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Biotechnol

,OUIIN*L

ELSEVIER

OF

Journal of Biotechnology 49 (1996) 83-93

Improvement
of the production of subtilisin Carlsberg alkaline
protease by Bacillus lichenformis
by on-line process monitoring
and control in a stirred tank reactor
A.B. van Putten,

bInstitute for

F. Spitzenbergerb,
G. Kretzmerb, B. Hitzmannb,
R. Simutisb, K. Schiigerlb,*

Fermentation
Technical Chemistry

Pilot Plant, Nouo Nor&k A/S, Hammer,


Germany
of the Uniwrsity qf Hannover, Callirrstr. 3, O-30167 Humover,

M. Dorsb,

Germany

Received 6 June 1995; revised 30 March 1996; accepted 2 April 1996

Abstract
The cultivation of Bacillus licheniformis
and the production
of subtilisin Carlsberg serine protease were investigated
in complex medium with starch and glucose, respectively, and Na-caseinate
as substrates to maximize the protease
concentration.
The turbidity and culture fluorescence were monitored in situ, the optical density on-line and the (dry)
sediment and (dry) cell mass concentrations
as well as the cell count and the DNA content were monitored
off-line.
These values are closely interrelated and were quantified by particular relationships.
By means of the six-channel flow
injection analyser (FIA) system, the following medium components
were monitored on-line: glucose, maltose, starch,
ammonium,
urea, phosphate
and protease activity. The same components
as well as protein, intracellular
phosphate
and.cc-amylase activity were evaluated off-line. The off-gas composition
was analysed on-line as well. Various control
strategies were tested in order to maximize the protease concentration:
On one hand, starch in various concentrations
was used as substrate. These runs were performed at non-controlled
starch decomposition,
at controlled and non-controlled
pH-values, respectively, and non-controlled
PO,-values. On the other hand, glucose was used as substrate in fed-batch
mode. These runs were performed with closed loop controlled pH- and PO,-valuesand
open-loop and closed-loop controlled
glucose concentrations,
respectively. The latter strategy yielded a higher protease concentration
than the former. With
complex medium and closed-loop controlled process, extremely high protease activities (24 000 EPE ml - ) were obtained.
Abbreviations:
A,, max. amylase activity [unit ml _ I; A,,,, max. amylase concentration [unit ml - 1; CAFCA, computer assisted
flow control & analysis; CDM, cell (dry) mass concentration [g 1~ I; CPR, CO, production rate [g I- h - I; DSM, German Collection
of Microorganisms;
EPE, esterolytic protease activity [unit ml I; F olu, glucose feed rate [g 1~ h - I; FIA, flow injection analysis;
G Gl, measured glucose concentration
[g 1~ 1 used for fed-batch control: GOD, glucose oxidase; OD, optical density; OTR, oxygen
transfer rate [g 1- h _ I; P,, maximum protease activity [unit ml - I; P,, maximum protease concentration
[g 1~ I; PO,, dissolved
oxygen concentration
with regard to its saturation
value [XI];R,, consumed glucose during the cultivation
[g 1~ 1; R,, consumed
protein during the cultivation [g I- I; Rs, consumed starch during the cultivation [g 1- I; RQ, respiratory
quotient [-_I; S,, substrate
(glucose) concentration
[g 1~ I; S,, substrate (protein) concentration
[g 1~ 1; S,, substrate (starch) concentration
[g I _ I; S,,,,
substrate
(glucose) concentration
at t = tPamdx [g I- I; S,,,, substrate (starch) concentration
at t = tPamaX [g I 1;SDM, sediment (dry) mass
concentration
[g I - 1; Y,;,, yield coefficient of cell count with respect to the substrate (starch and glucose, respectively) consumed;
YCDM:s, yield coefficient of growth with respect to the substrate (starch and glucose, respectively) consumed; Y,:,,,,
yield coefficient
of the product formed with respect to cell (dry) mass formed; Y p ,,, yield coefficient of product formed with respect to the cell count;
Y,,,, yield coefficient of the product formed with respect to the substrate (starch and glucose, respectively) consumed; Y,!,,, yield
coefficient of product formed with respect to protein substrate consumed.
* Corresponding
author.
016%1656/96/$15.00
PII SO 16% 1656(96)0

0 1996 Elsevier
1524-6

Science

B.V. All rights

reserved

84
Keywords:

A.B. Puttrn

et al.

