On-line analysis of yeast growth and alcohol production

Journal Elsevier

of Biorechnology,

1 (1984)

171-185

171

JBT 00113

On-line analysis of yeast growth and alcohol production W. Kuhlmann ’ lnstirur

ftir

‘**, H.-D. Meyer ‘, K.H. Bellgardt

Technische

‘*** and K. Schiigerl ’

Chemie der Universitlit Hannover, Callinstr. 3, D-30&? Regelungsrechnik der Universiliit Hannover, FAG. (Received

2 March

1984;

accepted

15 June

Hannover.

and ’ Institut

fiir

1984)

Summary

For state estimation of yeast growth and alcohol production in batch stirred-tank reactors the following process variables were measured on line: glucose (with a polarimeter or chemical reaction), orthophosphates (photometer), ammonium (ion selective electrode and NaOH/EDTA reagent), dissolved ethanol and CO, (mass spectrometer), ~0, (Clark electrode) in medium as well as CO, (IR) and O2 (paramagnetic effect) in exhaust gas. The sterile sampling procedures and transient functions of the analyzer systems are explored. The on-line evaluated data and a mathematical model were used to develop a state estimator and adaptive control, which are discussed in a separate paper. on-line analysis, Saccharomyces cereoisiae, yeast, alcohol

Introduction

On-line process analysis is a prerequisite for reliable data monitoring and computer control of bioreactors. One of the key parameters, the cell mass concentration, cannot be measured on line with sufficient accuracy. Therefore, it is usually calculated from data measured on line by an observer. However, the precise on-line measurements of other parameters are not satisfactory either. This is due partly to the lack of long-term stability of aseptic sampling devices (membrane fouling) and Present * Present l l

address: address:

0168.1656/84/$03.00

B. Braun Melsungen AG, D-3508 Gesellschaft fir Biotechnologische 0 1984 Elsevier

Science

Publishers

Melsungen, Forschung B.V.

F.R.G. mbH. D-3300

Braunschweig,

F.R.G.

172

partly to the lack of long-term stability of the instruments. Our aim was to establish reliable computer control. Therefore an extensive on-line process analyzer system was developed in our laboratory. This paper reports on some of the experiences gathered.

Materials and Methods

Succhuromyces cereuisiae LBG H 1022 was donated by the Institute of Biotechnology of ETH, Zurich. This strain was characterized in a series of excellent investigations carried out at the institute (e.g. von Meyenburg, 1969; Knoepfel, 1972; Schatzmann, 1975; Fiechter et al., 1981; Rieger, 1983). The nutrient medium composition of D 3% closely corresponded to that of Knoepfel(1972) and Schatzmann (1975) (Table 1). A stirred tank reactor (workshop Institut fti Techn. Chemie) with 3.5 1 overall and 2.5 1 working volumes (Fig. 1) was used. The medium was sterilized by filtration (Sartorius SM 162 and SM 113) and pumped into the fermenters by membrane dosing pumps (FE 211, B. Braun, Melsungen, F.R.G.). (For more experimental details, see Kuhlmann, 1983.) Process analysis

For continuous sampling of liquid medium a sterilizable filtration unit with a flat nylon microfiltration membrane (Sartorius, mean pore diameter 0.2 pm) was used. This unit was positioned close to the stirrer to avoid its mechanical fouling and from time to time it was backflushed. The necessary driving force (pressure difference) was maintained by a peristaltic pump (Cenco). Aseptic sampling.

TABLE 1 COMPOSITION

OF NUTRIENT

MEDIUM

D 3%.

The synthetic medium contains 30 g I-’ glucose and it consists of the following components per g glucose (Knoepfel, 1972; Schatzmann, 1975): PJW2SQ

(NH,),HW KC1 MgSO,.7 H,O CaCl,.2 H,O FeCIs.6 H,O ZnSO.,.7 H,O MnSO,.H,O CuS04.5 H,O m-Inosite Calcium pantothenate Vitamin B, (thiamine-HCI) Vitamin $ (pyridoxal-HCI) Biotin

150 mg 48 mg 30 mg 11.3 mg 14 mg 0.5 mg 0.3 mg 0.35 mg 0.08 mg 2mg 1 m3 0.2 mg 0.05 mg 0.001 mg

173

The sampling for mass spectrometric analysis was carried out by means of a sterilizable probe (Fig. 2) with a thin (0.1 mm) silicon film (Dipropylpolysiloxane) prepared from a silicon paste (Silopren Paste E/AC-VP 3011 transparent, Bayer AG) on a ceramic grit. The probe was connected to a four-channel mass spectrometer (GD 150/4 Varian MAT) by flexible Dekabon tubing. The volatile components were pervaporated into the vacuum, ionized in the ion source, analyzed by the magnetic field and detected by Faraday cups. Ammonium analysis.

