,OUIIN*L OF

Biotechnology ELSEVIER 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”, F. Spitzenbergerb, G. Kretzmerb, B. Hitzmannb, M. Dorsb, R. Simutisb, K. Schiigerlb,*

“Fermentation Pilot Plant, Nouo Nor&k A/S, Hammer, Germany bInstitute for Technical Chemistry of” the Uniwrsity qf Hannover, Callirrstr. 3, O-30167 Humover, 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 0 1996 Elsevier Science B.V. All rights reserved

PII SO 16% 1656(96)0 1524-6

84 A.B. Puttrn et al. : Journal of Biotecllnolog?: 49 (1996) 83--93

Keywords: High protease activity; Complex medium; On-line process monitoring; Various control strategies

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 alka- line serine protease subtilisin Carlsberg formed by Bacillus licheniformis and subtilisin Novo by Bacil- lus 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 conditions

Bacillus 1icheniJbrmis P300 was received from DSM, Braunschweig; 15 ml aqueous solution (con- sisting 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 - 78°C.

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 (Optitherm- L420) 0.2, Na-caseinate 4.0, soy flour 2.0, corn steep liquor 2.4, (NH,),HP04 1.6 for 16 h at 39.5”C.

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 inocu- lated with the preculture. The cultivation was performed at 39.5”C 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), fluorosen- sor (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 Soft- ware Platform) developed in the Institute for Tech- nical Chemistry.

2.3. On-line process 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-phos- phor-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 8.5

measured by the esterase side activity of protease using the model substrate of N-CBZ-valinep-nitro- phenylester, 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 automa- tion software package CAFCA (Computer As- sisted 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).

eter (Fluorosensor, Ingold) measurements and on-

line 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 addi- tion 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 derivatisa- tion.

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 concen- tration was not possible. The sediment (cells + solids) was separated from the sample by centrifug- ing 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 concen- tration (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 ~ ‘), sedi- ment (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)

86 A.B. Puttrn et al. /Journal of Biotechnology 49 (1996) 83-93

CDM = c (cell count) + d (2)

CDM = fSDM + g (3)

CDM = hDNA + j (4)

and the quality of these relationships is given by the particular correlation coefficients, R (see Sec-

tion 3.1).

2.5. Operation conditions.

From the large number of the cultivations per- formed, five different examples are presented: _ Standard medium (100 g 1~ ’ starch) no pH-

and 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).

3. Results

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)

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 relation- ships between different process variables. Interre- lation between the different cell variables were observed (Fig. 2a), which were quantified by Eqs. (1) and (2), and Eq. (3) and the following coeffi- cients:

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 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 1O’mll inRun Aand 150 x 10”mll’ in Run F), the coefficients differ only slightly.

The on-line monitored cell variables were influ-

enced by the variation of the cultivation medium properties. The turbidity and optical density de- pend 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 pH- variation, 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 concentra- tion, but for casein concentration.

3.2. Cultivation ,tYth starch as substrate

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 ex- hibited 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). Obvi- ously, the amylase was decomposed by the protease. The extracellular phosphate concentra-

(a) 70, 60

60- 70

_- 50- -60 L .h

b” 40- 950

r: .x

e 30- is 40

t .I *o- ) 30

10. 20

9.

6-

7-

e-

5-

cultivation time Ihl

0 10 20 30 40 60

cultivation time [h]

cultivation time Ihl

Cd) 9,e , , r , z

8.5 -

6-5

6.0 -

5,5- g ’ n c c 0 IO 20 30 40

cultivation time [h]

protein COIIC.: --a- protein total

-m- wilhout

0 10 20 30 40 50 I

cultivation time [h] ‘_I 16 1 100

.-0

I - -20 0 10 20 30 40 50

cultivation time [h]

500 , . , , , , .50 ,T’

(h) 60,

cultivation time [h]

s . I

60 -

F L

3 40-

I 9

20 -

0 10 20 30 40 60

cultivation 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 et al. 1 Journal oJ’ Biotechnology 49 (1996) 83-93

(i) 4wO , . , . , . , . , ,

I - IO0

L

E 3000. 2 - 60

0 :z 2000_ - 60

'", 8 - 40 L? x 'OrlO-

3 - 20

d

I O- -0 * I 18.. . I. a. I. I

0 10 20 sa 40 60

cukivation time [II]

ci) 0,15 0.15

F a12 .

it

I 0.66 .