: Journal of Biotecllnolog?: 49 (1996) 83--93

High protease activity; Complex medium; On-line process monitoring;

1. Introduction
Detergent enzymes are important
commodities
which are produced in large amounts (1300-1500
tons in 1990) and make up about 40% of the selling
price (US$300 million in 1990) of industrial enzymes
(Uhlig, 1990). The detergent enzymes are the alkaline serine protease subtilisin Carlsberg formed by
Bacillus licheniformis and subtilisin Novo by Bacillus amyloliquef aciens (Aunstrup,
1979; Aunstrup et
al., 1979). In the following the cultivation
of B.
lichenzformis and the production
of alkaline serine
protease subtilisin Carlsberg is investigated. In spite
of the industrial
importance
of this enzyme, only
a few papers have dealt with this system and they
only investigated the growth and product formation
in synthetic medium, where only very low enzyme
activities (4- 15 U ml ~ ) were obtained (Bierbaum
et al., 1991; Bulthuis et al., 1989; Frankena
et al.,
1985, 1986, 1988; Giesecke et al., 1991; Hanlon and
Hodges, 1981; Hanlon et al., 1982; Ward, 1985;
Wouters and Buysman,
1977). Only Kroner and
Kula (1984) reported
on the investigation
of
protease monitoring
and Hiibner et al. (1993) on
process monitoring
including protease analysis in
complex medium. The present investigation
is a
continuation
of those of Hiibner et al. (1993) and
has the aim of increasing the protease activity by
better process monitoring
and control.

2. Materials

and methods

2.1. Organisms

and growth

Various control strategies

The preculture was prepared in 2-l shake flasks


(125 rev./min) in 500 ml complex medium (g l-
in tap water) potato starch 40, amylase (OptithermL420) 0.2, Na-caseinate 4.0, soy flour 2.0, corn steep
liquor 2.4, (NH,),HP04
1.6 for 16 h at 39.5C.
The standard main culture consisting of (g 1- ):
corn starch 100, amylase (Optitherm-L420)
0.4,
Na-caseinate
27.0, soy flour 23.0, (NH,),HPO,,Q.S,
Na,HPO,.
2H,O 0.3, corn steep liquor 7.0, antifoam
agent
4.0, KH,PO,
0.3, MnSO,.H,O
0.02,
FeSO,. 7H,O 0.05, MgSO,. 7H,O 0.05 was inoculated with the preculture.
The cultivation
was
performed at 39.5C at an initial pH 6.8.
2.2. Bioreactor

und associated

instruments

The stirred tank reactor with a 20-l working


volume (B. Braun Melsungen)
was equipped with
a mechanical
foam destroyer
(Fundafoam
00,
Chemap), head pressure meter (Buster Type 9832)
mass flow meter (Brooks), off-gas analyser (Types
UNOR 6N and Oxygor AS 6N, Maihak), fluorosensor (Ingold), turbidity sensor (Type Mex 2 OD 10/S
EUR Control,
BTG), instruments
for pH, PO,,
temperature
and stirrer speed measurement,
and
on-line sampling
system (ABC Puchheim).
The
automatic reactor operation and evaluation
of the
measured
data were performed
by a front-end
computer
system with a VAX-computer
and a
software package RISP (Real time Integrated Software Platform) developed in the Institute for Technical Chemistry.