Medium was diluted with water at a ratio of 1: 11, mixed with a reagent solution (1 N NaOH, 0.025 M EDTA) in a thermostated stirred cell and pumped through a channel along an ammonium-sensitive electrode (Philips IS-570) (Fig. 3). In the alkaline solution (pH > 11) the electrode potential depends on the

8

to vacuum -

pump

Fig. 1. Stirred tank bioreactor (first stage of the cascade). 1, sterile filtered glucose solution inlet; 2, sterile filtered nutrient solution inlet; 3, pH probe with pH control; 4, heat exchanger fastened on the four baffles; 5, temperature measurement and control; 6, aseptic mass spectrometer sampling probe for dissolved gases and volatile components; 7, connection to mass spectrometer; 8, connection to vacuum; 9, air inlet; 10, thermostate; 11, aseptic sampling probe with microfiltration membrane for autoanalyzer; 12, sampling valve with steam coverage; 13, steam supply for sampling valve; 14, liquid outlet, connection to the second stage; 15, probe for dissolved oxygen; 16, 4 N NaOH to pH control; 17, 2 N HaSO, to pH control; 18, outlet gas cooler; 19, outlet gas, connection to exhaust gas analyzers; 20, stirrer (two-stage Rushton turbine).

174

partial pressure of NH,. In Fig. 4, the calibration lines are shown. The slope exhibits long-term stability. However, the lines drifted with time due to the aging of the hydrophobic microporous teflon membranes. The analyzer was recalibrated regularly by means of the minicomputer. Phosphate analysis. A modified method of Gohla et al. (1979) was used to measure the orthophosphate concentration. One part sample solution was diluted with 10 parts of sulphuric acid-vanadate-molybdate reagent (Table 2). A yellow phosphovanadomolybdene acid was formed, the extinction of which was measured in a thermostated flow-through cuvette at 450 nm (Photometer Beckman). Zmm0

il

30mm

4

Y

1 2

-It+

T

I I I I I

I I I 1 I

I I I I

I I I I

f=%

LmmQ

3

K-0

SSmm------j(

Fig. 2. Aseptic mass spectrometer sampling probe for dissolved gas and volatile components based on pervaporation. 1, silicone membrane; 2, ceramic grit; 3, O-ring. Fig. 3. Themostated stirred cell for potentiometric analysis of ammonia. 1, two inlets: one for sample, another for NaOH/EDTA; 2, magnetic stirred slab; 3, teflon membrane; 4. outlet; 5, NH,-selective electrode and its support; 6, thermostate.

175 Glucose analysis. When the glucose concentration was high enough a 1 ml flowthrough polarimeter cuvette (241 Perkin Elmer) was used to measure the polarization angle (Yat 385 nm: a =

[ a]yJc

being the length of the cuvette and c the glucose concentration. In Fig. 5, the calibration lines are plotted for glucose. Below 2 g 1-l glucose concentration, the relative error was too high. In this concentration range, a chemical reaction of glucose (with p-hydroxybenzoe acid hydrazide, ( p-HBAH) in a slightly alkaline medium) according to Lever (1972) was used with a photometer (Beckman) which was connected to a microprocessor system based on Motorola 6802 (Fig. 6). The microprocessor sets the proper wave length for the glucose or the orthophosphate analysis. In Fig. 7, the calibration lines for glucose and orthophosphate are shown.

J

UlrnVl

If o-

-SO-

-100 -

-1sol

I

I

I

I

I

I

20

SO

100

500

10cla

2ow

> CN lmg4-9

Fig. 4. Calibration lines of the NH,-selective electrode (Philips IS-570) at 25 o C. Voltage as a function of ammonia concentration. 0.1 ml min-’ medium feed rate; 1.0 ml min-’ H,O; 0.1 ml min-’ 1 N NaOH/0.025 M EDTA.

TABLE

2

REAGENT Part

A:

Part B: Parts

SOLUTION 1 g NHoVO, added to it.

FOR

PHOSPHATE

is dissolved

20 g (NH,),MgOz4.4

A and B are mixed

in 400 ml boiling H,O

and filled

ANALYSIS

are dissolved

to 1 1.

water,

cooled

down

in 600 ml water.

and 30 ml cont.

HISO,

are

176

This photometric glucose analysis was compared to an off-line enzymatic analysis (Glut-DH method, Merckotest 3389). The response functions for concentration steps in the first stirred tank of the

cGlucasc

[g ’ 1-l 1

Fig. 5. Polarimeter calibration lines for d-glucose. Angle of rotation [a]:’ at 25 o C as a function of glucose concentration at different wave lengths h. A, pure ghtcose sohrtion; + , ghtcose in nutrient salt medium; 0, glucose in nutrient salt medium+ 5% CsHsOH; 0, glucose in nutrient salt medium+ 10% C,H,OH.