1

f.& we-

b.

I

0.02 -

0.w .

I

(ml

I. 8.

9.0 -

1 .‘. 0 10 20 30 40 50

cultivation time [h]

i 0 I QOO

0 2 4 6 6 10 12

cultiwtion time [h]

(1) , ‘ , I ’ I ’ I ’ 1 I loo.

‘3 w 60 4 e . 60 8

8 0" 40

B 2 20

b B 0

I

I . I . , .

1, 8. 1 ‘1. ‘. 1 0 10 20 30 46 60

cultivation time [h]

0.5 -

6.0 -

50 5.5 6.0 6.5

cultivation time [h]

7.0 7.5

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 of Biotechnology 49 (1996) 83-93 89

h and during the production phase it increased gradually to 100% again (Fig. 21).

3.3. Cultivation with reduced starch concentration

To avoid oxygen limitation during the cultiva- tion, 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 con- sumed, 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 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 moni- tored 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.

Fig. 3. Comparison of the in situ monitored turbidity and

off-line measured cell count as well as the pH-value as a function of the cultivation time for Run C.

3.5. Cultivation with glucose as substrate and

with pH control

To avoid the limitation by the starch decompo- sition, 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 proportional- closed-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 de- creased below 2 g l- ’ and was stopped when it increased above this value. On account of the slugginess of the system, strong concentration fluc- tuations 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 limita- tion of a duration of 6 h and the glucose concen- tration 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 F&, feed rate of pure oxygen at the time

t; k, gain; PoZrequ, required dissolved oxygen con- centration (% 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.

90 A.B. Putten er al. ~Journal of Biotechnology 49 (1996) 83-93

Fig. 4. (a) Program layout and (b) equipment 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 h ‘1 I II

(6)

k* = go,:,,, vR $ZPR (12 g ~ ’ h -- ‘) (7)

I”

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 concentra- tion in the feed solution (g 1 - ‘); cSiCPR, specific glucose uptake rate with regard to the CO1 pro- duction rate (g g-l); k*, CPR-dependent vari- able (12 g ’ h ~ ‘); g, gain (1 g ~ ‘); Greyur required glucose concentration (g 1 - ‘); G,,,_, measured glucose concentration (g 1~ ‘).

If rJ sjCPR is not known, the following relation- ships were used:

F G,u = fCPR + f(Grcqu - G,,,,)CPR (1 h - ‘)

(8)

where f is a variable now. Eq. (8) can be used to determine ~s!~~,,,.

The feed was started when the glucose con-

centration 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 glu- cose, 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 feed-

rate (F,;,, = 3.56 g 1 ’ h ~ ‘) and the estimated number of cells in the reactor, the specific glu- cose consumption rate with regard to the cell

count us,,, could be directly determined. g-S,” gradually decreased with increasing cultivation time (Fig. 5~). The protease activity attained ex- tremely high values (maximum 24 000 EPE ml ~ ’ at 20 h) and its decrease after 20 h was moder-

ate (Fig. 5d). Therefore, the ammonium concen- tration was practically zero during the entire cultivation.

