©2000 Elsevier Science B.V. All rights reserved.

Biotechnology Annual Review.

Volume 5.
M.R. E1-Gewely,editor.

Fuzzy control of bioprocess in Japan

H i r o y u k i H o n d a a n d Takeshi K o b a y a s h i
Department of Biotechnology, Graduate School of Engineering, Nagoya University,Nagoya, Japan

Abstract. Process control of bioprocess has been carried out by the judgment of the experts, who are
the skilled operators and have lots of experiences for the control of the process. In almost all cases,
those experiences are described linguistic IF-THEN rules. Fussy inference is one of the powerful
tools to incorporate the linguistic rules to the computer for process control. Fuzzy control are
divided into two types; one is the direct fuzzy control of process variables such as sugar feed rate in
fed-batch culture and fermentation temperature in batch operation. The other is the indirect control
of bioprocess, in which at first the phase recognition is carried out by fuzzy inference and the con-
trol strategies constructed in each phase are used for the control of process variables. In Japan, the
fuzzy control has already been applied to practical industrial productions, such as pravastatin pre-
cursor, vitamin B2, and Japanese sake mashing process. In this review, these industrial approaches
of fuzzy control are introduced.

Keywords: bioprocess, fuzzy inference, IF-THEN rule, Japan, process control.

Introduction

From the view point o f large-scale production in bioprocess, the optimization

and automatic control o f bioprocesses such as fermentation process have been
strongly desired. Various optimization and control methods based on determinis-
tic mathematical models were proposed and attempted to meet the requirements.
However, this approach has never succeeded in practical industrial production,
because it is difficult to develop a mathematical model which can precisely
describe the complex intracellular reactions o f microorganisms in bioprocess.
T h e operational conditions in industrial bioprocesses are often optimized by uti-
lizing the knowledge acquired during repeated operations u n d e r the control o f
the experts, who are highly skilled operators. Such empirical knowledge-based
operations can be considered to be useful from a practical view point. Although
the knowledge c a n n o t be quantified, it is necessary for automatic control o f bio-
process to bring them u n d e r c o m p u t e r control and incorporate into traditional
control system.
Fuzzy set theory was developed [1]. Fuzzy control based on fuzzy set theory is a
mathematical tool for dealing with qualitative information and linguistic expres-
sions, and has been applied in the biotechnology field for several years. Fuzzy

Addressfor correspondence: Hiroyuki Honda and Dr Takeshi Kobayashi, Nagoya University,Depart-

ment of Biotechnology,Graduate School of Engineering, Furo-Cho, Chikusa-Ku, Nagoya 464-8603,
Japan. Tel.: +81-52-789-3215 Fax: +81-52-789-3214. E-mail: honda@proc.nubio.nagoya-u.ac.jp
control can easily incorporate empirical knowledge gained from skilled operators
by employing membership functions and IF-THEN rules, and it has proved to
be effective for the simulation [2--4], the expert system [5] and the on-line control
for bioprocesses [6-10]. Here, scientific papers published by Japanese research-
ers are mainly reviewed as shown in the title of this review. Studies on fuzzy con-
trol can be categorized into two types. The first type utilizes direct inferencing,
in which the fuzzy inference determines directly the outputs from the knowledge
base and on-line data. This method allows bioprocess control to be easily auto-
mated using the knowledge of expert operators, and it simply transfers the opera-
tors know-how into the control system. Nakamura et al. [6] reported the fuzzy
control of sugar feeding rate in glutamic acid fermentation. Hosobuchi et al. [11]
applied fuzzy control to the production of the pravastatin precursor. Horiuchi et
al. [12] reported the fuzzy control of vitamin B2 production. Those are examples
in large-scale fermentation. Alfafara et al. [13] presented a method of applying
fuzzy logic on a compensator in a scheme of feedforward/feedback controller to
realize utilization of nonlinearity for robustness of control, flexibility in use of
multiple measurable variables, and easy and effective use of expert knowledge.
The second type is indirect inferencing in which fuzzy inferencing is at first
used to estimate the culture phase or physiological state, after which empirical
control strategies are performed in each phase or state [7,14-16]. Control values
are determined on the basis of the inferred culture state. Shimizu et al. [17] devel-
oped a method for culture state diagnosis and parameter estimation utilizing
the fuzzy inference procedure in combination with molar-flux calculations based
on a metabolic reaction model. These second type studies suggest the possibility
of more flexible bioprocess control based on qualitative fermentation characteris-
tics. Horiuchi et al. [18,19] have also reported several laboratory-scale applica-
tions of phased control using fuzzy inference to fed-batch cultures for m-amylase
production and recombinant 13-galactosidase production.
In this review, characteristics of these fuzzy control systems and the usefulness
of fuzzy control for automatic control of bioprocess are described after the out-
line of fuzzy reasoning or control was mentioned. In Japan, large-scale industrial
applications of fuzzy control to bioprocesses have been reported on the [11,12]
and they are also reviewed.