conditions
2.3. On-line process

Bacillus 1icheniJbrmis P300 was received from


DSM, Braunschweig;
15 ml aqueous solution (consisting of 5 g 1~ yeast extract, 5 g l- glucose, 5
g 1- NaCl and 10 g 1~ Na-caseinate
at pH 7) and
25 ml aqueous solution (containing 250 mg glucose)
were mixed and sterilized separately. This mixture
was inoculated
with the bacteria and after 10 h
cultivation,
1 ml bacterium suspension was added
to each of 40 kryotubes,
filled with 0.5 ml 30%
glycerol solution. These tubes were stored at 78C.

monitoring

The on-line medium composition monitoring was


carried out with a six-channel flow injection analyser
(FIA) system consisting of the following channels:
glucose (GOD), starch and maltose (a-glucosidase,
glucoamylase),
ammonium
(Orion electrode), urea
(urease and NH, electrode), phosphate
(a-phosphor-molybdenumblue,
absorbance at 825 nm) and
protease (Fig. 1). The enzymes were immobilized on
a VA Epoxy Biosynthe carrier (Riedel de Haen;
Jiirgens et al., 1994). The protease activity was

A.B. Putten et al. 1 Journal of Biotechnology 49 (1996) 83-93

measured by the esterase side activity of protease


using the model substrate of N-CBZ-valinep-nitrophenylester,
which was converted by the protease
top-nitrophenol
and the concentration
of the latter
was measured at 340 nm and pH 6. This analysis
was performed by stopped FIA. The esterolytic and
proteolytic
activities
of the subtilisin
Carlsberg
protease are the same (Hiibner et al., 1993). The
multichannel
FIA was operated with the automation software package CAFCA
(Computer
Assisted Flow Control & Analysis; Hitzmann
et al.,
1993), which is connected to an expert system for
knowledge-based
fault detection and diagnosis.
The determination
of the cell concentration
parameters
was performed
by means of three
different methods: in situ turbidity measurements
(MEX 2, OD 10/S, EUR Control), in situ fluorom-

-la+
maltose,
total sugar

Fig. 1. Six-channel
on-line FIA system for monitoring
the
concentrations
of glucose, maltose, starch, ammonium,
urea,
phosphate
and protease activity (Putten et al., 1995).

8.5

eter (Fluorosensor,
Ingold) measurements
and online optical
density
(OD)
measurement
(by
photometer
P 6000 Skalar).
For more details of the on-line measurements
see
Putten et al. (1995).
2.4. Off -line process analyses
The medium components,
which were monitored
on-line, were determined
off-line as well. In addition protein was determined according to Bradford
(1976) modified by Sedmak and Grossberg (1977).
Intracellulary
phosphate
was analysed
after cell
disintegration
and treatment
with 0.5 M HClO,
with the same method as the extracellulary
one. The
concentrations
of the amino acids were determined
by HPLC with OPA-MCE
pre-column
derivatisation.
Total cell count was determined microscopically
with the Neubauer counting chamber. On account
of the solid content of the cultivation
medium, the
direct determination
of the cell (dry) mass concentration was not possible. The sediment (cells +
solids) was separated from the sample by centrifuging at 12 500 rev./min. After washing and drying,
the sediment (dry) mass concentration
(SDM) was
determined by weight. The wet sediment of another
sample was incubated
in a shaker with 0.25 M
HClO, for 40 min, washed, dried and weighed. This
was the cell free solid content in the sample. The
difference of the (dry) sediment concentration
and
the (dry) sediment residue concentration
after the
HClO, treatment is called cell (dry) mass concentration
(CDM).
The optical density (OD) was
measured at 550 nm and the DNA content with the
Dische reaction (Dische, 1930).
The various cell variables were well correlated
during the exponential
growth phase. During the
stationary growth phase the scattering of the data
was larger. The close relationships
between the cell
(dry) mass concentration
(CDM; mg ml ~ ), sediment (dry) mass concentration
(SDM; mg ml-)
measured by weight, the optical density (OD), the
cell count ( lo9 ml - ) measured
in the counting
chamber
by microscope
and the DNA content
(DNA; mg ml - ) were quantified by:
CDM

= aOD

+ b

(1)

A.B.

86

Puttrn

+ d

et al.