3.7. Comparison of the runs A und E

In Table 1 the process parameters of the runs A and E are compared. The difference be- tween 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 perfor- mance glucose was used as substrate and its con- centration was closed-loop controlled by fed-batch operation. Furthermore, pH- and Po2- values 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 of Bioterhtzology 49 (1996) 83-93 91

(a) 1

Z- L zo- 64

B v IO-

I i

O-

(b) 7.5 -

z TO- _;;; ’ B ‘a ,x 6.5 -

B B .5 6.0 -

5.5 -

5.0 -

cultivation time [h]

o.ooo~ * . 3 ’ 0 . c 1 4.0 4.5 5.0 5.5 6.0 6,

cultivation time [h]

250

I I I. I., I,

0 1

10 20 30 40 50

cultivation time [h]

Fig. 5. Process performance of Run E: (a) Cell count and concentrations of (dry) cell mass (CDM), (dry) sediment (SDM); (b) cell

count and in situ monitored turbidity; (c) Specific glucose uptake rate with respect to the cell count; (d) Protease activity.

without control of pH- and PO,-levels. The yield coefficients markedly increased: Y,,,,, by 141%

and Ypfs by 137%. The maximum protease activ- ity was higher by 50% and the protease productiv- ity (EPE h ~ i) by 187%.

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 en- zymes occur, which cause a deterioration of the carbon balance.

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 compo-

nents monitored by a six-channel FIA on-line

agreed well with the off-line measured data. How-

ever, 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 reduc- tion of the starch 'concentration' to avoid oxygen

limitation was not successful, and it reduced the

protease, concentration considerably.

92

Table I

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

Comparison of the process parameters of the runs A and E

Abbreviation Run A

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.

CDM

n

ii,,,

P*

P,

A,

Ss

S, S spm Sc;,,

Rs

R,

S, S Pm R Pm Y I”S

Y,V, Y ,‘,C”M

Y”p.” Yield coeff. of growth YCDM,S Yield coeff. of growth Y “,‘S

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

13.5 g lo_’

86.5 g I-’

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’

Run E

30 g I- ’ 195 X IO’” I-’

0.56 hh’

24 000 EPE ml ’ IO g 1-l

Change (‘AI)

-36”

+ 59”

+7

f50

+50

6 I-’ g ._.

2gl-’

54 1-1 g

39.0 I-’ g

2.0 I-’ g

37.0 g I-’ +28 0.19 g gg’ +I37

0.27 g g-’ +17 0.33 gg’ g 1136

0.05 X lo--” g

0.56 g g-’ +4 3.61 x IO” g-’ + 141

The yield coefficients of growth YCDM..s are related to the substrate (starch or glucose, respectively) consumed. The yield coefficients of the product are related to the substrate (starch and glucose, respectively) consumed: Y,,,, related to the protein consumed: Y,:,,,

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 concentra-

tion at about 2 g I- ’ and constant pH at 6.8 and constant non-limiting PO,-level, the protease ac-

tivity 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).

Acknowledgements

The authors gratefully acknowledge the finan- cial support of AIF (Project Nr. 8959) and DECHEMA, Frankfurt.

References

Aunstrup, K. (1979) Production, isolation and economics of

extracellular enzymes. In: Wingard, L.B., Katalski-Katzir

E. and Goldstein, L. (Eds.), Biochemistry and Bioengineer- ing, Vol. 2. Academic Press, New York, pp. 27-69.

Aunstrup, K., Andresen, 0.. Falch, E.A. and Nielsen, T.K. (1979) Production of microbial enzymes. In: Peppier. H.J.

and Perlman, D. (Eds.), Microbial Technology, Vol. I, 2nd

edn. Academic Press, New York, pp. 281-309. Bierbaum, G., Giesecke, U.E. and Wandrey, C. (1991) Analy-

sis of nucleotide pools during protease production with Bacillus lichenif’ormis. Appl. Microbial. Biotechnol. 35,

7255730.

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

Bradford, M.M. (1976) A rapid and sensitive method for the

quantification of microgram quantities of protein utilizing

the principle of protein-dye binding. Anal. Biochem. 72,

248-254.

Buhhuis, B.A., Koningstein, G.M., Stouthamer, A.H. and

Versefeld, H.W. van, (1989) A comparison between anaer-

obic growth of Bacillus licheniformis in continuous culture

and partial recycling fermenter, with contributions to dis-

cussion on maintenance energy demand. Arch. Microbial.

