiol. Biotechnol., 20, 365-370 (1984). 15. Horitsu, H., Adachi, S., Takahashi, Y., Kawai, K., and Kawano, Y.: Production of citric acid with immobilized Aspergillus niger. Appl. Microbiol. Biotechnol., 22, 8-12 (1985). 16. Chung, B. H. and Chang, H. N.: Aerobic fungal cell immobilization in a dual hollow-fiber bioreactor: continuous production of a citric acid. Biotechnol. Bioeng., 32, 205-212 (1988). 17. Roehr, M., Zehentgruber, O., and Kubicek, C.P.: Kinetics of biomass formation and citric acid production by Aspergillus niger on pilot plant scale. Biotechnol. Bioeng., 23, 2433-2445 (1981).