/Journal of

CDM

= c (cell count)

CDM

= fSDM

+ g

(3)

CDM

= hDNA

+ j

(4)

and the quality of these relationships


the particular
correlation
coefficients,
tion 3.1).
2.5. Operation

(2)

is given by
R (see Sec-

conditions.

From the large number of the cultivations


performed, five different examples are presented:
_ Standard
medium (100 g 1~ starch) no pHand PO,-control
(Run A and F)
_ Medium with 50 g l_ starch and no pH- and
PO,-control
(Run B)
~ Medium with 50 g 1~ starch and pH-control,
but no PO,-control
(Run C)
_ Glucose
fed-batch
operation
with pH- and
PO,-control
(Run D)
~ Glucose
fed-batch
operation
and pH-, PO,and close glucose concentration
control (Run
E).

Biotechnology

49 (1996)

83-93

A comparison
of these coefficients with those
evaluated with another cultivation
(Run F):
a = 0.154, b = 9.58, with R = 0.99
c = 0.232, b = 14.0, with R = 0.99
f = 0.899, g = - 6.95, with R = 0.99
h = 26.01, j = 13.5, with R = 0.98
indicates that, in spite of the different cell counts
(120 x 1Omll
inRun
Aand
150 x 10mll
in Run F), the coefficients differ only slightly.
The on-line monitored
cell variables were influenced by the variation
of the cultivation
medium
properties.
The turbidity
and optical density depend on the pH-value of the cultivation
medium.
In Fig. 2b,c the turbidity
and OD values are
shown. They exhibit a maximum/minimum
during
5- 10 h and increase after 25 h, caused by pHvariation,
in contrast to the off-line cell variables
(Fig. 2d). The course of the culture fluorescence
differs considerably
from those of the other cell
variables. This is due to the presence of a large
amount of the fluophore casein pepton, which is
gradually
consumed
by the cells and exhausted
within 15 h (Fig. 2e). Therefore, the fluorescence
intensity decreased during the exponential
growth
phase and had no relevance for the cell concentration, but for casein concentration.

3. Results

3.2. Cultivation

3.1. Interrelationships
between the concentrations
of the cell (dry) mass (CDM), the sediment (dry)
mass (SDM), the DNA, the cell count and the
optical density (OD)

The decomposition
of the starch was probably
the rate-limiting
process for the glucose uptake.
After 8 h, no glucose could be detected, but after
40 h, starch and maltose were still present in the
cultivation
medium
(Fig. 2f). The ammonium
(Fig. 2g) and urease (Fig. 211) appeared after 30 h,
when the protease started to decompose
due to
self-digestion
(Fig. 2i). The protease activity exhibited a maximum
at 25 h and attained a fairly
high value of 16 000 EPE ml ~
The r-amylase
activity displayed
a maximum
(3450 U ml at 12.5 h) as well, but occurred
earlier, and was lower than that of the protease
activity. After the maximum it reduced to zero at
25 h (Fig. 2i), and the starch conversion
into
maltose and glucose was stopped (Fig. 2f). Obvithe amylase
was decomposed
by the
ously,
protease. The extracellular
phosphate
concentra-

Several cultivations
were carried out with the
standard
medium composition
with and without
pH and PO, control. Only one typical cultivation
(Run A) is presented here to show the relationships between different process variables. Interrelation between the different cell variables
were
observed (Fig. 2a), which were quantified by Eqs.
(1) and (2), and Eq. (3) and the following coefficients:
a = 0.20, b = 10.3, with R = 0.99
c = 0.25, d = 15.4, with R = 0.97
f = 0.90, g = - 6.99, with R = 0.99
h = 24.3, j = 7.21, with R = 0.97

,tYth starch as substrate

(a)

70,

60

60-

70

_L

50-

40-

-60
.h

r:

950
.x

30-

*o-

10.

is

.I

40

30

20

cultivation

time

Ihl
Cd)

9.