Fuzzy inference

Fuzzy set theory was developed by Zadeh [1]. In this theory, the numerical data
of state variables are transferred to the fuzzy number using membership func-
tions. State variables are categorized by fuzzy sets such as "Small (S)", "Medium
(M)", and "Big (B)". Fuzzy number is a grade of categories, and corresponds to
the possibility belonging to each sets. From the combination of grade of state
variables (output), many production rules concerning control variables are con-
structed. Table 1 shows an example of production rules for fuzzy control. This
control system was constructed for fuzzy control of feed rate in submerged cul-
Table 1. Production rules of fuzzy control.

DO concentration: B DO concentration: M

Glucose Glucose
Ethanol Ethanol
S M B S M B

S PB PM ZE S PM PS NS
M PM ZE NS M PS ZE NM
B ZE NS NB B NS NM NB

S: small, M: medium, B: big, P: positive, N: negative, ZE: zero.

ture of yeast [20]. Therefore, production rules are used for determination of
alteration of feed rate, DE Fuzzy inference is carried as follows:

IF DO concentration is "B" and glucose concentration is "S" and ethanol con-

centration is "S", THEN DF is "PB".

Membership functions for inputs and outputs are established as in Fig. 1. "S",
"M", and "B" concentrations were set as 0.1, 0.2, and 0.3 g/1 for glucose and 1,
2, and 3 g/1 for ethanol, respectively. Concerning DO concentration, only two
memberships as "M" and "B" were set in this research since it was not much var-
ied throughout fermentation. The inference procedure is shown in Fig. 2, which
follows Mamdani's min/max algorithm [21]. The grade of fuzzy set outputs is
determined as the minimum value among the grades of fuzzy set inputs by each

S S s

0 2 5 00 011 012 013 0 1 2 3

a-1 DO (ppm) a-2 Glucose(g/l) a-3 Ethanol (g/l)

NB NM NS ZE PS PM PB

J I
S J f
-g J i
I t
o f
o ~ f
I f

0 ,.~
-1F* 0 3F*
AF
(l/h)

Fig. 1. Membership functions of DO (a-l), glucose (a-2), and ethanol (a-3) and output (b). Simplified
characters: B, big; M, medium; S, small; PB, positive big; PM, positive medium; PS, positive small;
ZE, zero; NS, negative medium; NB, negative big.
4
Input'sfuzzyset Output'fuzzy
s set
..&
f "x ~ '
DO Glucose Ethanol

I ° / ,,

Rule3 ~

DO G E t

2
AF

Fig.2. Fuzzy inference for the determination of AE DO, G and E in the horizontal axis indicate on-
line measured concentrations of DO, glucose and ethanol, respectively.

selected rule. Using production rules, corresponding fuzzy set and grades of out-
put variable are determined. Defuzzification is then carried out using a simpli-
fied center of gravity method (see Fig. 2).

DF = f DF#(DF)aDF
f #(DF)dDF

Here, # shows membership function of output variable. In this case, the value of
D F is bound by - I F * (DFmin) and +3F* (DFmax) (Fig. lb).

Fuzzy control system

Direct inference of process variables

In many cases, various process variables are determined directly from fuzzy infer-
ence. As a process variable, glucose feeding rate in fed-batch culture has received
much attention. Nakamura et al. [6] reported the fuzzy control for determination
of sugar feeding rate in glutamic acid fermentation. Consumption rate of ammonia
in glutamic acid fermentation was correlated with consumption rate of sugar, and
control of sugar feeding rate was important for glutamic acid production at low
sugar concentration in fed-batch culture. Fermentation was done using 1 kl bench-
scale fermenter. This is the first application of fuzzy control in Japan, in which the
effectiveness of fuzzy control was proven in a large-scale fermentation. As another
variable, temperature in batch alcohol fermentation is also introduced [3,10,22].

Feed rate

Since catabolite repression of glucose and inhibitory effect of by-product such as

ethanol or lactate cannot be ignored in fermentation of Saceharomyces cerevisiae,
it is important to maintain glucose and inhibitor concentrations at low level. Fig-
ure 3 shows the effect of ethanol concentration on specific growth rate, #, and
the productivity. For this purpose, fuzzy control strategy has been often used for
feed rate control.
Schematic feature of the control strategy is shown in Fig. 4. For this purpose, in
general, the control strategy consisted of a feed forward and a feedback control
described below [20].

