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1 s2.0 0168165696015246 Main
1 s2.0 0168165696015246 Main
1 s2.0 0168165696015246 Main
,OUIIN*L
ELSEVIER
OF
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
M. Dorsb,
Germany
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
reserved
84
Keywords:
A.B. Puttrn
et al.
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
und associated
instruments
conditions
2.3. On-line process
monitoring
-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)
(2)
is given by
R (see Sec-
conditions.
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
(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
-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]
88
A.B. Putten
(i)
4wO
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]
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.
1
20
1.
30
(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).
A.B.
it increased
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
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
of Biotechnology
A.B. Putten
90
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 -- )
+ f(Grcqu -
G,,,,)CPR
(1 h - )
(8)
where f is a variable
determine
~s!~~,,,.
49 (1996) 83-93
(7)
of Biotechnology
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)
ZL
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
time [h]
I.
I.,
10
20
cultivation
I,
30
1
50
time [h]
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
of the process
parameters
of Biotechnology
49 (1996) 83-93
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.
Change
+28
+I37
+17
1136
+4
+ 141
Acknowledgements
The authors gratefully acknowledge
cial support
of AIF (Project
Nr.
DECHEMA,
Frankfurt.
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