9,e

g
0

IO

cultivation

time Ihl

8.5-

6-

7-

6-5

e-

6.0
5-

10

20

30

cultivation

protein COIIC.: --a-

-m-

40

5,5-

60

time [h]

n
20

cultivation

c
30

c
40

time [h]

protein total
wilhout
500

.50
,T

10

20

cultivation

30

40

time [h]

50

_I

1100

16

cultivation
(h)

60,

time [h]

60 F
L
3

40-

I
9
20 -

.-0

10

20

cultivation

30

time [h]

40

I
50

- -20
0

10

20

cultivation

30

40

60

time [h]

Fig. 2. Process performance


of run A: (a) Cell count and concentrations
of the (dry) cell mass (CDM), (dry) sediment (SDM); (b)
Cell count and in situ monitored
turbidity;
(c) Cell count and on-line monitored
optical density (OD); (d) pH-value;
(e)
Concentration
of protein with and without protease;
(t) On-line monitored
concentrations
of glucose, maltose and starch; (g)
Concentration
of ammonia (left: entire concentration
course, right: the concentration
course in the first 30 h plotted with expanded
scale); (h) Concentrations
of urea and cell count; (i) On-line measured concentration
of starch, and off-line measured activities of
protease and a-amylase; (j) Oxygen transfer rate (OTR) and CO, production
rate (CPR); (k) Comparison
of the courses of cell count
and CPR during the exponential
growth phase; (I) Dissolved oxygen concentration
with respect to its saturation
value (PO& (m)
Yield coefficient of glucose uptake rate with regard to the CO, production
rate (CPR) at the beginning of the cultivation.

88

A.B. Putten

(i)

4wO

et al. 1 Journal oJ Biotechnology

49 (1996) 83-93

,
- IO0

I
L
E
2

3000.

0
:z

- 60

2000_

- 60

'",
8
L?
x

- 40
'OrlO-

- 20

d
I
*

O-

-0

18..

10

I.

a.
sa

I.

20

QOO

40

60

ci)
F

a12

(1)

0.15

0,15

10

12

.
46

60

loo.

w 60

it
I

cultiwtiontime [h]

cukivation time [II]

4
e

0.66 .

60

8
0"

40

0.02 -

B
2

20

0.w

bB

f.& web.

I
.

0
I.

10
8.

20

..

40

30

50

10

1,

8.

cultivation time [h]

1
20

1.

30

cultivation time [h]

(ml

9.0 0.5 -

6.0 -

50

5.5

6.0

cultivation

6.5

7.0

7.5

time [h]

Fig. 2. (i)-(m).

tion decreased from 11 mM 1~ to zero after 10 h,


but the intracellular
phosphate
concentration
closely followed the cell mass concentration.
Thus
the phosphate
content
in the cells was nearly
constant.
The oxygen uptake rate OUR, which is about
equal to the oxygen transfer
rate OTR under
balanced
growth, and the CO, production
rate
(CPR) exhibited sharp maxima at 13 h (Fig. 2j).
Their exponential
increase corresponded
to the
exponential
growth phase (Fig. 2a,b). The respira-

tory quotient RQ = CPRjOTR


= 1 during the
entire cultivation
time.
During the exponential
growth phase there was
a close relationship
between the CPR and the cell
count (Fig. 2k). Therefore, the glucose uptake rate
was connected to CPR. This specific glucose uptake
rate gSICPR is shown in Fig. 2m. It attained
a
maximum value (8.79 g g-l) at 5.7 h. During the
exponential
growth phase, the dissolved oxygen
concentration
with respect to its saturation
value
(PO*) quickly dropped from 100% to zero at 12.5

A.B.