Feedforward control To maintain the exponential growth phase in a fed-batch cul-

ture of yeast, glucose must be fed exponentially while the other nutrients will be
kept sufficiently. To achieve this, the mass balance equation between feed and
consumption of glucose should be considered. The feedforward glucose feed rate
(normal glucose feed rate, F*) can be determined in accordance with increase
of the cell concentration as follows:

F * - #XV (1)
Yx/sSo

m
m
0

0.10 ,L,',8 . . . . . 30 r= 5~

0.08 ' ~ 6 4~
0 2o~-
0.o6 • :,=

c
:~ 0.04 • • W 0
O o 10 -m
0.02 " °o
C
2 E 2~_
|

0.00 " 0 I i

10 20 30 ~
E t h a n o l (g/I)

Fig. 3. Effects of ethanol concentrations in batch ctdture of S. cerevisiae 20B--12/pNA3. Symbols:

(C)), specific growth rate (h l); (O), cell concentration (g/L); (A), m-amylase (U/mL); (A), specific
s-amylase activity (U/mg dry cells).
 I Feedforward I -
Setooints I controller I" I/1"~'
Glucose / F. I r,~
Ethanol ~ / ' ","
DO ^ _ I Fuzzy . I aF ,~+ F , _ , _ -- 1 ' x/s
iJ-'-"=='l t'eeclbacK ~ioreactor
+ ~- IEd~rdi(e;I + - ' ' I r,, ]A
I Glucose
ID%ano'

Fig. 4. Schematic representation of the fuzzy control strategy: F*, normal glucose feed rate; F, real
glucose feed rate; AF, corrected glucose feed rate; X, cell concentration; I~, specific growth rate; rs,
specific glucose consumption rate; Yx/s, cellular yield; DO, dissolved oxygen concentration.

where #, X, So, and Yx/s denote the specific growth rate, concentrations of cell
and feed glucose, and cellular yield from glucose, respectively.
In order to obtain those data, computer controlled fermentation system should
be established as illustrated in Fig. 5. Concentrations of cell and dissolved oxygen
(DO) in culture broth were measured on line by a turbidimeter and a dissolved
oxygen sensor which were interfaced with A / D converters to transmit signals to
a personal computer. Glucose and ethanol concentrations in broth were meas-
ured on-line every 5 min by a glucose-ethanol analyzer. For simultaneous meas-
urement of glucose and ethanol concentrations, immobilized enzymes (glucose
oxidase for glucose and alcohol oxidase for ethanol) were used as the biosensors.
The specific growth rate was established from cell concentration which was
estimated by the turbidity values measured on-line by the turbidimeter.

Air
cPu
(Pc~ol)

Ip.,~=.ol~,.I,
i
HCl

Fig. 5. Schematic diagram of a computer-controlled fermentation system.

 Then, the values of # and Yx/s were calculated from data of a serial cell concen-
tration and glucose consumption within 20 min.

Feedback control. The previous determination of normal glucose feed rate F* is

just a rough approximation. The feedfoward control can correctly control the
nutrient concentration when equation [1] is correct during fermentation. Due to
changes of cell activity and environmental conditions, the feed rate has to be cor-
rected. This correction was noted as DF which was determined by the fuzzy con-
trol algorithm. As a result, the real glucose feed rate was adjusted as
F = F* + D F (2)

Determination of DF was carried out by fuzzy inference as mentioned above.

Cultivation results are shown in Fig. 6. Ethanol and glucose concentrations
were kept at the almost constant level during cultivation.
In the case of mammalian cell culture, concentrations of glucose, glutamine
and lactate are used as state variables. Lenas et al. [23] reported the establish-
ment of the fuzzy controller. In this paper, a fuzzy adaption strategy was com-
bined with a static PI-like fuzzy controller in an hierarchical structure. Maximum
and minimum values of DF (FTMAX and FTMIN) were decided by another
fuzzy controller. As shown in Fig. 7, F T M A X and FTMIN shifted toward posi-
tive direction in growth phase from 4 0 - 1 0 0 h. From this graph, it was found
that adaptive control during cultivation time was carried out.

Temperature

Japan's traditional sake brewing technology has been formulated through the

6
"6
4
e-
ra -~
108~ : "-- " -I 20o0= 11°
' .48v.
~ = 10
~I 6
~oo ~ 1 :~
¢~-14
1~1~~,
50 2 'o
Q.
• 01,= " ~ -- , , o
0 10 20 30
Time(h)

Fig. 6. Expression of s-amylase gene of S. cerevisiae 20B- 12 harboring plasmid pNA3 by controlling
both the glucose and ethanol concentration using the fuzzy controller.
 3.0
d "7
2.5 ~.
2.0
8
1.5
(9
0
0.4
°41 b ,, mAAAA,~-~----fl6o e
0.3 ~
~ 0a
~ 02 40 0.2 .~
E
0.1

0r~l', , , , z.~ , I 0 J 0 (.9

20
f
2o
--nj L
0 ....................... 0
w ~ r--"
z -10

-20
-30
20 40 60 80 100 120 140 20 40 60 80 100 120
Time (h) Time (h)