152, 4999507.

Dische, D. (1930) Calorimetric estimation of DNA. Micro-

chemie 8, 4-32.

Frankena, J., Versefeld, H.W. van and Stouthamer, A.H.

(1985) A continuous culture study of bioenergetic aspects

of growth and production of exocellular protease in Bacil-

lus licheniformis. Appl. Microbial. Biotechnol. 22, 169-

176.

Frankena, J., Koningstein, G.M., Versefeld, H.W. van and

Stouthamer, A.H. (1986) Effect of different limitations in

chemostat cultures on growth and production of exocellu-

lar protease by Bacillus licheniformis. Appl. Microbial.

Biotechnol. 24, 106- 112.

Frankena, J., Versefeld, H.W. van and Stouthamer, A.H.

(1988) Substrate and energy costs of the production of

exocellular enzyme by Bacillus licheniformis. Biotechnol.

Bioeng. 32, 8033812.

Giesecke, U.E.. Bierbaum, G., Rudde, H., Spohn, U. and

Wandrey, C. (1991) Production of alkaline protease with

Bacillus licheniformis in a controlled batch process. Appl.

Microbial. Biotechnol. 35, 720-724.

Hanlon, G.W. and Hodges, N.A. (1981) Bacitracin and

protease production in relation to the sporulation during

exponential growth of Bacillus lichenifbrmis on poorly

utilized carbon and nitrogen sources. J. Bacterial. 147,

427-43 1.

Hanlon, G.W., Hodges, N.A. and Russel, A.D. (1982) The

influence of glucose, ammonium and magnesium availabil- ity on the production of protease and bacitracin by Bacil-

lus licheniformis. J. Gen. Microbial. 128, 845-854. Hitzmann, B, Lammers, F., Weigel, B. and van Putten, A.B.

(1993) Die Automatisierung von FheRinjektions-Analysen- Systemen. BIOforum 16, 450-454.

Htibner, U., Bock, U. and Schtigerl, K. (1993) Production of alkaline serine protease subtilisin Carlsberg by Bacillus

licheniformis on complex medium in a stirred tank reactor. Appl. Microbial. Biotechnol. 40, 182-188.

Jiirgens, H., Kabus, R., Plumbaum, T. Weigel, B., Kretzmer, G., Schiigerl, K., Andres, K., Ignatzek, E. and Gitfhorn,

F. (1994) Development of enzyme-cartridge flow-injection analysis for industrial process monitoring. Part I. Develop- ment and characterization. Anal. Chim. Acta 298, 141-

149. Kroner, K.H. and Kula M.R. (1984) On-line measurement of

extracellular enzymes during fermentation by using mem- brane techniques. Anal. Chim. Acta 163, 3-15.

Putten, A.B. van, Spitzenberger, F., Kretzmer, G., Hitzmann, B. and Schiigerl, K. (1995) On-line and off-line monitoring

of the production of alkaline serine protease by Bacillus

licheniformis. Anal. Chim. Acta 317, 247-258. Sedmak, J.J. and Grossberg, SE. (1977) A rapid, sensitive and

versatile assay for protein using Coomassie Brilliant Blue

G250. Anal. Biochem. 79, 544-552~. Uhlig, K. (1990) Enzyme arbeiten fur uns. Carl Hanser, Mu-

nich. Ward, O.P. (1985) Proteolytic enzymes. In: Moo-Young, M.

(Ed.), Comprehensive Biotechnology, Vol. 3. Blanch,

H.W., Drew, S. and Wang, D.I.C., (Eds), The Practice of Biotechnology; Current Commodity Products. Pergamon,

Oxford, pp. 7899818. Wouters, J.M.T. and Buysman, P.J. (1977) Production of

some exocellular enzymes by Bacillus licheniformis 749/C

in chemostat cultures. FEMS Lett. 1, 1099112.