Putten et al. /Journal

h and during the production


phase
gradually to 100% again (Fig. 21).
3.3. Cultivation

it increased

with reduced starch concentration

with pH control

To eliminate
this negative effect, the pH was
controlled and kept at its initial value 6.8 (Run C).
This had a positive effect on the turbidity
monitored in situ (Fig. 3) and OD values measured
on-line, which now agreed well with the off-line
measured cell mass parameters.
More important
was the positive effect on the protease activity. Its
maximum
(11000 EPE ml - ) was an order of
magnitude
higher than in the cultures
without
pH-control.

and
as a

89

and

To avoid the limitation


by the starch decomposition, the starch was replaced by glucose, the
amount of which corresponded
to the 100 g 1-
starch (Run D). The intention
was to try to keep
the glucose
concentration
by a proportionalclosed-loop control and by fed-batch operation at
a constant value. The pH-value was maintained
at
6.8 during the cultivation.
However, the control of
the glucose feeding was not perfect. The feeding
was started when the glucose concentration
decreased below 2 g l- and was stopped when it
increased
above this value. On account
of the
slugginess of the system, strong concentration
fluctuations in the range O-5 g 1- glucose occurred,
which caused considerable
fluctuations
of PO, as
well. It was not possible to avoid the oxygen
limitation by stirrer speed control. Between 17 and
23 h, the PO, dropped to zero. This oxygen limitation of a duration of 6 h and the glucose concentration fluctuation
reduced the protease activity to
6000 EPE ml-

3.6. Cultivation with glucose as substrate and


with closed loop control of glucose concentration
and pH- and PO,-values
To maintain
the required
constant
dissolved
oxygen concentration
PoZreqU (% of saturation)
a
P-controller
was used and pure oxygen was added
to the pressurized air (Run E) (Fig. 4). The feed rate
of oxygen
F,,
was calculated
by: F& =
k(PoZrequ - PoZmeas) + F& + fCPR (1 min - )
(5)
Where

Fig. 3. Comparison
of the in situ monitored
turbidity
off-line measured
cell count as well as the pH-value
function of the cultivation
time for Run C.

49 (1996) 83-93

3.5. Cultivation with glucose as substrate


with pH control

To avoid oxygen limitation


during the cultivation, the starch concentration
was reduced from
100 g 1- to 50 g 1- (Run B). However, a reduction
of PO, to zero at 12.5 h could not be avoided. The
courses of the process variables did not change
considerably
due to the reduction
of the starch
concentration,
but the starch was completely consumed, which caused a substrate
limitation
and
reduction of the maximum
protease activity. The
variation of the pH-value was more pronounced
as
well. This added some inhibition
due to higher
H +-ion concentration
and caused the decrease of
the maximum protease activity to 700 EPE ml - i.
3.4. Cultivation

of Biotechnology

F&, feed rate of pure oxygen at the time


t; k, gain; PoZrequ, required dissolved oxygen concentration
(% of the saturation
value); Pozmeasr
measured dissolved oxygen concentration
(! of the
saturation);
F& , feed rate at the time t - 1; f,
factor for a constant
pure oxygen feeding rate
(bias).
As soon as the PO, was approximately
5% above
the required value, only the basic value (f x CPR)
was fed and when it was 30% above the required
value, no pure 0, was fed to the system.

A.B. Putten

90

Fig. 4. (a) Program

layout

and

(b) equipment

er al. ~Journal

for dissolved

oxygen control.
To maintain
a constant
glucose concentration
by P control and fed-batch
operation,
the feed
rate was calculated by the following relationship:
FCjlu = crs,,,,s S_ CPR + k*(G,c4, - G,,,,)(l
III

1
(6)

k* =

vR

go,:,,,

$ZPR
I

(12 g ~ h -- )

The feed was started when the glucose concentration


decreased below 3 g l- . k* and f in
Eqs. (7) and (8) were varied. When the glucose
concentration
increased
quickly, and a negative
feed rate was evaluated
in Eq. (8), Fa,,, = 0
was set. As a result of this process control, the
glucose concentration
was maintained
at 2 g 1
f 9%. Under these conditions
the off-line and
on-line cell mass variables
agreed satisfactorily
(Fig. 5a,b). Since during
the cell growth,
0.6
mol COZ and during
the protein
production
only 0.18 mol CO, is formed from 1 mol glucose, the specific CPR (qcoJ dropped during the
cultivation,
which was taken into account by the
factor f. On account
of the constant
glucose
concentration
(G,,,,
= 2 g 1 _ ), glucose feedrate (F,;,, = 3.56 g 1 h ~ ) and the estimated
number of cells in the reactor, the specific glucose consumption
rate with regard to the cell
determined.
g-S,
count
us,,, could be directly
gradually
decreased
with increasing
cultivation
time (Fig. 5~). The protease activity attained extremely high values (maximum
24 000 EPE ml ~
at 20 h) and its decrease after 20 h was moderate (Fig. 5d). Therefore,
the ammonium
concentration
was practically
zero during
the entire
cultivation.
3.7. Comparison