Fig. 7. (a,b, and c) Static fuzzycontroller performance in simulations.(a) Glucose concentrations dur-
ing the cultivation (set point 2.0 g 1-1). (b) Glucose feeding. (c) FTMAX(MIN) parameter values
and membership functions of AF during the cultivation. (d, e and f) Adaptive fuzzycontroller perfor-
mance in experiments. (d) Glucose concentration during the cultivation(set point 2 g 1 l). (e) Gluta-
mine concentration during the cultivation (set point 0.6 g 1-1). (f) FTMAX(MIN) parameter values
of glucose AF membershi function. (Controller parameters values: A = 0.16 (gluc.),B = 0.08 (gluc.),
L1 = 0.7, L2 = 4.2, S = 0.3 (gluc.), AD = 2.5, A = 0.03 (glut.), B = 0.015 (glut.), S = 0.06 (glut.).

accumulation of various kinds of technical knowledge by sake-brewing experts

(Tbji). Steamed rice is added in mashing Onoromi) tank. Fungi and yeast are
inoculated and both of saccharification and fermentation is carried out simulta-
neously. In the early moromi phase, temperature increases due to fermentation
heat and the expert gradually decrease temperature in the late phase according
to moromi state in order to increase ester component. In moromi, measurable
state variables are alcohol concentration, specific gravity (Baumt), and tempera-
ture. To brew a sake of a desired quality, only temperature is precisely controlled
by Toil. There are many reports on temperature control of moromi by fuzzy infer-
ence [3,22,24-26]. We also reported the fuzzy control of Ginjo sake mashing
[10,27]. Ginjo sake has extremely rich flavor and it is categorized as a special
grade.
Oishi et al. [22] reported the fuzzy control ofmoromi process. The expert usual-
ly judge a change in moromi temperature (CT) by evaluating subjectively the fol-
lowing three parameter (DB, DD, At,): the difference between estimated and
reference Baum+ values at a future target time (DB), the difference between the
decreasing rate of Baum6 and that of the reference Baum6 up to the present
time (DD), and the present alcohol concentration (AL). The following linguistic
rules are used for the judgment.
 9

"It is better to keep the moromi temperature, because the rate of decrease in the
Baum6 up to this time has been relatively large"
"It is better to decrease slightly the moromi temperature, because the alcohol
concentration is high"
They constructed the fuzzy rules for temperature control by using CT, AL, DB,
and DD. As shown in Fig. 8, membership functions of DB were changed based
on moromi period. In addition, production rules were also changed depending
on alcohol concentration (Fig. 9).

Other approach

Expert system has been applied for the control of bioprocesses to provide a
means of imitating the decisions by a skilled operator. Sophisticated expert sys-
tems have been realized by a significant development of the computer technology.
Expert system consists of an editor for editing various knowledge-base and infer-
ence engine for utilizing knowledge-base. There are various reports for expert
system [28--30]. Construction of expert system using fuzzy inference was
reported by Kishimoto et al. [5]. They studied the optimization of penicillin feed-
ing in glutamic acid production. Fuzzy inference is useful for construction of
inference engine in expert system, because the knowledge-base of the skilled
operator is often collected as linguistic rules from interview.

(A)

0
- 0.35 - 0.25 0 0~2 0.35

(B)

0
- 0.35 - 0.12 0 0.16 0.35

(c)
(3

0
- 0.35 -0.08 0 0.11 0.35

Fig. 8. Membership functions of DB at the different brewing periods; the 5 th day (A), the 12th day (B),
and the 15th day (C).
 I0

(i) A L LO
DB
NB NM NS ZE PS PM PB
NB NS* ZE PS PS PM PB PB
NM NS ZE ZE PS PM PB PB
NS NS ZE ZE ZE PM PB PB
DO I ZE NS ZE ZE ZE PS PM PB
PS NS NS ZE ZE PS PM PB
PM NS NS ZE ZE ZE PS PM
PB NS NS ZE ZE ZE ZE PS

(2) A L = MI
DB
NB NM NS ZE PS PM PB
NB NS ZE PS PB PB PB PB
NM NS NS ZE PM PB PB PB
NS NB NM NS PS PM PB PB
DO ZE NB NM NS ZE PS PM PB
PS NB NB NM NS PS PM PB
PM NB NB NB NM ZE PS PM
PB: NB NB NB NB NS ZE PS

(2) AL = HI DB
NB NM NS ZE PS PM PB
NB NB NS ZE ZE ZE ZE ZE
NM NB NS ZE ZE ZE ZE ZE
NS NB NM NS ZE ZE ZE ZE
DO ZE NB NM NS ZE ZE ZE ZE
PS NB NB NS NS ZE ZE ZE
PM NB NB NM NM ZE ZE ZE
PB NB NB NB NB NS ZE ZE

Fig. 9. Rules for fuzzycontrol.NS* means "if (DB = NB) & (DD = NB) & (,4L= LO) then (CT -- NS)."

Identification of culture phase

Phase recognition is one of the most important things for precise control of pro-
cess variables, because the nutrient consumption rate or the product formation
rate depends on the physiological situation of microorganism. Therefore, fuzzy
control strategy should be also changed in each culture phases such as lag phase,
early logarithmic phase, late logarithmic phase, production phase, and so on.
Identification of culture phase is done by using some state variables.

Pravastatinprecursorproduction
ML-236B is the precursor of pravastatin sodium (trade name Mevalotin), a 3-
hydroxy-3-methylglutaryl-CoA reductase inhibitor clinically applied in the treat-
ment of hypercholesterolemia, which is produced by Sankyo Co. ML-236B is a
fermentation product generated by Penicillium citrinum. Production of ML-
236B depends strictly upon the culture broth pH, which could be regulated by
sugar concentration. Hosobuchi et al. [11] constructed the fuzzy controller for
determination of the sugar feed rate. Cultivation period could be divided into
five clusterized phases. The feeding control strategy was altered for each phase.
The rules for the skilled operators for controlling the sugar feed rate in the pro-
duction culture are given in Table 2. These rules were expressed linguistically,
and the control of feed rate by skilled operators was carried out sequentially.
 11

Table 2. Rules for culture phase identification and selection of sugar feeding.

1. If the concentration of the reducing sugar is more than 1%, then the culture phase is 1 and no su-
gar is to be fed.
2. If the concentration of the reducing sugar is less than 1% during the period from 30-50 h after
starting the cultivation, then the culture phase is 2 and the sugar feeding is to be maintained at a
constant feed rate.
3. If the pH value is less than 4.3, then the culture phase is 3 and the feed rate is to be gradually re-
duced.
4. If the pH value is less than 4.0 and the slope of the pH begins to rise, then the culture phase is 4
and the feed rate should be gradually increased.
5. If the pH value is around the set point, then the feed rate should be decided upon according to the
following relationship.

pH value Sugar feed rate

High Increase
Low Decrease

Slop of pH Sugar feed rate

Up Increase
Down Decrease

Figure 10 shows the algorithm of fuzzy control, based on the rules for classifica-
tions of culture phases and control strategies shown in Table 2 and also by use

Measurement of state
variables of culture
I
[ C'almllationof level of membership
function for each state variables i I
I

I
I Estimation of culture phase from I
the pattern of the level of the membership
function for each state variable
I
Defuzzyfication I
(Sugeno's estimation method 3) [
i
[
[ Control of sugar feeding rate [

Fig. 10. Algorithm of the fuzzy control system for ML-236B production culture.
12

of the knowledge of skilled operators, the membership function for each state
variable and the method of control in each phase were constructed (Fig. 11). In
this case, TCO (total CO2 evolved) and PHV (pH value) are used for phase recog-
nition. For example,

If TCO = B and PHV = M, then phase = III

The probability that the measured state falls into the phase i (i = I, II, III, IV or
V) was calculated as follows:

If p(TCO = B) = 1 and p(PHV = M) = 0.6, then p(phase III) = 0.6

The value of the operative variable (sugar feed rate), E was calculated for each
situation. Sugenos inference method III [31] was used for defuzzification. For
example, the sugar feed rate in phase III, Fin was defined as follows:

If pH slope is B, then FIII -- 7.0

If pH slope is MB, then FII I = 6.0

When the value of pH slope was classified as intermediate of B and MB, then
FII I w a s defined as follows:

Fm = (pB x 7.0 + pMB x 6.0)/(pB+pMB)

o~-Amylaseproduction
Since s-amylase production by Bacillus species is regulated by catabolite repres-

Identificationof Culture
Membership function Phase from the pattern of Control method
membershipfunction level

1~ B No
1 = feeding
F=0

o I I
0 0.25 0.5 0.75 1.0 1.25 Constant sugar
Total CO2 evolved (mol/L) 2 ~ feeding
P = constant

1~ Sugar feeding according

3
0 I \£A'A/]( I
3.4 3.6 3.8 4.0 4.2 4.4
D- to pH slope as an index
F = f(pH slope)

pH value Sugar feeding according

1 4 ~ to pH value and pH slope
as indexes - 1
F = g(pH, pH slope)

Sugar feeding according

0 5 .~ to pH value and pH slope
-16 -9 0 9 16 as indexes - 2
pH slope ( X 100pl-I/hr) F = h(pH, pH slope)

Fig. 11. The membership function for each state variable and the method of control in each phase.
 13

sion by glucose, the glucose concentration must be maintained at a low level for
u-amylase production. On the other hand, glucose starvation initiates the cell
cycle, leading to the sporulation phase where u-amylase production completely
stops. Therefore, to maximize u-amylase production, the system is requested to
optimally control the glucose feed rate so that the glucose concentration is low
enough to prevent catabolite repression yet high enough to prevent acceleration
of the cell cycle to the sporulation phase. To overcome this problem, Horiuchi et
al. [18] constructed the fuzzy controller so as to feed glucose at optimal feeding
rate. The culture phases and substrate feeding strategies are summarized in Table
3.
In this case, culture phase transitions were characterized using the following
variables: culture time, cell concentration and CO2 content in the exhaust gas.
In total, as shown in Table 3, 14 rules including five rules for describing each cul-
ture phase (used to identify the phases), were constructed for the system using lin-
guistic expressions and membership functions for the state and control variables.
Examples of production rules are shown below:

Rule for lag phase

IF culture time is very short
and CO2 evolution rate is very low
and cell conc. is very low,
THEN culture is in the "lag phase"
and feed rate should be very low
Rule for high u-amylase production phase

Table 3. Production rules for fuzzy inference system.