+ f(Grcqu -

G,,,,)CPR

(1 h - )
(8)

where f is a variable
determine
~s!~~,,,.

49 (1996) 83-93

(7)

Where F G,u, glucose feed rate (1 h ); CPR,


CO, production
rate (g 1~ h - ); V,, working
volume of the reactor (1); Sin, glucose concentration in the feed solution (g 1- ); cSiCPR, specific
glucose uptake rate with regard to the CO1 production
rate (g g-l);
k*, CPR-dependent
variable (12 g h ~ ); g, gain (1 g ~ ); Greyur required
glucose concentration
(g 1 - ); G,,,_,
measured
glucose concentration
(g 1~ ).
If rJsjCPR is not known, the following relationships were used:
F G,u = fCPR

of Biotechnology

now. Eq. (8) can be used to

of the runs A und E

In Table
1 the process
parameters
of the
runs A and E are compared.
The difference between the (dry) cell mass: - 36% and the cell
count: + 59% of the runs 705 and 1221 is due to
the considerable
difference
in the cell size. The
cells in run E were very small and in run A
large.
According
to the industrial
praxis starch is
used as substrate and the pH- and PO,-values are
not controlled.
To improve the process performance glucose was used as substrate and its concontrolled
by
centration
was
closed-loop
fed-batch operation.
Furthermore,
pH- and Po2values were closed-loop
controlled
as well.
With 2 g l- glucose concentration
and with
PO, > 50% and pH 6.8 a much higher protease
concentration
was obtained than with starch and

A.B. Putten et al. /Journal

(a)

ZL

of Bioterhtzology 49 (1996) 83-93

91

zo-

64

I
i

IO-

O-

o.ooo~
*

4.0

4.5

5.0

cultivation

5.5

6.0

6,

time [h]

(b)
250

7.5 -

z
_;;;

a
,x

TO-

6.5 -

B
B

.5 6.0 5.5

5.0 -

cultivation

Fig. 5. Process performance


count and in situ monitored

time [h]

I.

I.,

10

20

cultivation

I,

30

3.8. Carbon balance


The carbon balance was 100% ( - 3.5%) during
the first 10 h of the Run A. After 10 h, it
gradually decreased to 78% at 35 h. During Run
E the carbon balance was 100% ( - 3.8%) much
longer in the first 20 h and it decreased to 78%
much later, namely at 43 h. This indicates that the
carbon
balance
was satisfactory
during
the
growth phase. However,
during the production
phase (with starch as substrate) and at the end of
the production
phase (with glucose as substrate) a
gradual cell lysis and later proteolysis
of the enzymes occur, which cause a deterioration
of the
carbon balance.

1
50

time [h]

of Run E: (a) Cell count and concentrations


of (dry) cell mass (CDM), (dry) sediment
turbidity; (c) Specific glucose uptake rate with respect to the cell count; (d) Protease

without control of pH- and PO,-levels. The yield


coefficients markedly increased: Y,,,,,
by 141%
and Ypfs by 137%. The maximum protease activity was higher by 50% and the protease productivity (EPE h ~ i) by 187%.

40

(SDM);
activity.