Culture time State variables CO2 Cell conc. Control variables

content feed rate

ss ss ss ss (= 0)
s ss ss ss (= 0)
S L S LL
S LL S LL
S L M LL
S M M LL
M L M L
M L L L
L L M M
L L L M
L M LL M
LL S LL S
LL M LL S
LL M L S

Abbreviations: SS, very low or very short; S, low or short; M, medium; L, high or long; LL, very high
or very long.
14

IF culture time is medium

and CO2 evolution rate is high
and cell concentration is medium,
THEN culture is in the "high a-amylase production phase"
and feed rate should be high.

Each identification of a culture phase was expressed as a value of the "adaptabil-

ity", which indicated how well the current data fitted with each production rule.
The feed rate was inferred using the min/max method and defuzzificated by cal-
culating the center of gravity of the inferred results.

Automatic fuzzy controller

Construction of the fuzzy controller takes usually a relatively long time to tune
membership functions by trial and error or by simulation and a lot of experts
experiences are necessary for identifying the rules.
Recently, fuzzy neural networks (FNNs) were studied as a tool for fuzzy mod-
eling [32-36]. The FNNs have neural network structures in which fuzzy produc-
tion rules and membership functions are automatically acquired and tuned from
the numerical data collected. Hanai et al. [34] constructed F N N model for tem-
perature control in the Ginjo sake mashings without manual tuning. Tuning and
Toji's experiences were not necessary for construction in this modeling method.
The acquired fuzzy rules of F N N can be linguistically compared with the experi-
ences of Toji.
Configuration of F N N "Type I" is shown in Fig. 12. The F N N realizes a simpli-
fied fuzzy inference of which the consequences are described with singletons. The
inputs are nonfuzzy numbers. In Fig. 12, the F N N has two inputs xl and x2 and
one output y* and three membership functions in each premise. The symbols of
the circle and square in Fig. 12 mean units of the neural network and the denota-
tion ~c, ~g, ~ 1 and -1 are the connection weights. The connection weights ~c
and ~g determine the positions and gradients of the sigmoid functions ' ~ ' in the
units in (Q-layer, respectively, where sigmoid functions ' ~ ' are defined as follows:
1
f ( x ) : l+exp-~g(X+Wc) (3)

Each membership function consists of one or two sigmoid functions. In the

FNN, the membership functions in the premises are tuned and the fuzzy rules
are identified by adjusting the connection weights we, ~g and wf through the
back propagation learning algorithm [37]. The connection weights ~c and a~g are
initialized so that the membership functions in the premises are appropriately
allocated equally. The value of ~f is initialized to be zero. This means that the
F N N has no rules at the beginning of the learning. After learning, acquired con-
nection weights ~j. can be described linguistically as follows:
 15
W¢ W

X1 m small]nedlu
nedlufv big
~ W f tsmal' 0"0 0'3 0.5
x= edlum-0.4 0.0 0.25
big .0.8 .0.4 0.0

I~ small medium big

1
I~ [ A ]~ (A) (B) (C) (D) (E) (F)
1 ~[ j \ ~K._. I II I
| I = a premises consequences
k x~

Fig. 12. Structureof fuzzyneuralnetwork.

If xl is big and x 2 is small, then output variables should be 0.5.

Using this tool, fuzzy rules of bioprocess can be acquired precisely without tun-
ing of membership functions and from the fermentation results obtained until
now, not interview with the expert. Moreover, the fuzzy control can be carried
out more easily.

Industrial applications in Japan

Nakamura et al. [6] reported the construction of fuzzy controller for determina-
tion of sugar feed rate in fed-batch culture of glutamic acid fermentation. In this
report, fermentation was performed in 1 kl bench-scale fermenter. This is the
first trial of fuzzy control in large-scale fermenter in Japan. Since this system is
not working now, the other system as mentioned below are reviewed.

Vitamin B2 (VB2) production

Horiuchi and Hiraga [12] reported the industrial application of a fuzzy control
system to large-scale recombinant VB2 production. The system was applied to
the on-line control of the feed rate and pH for the fed-batch cultivation of Bacil-
lus species to produced VB2.
Microbial VB2 production is a well-known process, but it has not been
employed commercially due to the high production costs compared with the con-
ventional two-step process, which comprises the microbial conversion of glucose
to D-ribose followed by the chemical conversion of ribose to riboflavin (VB2).
Recently, however, Nippon Roche, Japan has developed and commercialized sin-
gle-step fermentative VB2 production using a recombinant Bacillus strain, which
16

effectively produces the VB2 directly from glucose in a fed-batch operation.