(b) cell

4. Conclusions
The cell mass concentrations
monitored
with a
turbidometer
in situ and by optical density on-line
in complex medium agreed well, if the pH-value
was kept constant.
The low molecular
components monitored
by a six-channel
FIA on-line
agreed well with the off-line measured data. However, the protease activities in the on-line sample
and reactor differed due to the fouling of- the
microfiltration
membrane
and partial retention of
the protease
by the membrane,
but the actual
protease
activity could be evaluated
from the
on-line monitored
data by means of a constant
factor. The enzymatic
decomposition
of starch
was probably
the rate limiting
step of the cell
growth during the exponential
phase. The reduction of the starch 'concentration' to avoid oxygen
limitation
was not successful, and it reduced the
protease, concentration
considerably.

92
Table I
Comparison

A.B. Putten et al. /Journal

of the process

parameters

Cell (dry) mass at t = tPmilx


Cell count at t = tFman
Max. specific growth rate
Max. protease activity
Max. protease concentration
Max. a-amylase activity
Starch cont. at t = 0
Glucose cont. at t = 0
Starch cont. at t = tPman
Glucose cont. at t = tPman
Consumed
starch at t = tPmax
Consumed
glucose at t = tPm.lx
Protein cont. at t = 0
Protein cont. at t = tPmiir
Consumed
protein at t = tPrnax
Yield coeff. of product form
Yield coeff. of product form
Yield coeff.
Yield coeff.
Yield coeff. of growth
Yield coeff. of growth

of Biotechnology

49 (1996) 83-93

of the runs A and E


Abbreviation

Run A

Run E

CDM
n

47 g I-
139 X lOI I-
0.52 h-
16 000 EPE ml-
6.1 g I-
3450 U ml-
100 g 1-l

30 g I-
195 X IO I-
0.56 hh
24 000 EPE ml
IO g 1-l

ii,,,
P*
P,
A,
Ss
S,
S spm
Sc;,,
Rs
R,
S,
S Pm
R Pm
Y IS

Y,V,
Y ,,CM

Yp.
YCDM,S
Y,S

(AI)

-36
+ 59
+7
f50
+50

6 g I-
._.

13.5 g lo_

2gl-
86.5 g I-
54 g 1-1
39.0 g I-
2.0 g I-
37.0 g I-
0.19 g gg
0.27 g g-
0.33 g gg
0.05 X lo-- g
0.56 g g-
3.61 x IO g-

39.2 g I -
10.4 g 1-i
28.8 g I-
0.08 g g -
0.23 g go
0.14 g g-1
0.05 x lOpI2 g
0.54 g gg
1.50 X IO gg

The yield coefficients of growth YCDM..s are related to the substrate (starch or glucose,
of the product are related to the substrate (starch and glucose, respectively) consumed:
related to the cell (dry) mass formed: Y PiCnM
_-... and to the cell count: Y,,,.
In Run A, the cells were considerably
larger than in Run E.
bProduct yield with respect to the cell count n.

The increase of the protease concentration


by
an improved
strategy was very successful.
The
replacement
of starch by glucose in the complex
medium and by maintaining
a glucose concentration at about 2 g I- and constant pH at 6.8 and
constant
non-limiting
PO,-level, the protease activity was increased from 16 000 ml ~ (at 100 g
starch concentration
and non-controlled
pH- and
PO,-values)
to 24 000 ml - . This is higher by a
factor of 5 x lo3 than those obtained
with
synthetic medium (Bierbaum et al., 1991; Bulthuis
et al., 1989; Frankena
et al., 1985, 1986, 1988;
Giesecke et al., 1991; Hanlon and Hodges, 1981;
Hanlon
et al., 1982; Ward, 1985; Wouters and
Buysman,
1977).
It is expected that the protease activity can be
further increased by reducing the glucose to about
0.5 g 1~ and PO, below 60% at the beginning
of
the protease production
(Frankena
et al., 1988).

Change

+28
+I37
+17
1136
+4
+ 141

respectively) consumed. The yield coefficients


Y,,,, related to the protein consumed: Y,:,,,

Acknowledgements
The authors gratefully acknowledge
cial support
of AIF (Project
Nr.
DECHEMA,
Frankfurt.

the finan8959) and

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