For the phase identification, four parameters (the culture time, CO2 evolution
rate, total CO2 evolution and DO) were selected as state variables on the basis
of operating experiences and a simulation study. The control variables were the
glucose feed rate and the culture pH. Based on repeated operating experiences,
the control variables were described as follows:

Feed rate f o r A phase : FA -- aFA + bFA x (4)

p H f o r phase : P H A = a P H A + b P H A x (5)
Here, a and b are the specific constants for each phase and t is the time from the
beginning of the A phase. Therefore, the accurate phase identification is desired.
Figure 13 shows a typical set of results from a fed-batch cultures for recombi-
nant VB2 production under fuzzy control. Figure 13a displays the results of cul-
ture phase identification as the time courses of the average adaptabilities of the
on-line data to the rules for each phase. The culture phase transitions from the
lag phase to production phase 2, which are regarded as fuzzy sets since they
have ambiguous and overlapping boundaries, were properly recognized by the
system. Identification of the culture phases by the inference system coincided
almost exactly with identifications made by the experienced operators. Figure
13b and c show the glucose feed rate and pH as they were inferred and controlled
by the system. Figure 13d shows the time courses of the optical density at 540
nm and VB2 concentration. As a result of appropriate feeding and pH control,
the final VB2 concentration reached the level of the maximum concentration
achieved in a the fed-batch culture manually controlled by experienced operators.
This fuzzy control system has been successfully operated in a large-scale main
fermenter for more than 2 years, during which time over 200 batches have been
completed. These operational results show that the system is able to successfully
carry out these tasks in commercial-scale production. Fuzzy inferencing is a use-
ful tool for realizing on-line control based on qualitative characteristics such as
culture phases and empirical information gained from operators, both of which
play significant roles in the daily operation of industrial fermentation processes.
 17

Phase (a)
1.0
Lag o~IL I
1.0
Growth 01 II1~,~ I

Production11'~l =I
= _
II I

~'~No~1.5f (b)

"~ 1.0
1.0

01 i i i i

? d (d)
~8
°-!
0
10 20 30 40
Time (h)

Fig. 13. Results of fuzzy control of a fed-batch culture for recombinant VB2 production (a); time
courses of average adaptability of on-line data to the rule for each phase. (b), (c) and (d); glucosefeed
rate, pH, and cell concentration and VB2 concentration.

ML-236B production under fuzzy control

As described above, fuzzy controller constructed in Sankyo Co. was applied to

the determination of sugar feed rate in the industrial production of ML-236B,
the Mevalotin precursor [11]. Figure 14 shows the time course of ML-236B pro-
duction culture under fuzzy control using a 30 1 fermenter. The time courses of
probability of the classified culture phases inferred by fuzzy rules are shown in
the upper portion of the figure. Sugar feeding was started when the maltose con-
centration was about 10 g/1. The pH value decreased gradually from 40--80 h
and was controlled at about 3.9 to 4.0 after 80 h of cultivation. This result indi-
cated that the control system using the fuzzy clusterization theory worked well
in ML-236B production culture.
The time courses of pH in cultures under manual control (12 examples) and
fuzzy control (20 examples) are presented in Fig. 15. The pH set point was 3.9
in all cultivations. Under manual control, the pH varied within the range from
3.6 to 4.2. While under fuzzy control, the pH was controlled within the narrow
range of 3.8 to 4.0. The time courses of ML-236B production under manual con-
trol and fuzzy control are shown in Fig. 16. ML-236B production under fuzzy
control was 10% higher than that under manual control.
18

Probability of culture phase

i I

7
~3 - Sugar
,~...~. - 60
6 40

~s
~2 ~ ~ = "lk I ~
."~!40
-5o
30 ~
•"w 30
.-~_~ 20 -~
4
1 20
10 []

3 o~ 0 0
0 40 80 120 160
Time (h)

Fig. 14. Time courses of probability of culture phases in ML-236B production culture with fuzzy con-
trol of the sugar feed rate using a 30 1 fermentor.

This control system has been scaled up to industrial scale in order to automate
pH control in the fermentation process.
In Sankyo Co., milbemycin production has been also performed by fuzzy con-

7
Manual control

3
7
Fuzzy control

~s

4
v

3 i I I I I I
1O0 200 300
Time (h)

Fig. 15. Comparison of time courses o f p H values under manual and fuzzy control.
 19

7 120

6 90A

==s 6o
113

4 3O '~

3 I I I I I I 0
7 Fuzzy control J " 120

6 90~
60

4 30

3 I I I i I I
0 1O0
Time
200
(h)
300

Fig.16.Time coursesof ML-236B productioncultureunder manual and fuzzycontrol.

trol. In this case, low productivity syndrome of milbemycin caused mainly by

insufficient oxygen supply in a commercial-scale fermenter. Okada et al. [38]
reported the syndrome could be overcome by the consistent and reliable actions
of fuzzy control system. Until now, over 1O0 cultivations in industrial scale were
performed under fuzzy control.

Automatic fuzzy modelingfor temperature controlof Ginjo sake mashing

As mentioned above, there are many reports on temperature control of sake

mashing [3,22,24-26]. It is considered that these results are actually applied for
practical sake mashing in Gekkeikan Co. and Ozeki Co. Here, temperature con-
trol of Ginjo sake mashing is described. For temperature control of Ginjo sake
mashing, F N N model was constructed. The accuracy of F N N model was con-
firmed from the results of the simulation, which were compared with the average
values of 25 Ginjo mashings.
Using the F N N model, Ginjo sake mashings using 1500 kg total rice were per-
formed in Sekiya Brewing Co. [36]. The time course pattern and Ginjo sake pro-
duced were compared with those produced by the conventional method operated
by a Toji.
Mashing results are described in Fig. 17. The time courses of Baum6 and alco-
hol concentration coincided well with those of the conventional method through-
out the mashing period. Temperature in the moromi mash using the F N N model
reached a maximum temperature at two days later and was a little lower during
20th to 28th day. As shown in Table 4, the mashing period and all of other values
20

5" oo °oo
o e
9 eo
~ °oee e

i
E
8 OO O •

.%
6
o
, , , , , , ~

5 0 ° ~ e ~ ~o • C. . . . . t i o n a l control
cont
0 F N N control
6 0
go
o
Q
4 o
¢D

2 Oo

. . . .
o

o
tO
_8

J 10 20 30 40
Mashing time (d)

Fig. 17. Time courses of temperature, Baum6 and alcohol concentration.

were in the same range as the other seven mashings performed conventionally by
the Toji. Therefore, it was confirmed that the mashing was performed well using
the FNN model.
Sake obtained from the mashing in Fig. 17 was assayed and assessed by sen-
sory evaluation by 13 expert panels. The score for flavor for the sake produced
by the FNN control was very similar to that of the conventional control. Scores
for taste, harmony and total evaluation were a little worse. The quality of the
sake produced by the F N N control was judged by Toji to be in the acceptable

Table 4. Comparison of mashes at the finishing day.

References a
FNN Conventional
control control Average Minimum Maximum

Mashing period (d) 36 35 34 33 36

Baum+ (-) 0.20 0.25 0.16 0 0.30
Alcohol (%) 17.2 17.3 17.1 17.0 17.3
BMD (d) 7.0 8.5 6.1 0 9.9
Acidity (ml) 1.5 1.5 1.5 1.4 1.6

a These data were obtained from other seven mashes operated under similar conditions.
 21

range when compared with that from 25 commercial Ginjo mashings by the
Sekiya Brewing Co.
The F N N model proposed by us has some benefits since the model can be
constructed automatically from data sets collected previously and does not need
much time for tuning membership function compared with conventional fuzzy
control. In addition, the rules acquired after learning can be described linguisti-
cally as IF-THEN rules and those are so explicit as to be understood. It should
be noted that the rules identified depend on the data sets utilized for FNN learn-
ing even though the same input variables were prepared.

Summary

Fuzzy control systems based on knowledge-base rules are categorized into two
types; direct inference of process variable and control after phase identification
based on fuzzy inference. Even in the case of direct inference, in almost all cases,
fuzzy rules constructed alter during culture period for realizing the precise con-
trol of process variables. The fact means that alteration of fuzzy rules influences
the difference of culture phase based on physiological state of microorganism.
In many cases, no clear structure for phase recognition is observed into fuzzy
controller. However, difference of physiological state is involving implicitly in
lots of fuzzy rules. As mentioned above, fuzzy controller constructed by Lenas
et al. [23], which was used for recombinant mammalian cell culture, varied maxi-
mum and minimum DF values according to change of cell activity. In the case
of fuzzy controller constructed by Oishi et al. [22], three tables on fuzzy roles
were constructed depending on alcohol concentration and in addition member-
ship function of DB, which is one of the state variables, was changed depending
on culture time. Hanai et al. [10] also constructed the fuzzy controller divided
culture period into four regions for temperature control of Ginjo sake mashing.
Construction of algorithm for phase identification plays important roles when
the control strategy is significantly different in each phases. In addition, identifi-
cation of phase and its indication during cultivation allow the system to be opera-
tor-friendly use and will bring the operator to the reliability.
In Japan, some industrial processes have been operated under fuzzy control. In
this articles, three cases were introduced as examples, that are Mevalotin precur-
sor production (Sankyo Co.), vitamin B2 production (Nippon Roche Co.), and
sake mashing (Sekiya Brewing Co., Gekkeikan Co. and Ozeki Co.). For precise
control of bioprocess, lots of knowledge of the skilled operators are very impor-
tant. In almost all bioprocess, lots of rules on process operation should be
acquired explicitly or implicitly. In order to incorporate those linguistic rules
into the computer control, fuzzy control is one of the useful tools. The fuzzy con-
trol systems will be established moreover, and the application of fuzzy control
will be proceeded widely in field of the bioprocess control.
22

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