Mictoprozessorsystem with CPU MC 6602

poratlcl data connections to PDP 11/3L switches

to

sense

Fig. 6. Microprocessor

system

connected

to the photometer.

E/A,

input/output;

M, engine.

177

/caucoscIg~l-11=0.369

Fig. 7. Calibration nm. 0, d-glucose

E-O.011

lines of the photometric at 410 nm.

analysis

of phosphate

IO,&-

: I i ; t

II

-

-

and glucose.

at 450

-.-

V

IV

0. orthophosphate

-

Polorimelcr

--

Phobmcta

--.

NH,-.lcktrode

range 5 110 1.0

LV

0.2m HClOt

IV

Glucose

I 103 0.6

3 90 0.6

3091.’

POE

l.LL91”

N

2.5L9.f

In

dilution

1:2

n

dilution

1:lO

2 60 04

I?---. I

1 70 0.2

0 60

Fig.

O0

10

20

CO

60

70

8. Transient functions of analyzers with nutrient medium (output signal at a step input change). electrode signal. Ranges: , polarimeter signal; - - -, photometer signal; * - * -. , NH,-selective 2.54 g 1-l N; III: dilution 1: 2 of the medium I. V: 0.2 ml HCIO,; IV: 30 g I-’ glucose, 1.44 g I -t PO:-, with water; II: dilution 1 : 10 of the medium with water.

178

cascade are shown in Fig. 8. The new steady-state value was attained after lo-16 min. Since in each of the stirred tanks of the cascade the concentrations of these components were measured by the same analyzers, the minicomputer provided for the periodic sampling of the tanks. The length of a period was usually 20 mm. The lag time was reduced by means of sampling pumps which were controlled by the minicomputer and by the separation of analyzer flow by gas slugs. Ethanol and dissolved CO,. The sampling for the mass spectrometer was carried out by pervaporation of the volatile components. For ethanol the fragment CHzOH+ (m/e = 31) and for COz the molecule peak CO: (m/e = 44) were used. We did not succeed in using the molecule peak HzO+ (m/e = 18) as an internal standard according to Pungor et al. (1983), since the fluctuation of this peak is large. For the calculation of ethanol and CO, concentrations polynomials of degree 2 were used for each sampling tube i: E [gl-‘1

=ai[l~~“]‘+bi[I~]

+Ci

C [W saturation] = di [ &,-~]‘+f.[c”-$1 where E is the concentration

+gi

of the component, and a, b, c, d, f and C are constants.

Corrections: - amplifier zero current I,: Z = Z, - I,; - temperature correction (28-32’ C): L-0.5!% per f O.l°C for Z3,,

relative height of signal

Fig. 9. Transient function of the mass spectrometer sampling-analyzer system. Coupling of fermenter and mass spectrometer with a 2 m connection: -, polyethylene tubing 8 mm in diameter; - - -, Dekabon tube 6 mm in diameter. Concentration step E = 0 to E = 10 g I-‘. Coupling of fermeter and mass spectrometer with a 3 m Dekabon tube 8 mm in diameter: -* .-. ., concentration step E = 0 to E~6.5 g I-‘; .-.-., switch from stirred tank II, 6.5 g 1-r ethanol to stirred tank I (H,O) and to stirred tank II again.

179

f0.02% per f O.l°C for I,. - background signal Ib.$.: Z’O” = z - I,.,; - influence of salt concentration

on the signal for ethanol: +3% for medium D 3% +5% for medium D 6%; - no correction was necessary for Desmophen 3600 antifoam agent; - no correction for the stirrer speed in the range 150-1400 min-’ was necessary; - no influence of liquid film at the membrane was detected; - no correction of m/e = 31 due to the background peak m/e = 32 was necessary. The transient responses to a step input function are shown in Fig. 9. 95% of the steady-state signal level was reached within 0.5 and 5 min depending on the operating conditions. Pungor et al. (1983) reported a 3 min response time for 90% of the steady state signal level for a concentration step of ethanol from 1.5% to 2.0%. The response times of the sterilizable oxygen were in the same order of magnitude: 1 min for 98% of the steady-state level after a step change from saturation to zero level of dissolved oxygen (Bier and Ingold, 1976). Analysis of outlet gas. An infrared analyzer (Unor 6, Maihak) was used for CO, and a paramagnetic analyzer (Oxygor 3, Maibak) for 0,. For reliable measurements the following precautions were taken: - the sampling gas was cooled by a dry ice/methanol mixture to remove the residue water; - an electrical conductivity foam detector was applied to prevent foam penetration into the instrument cuvettes; - a glass-fiber filter was used to remove fine particles from the gas; and - flow meters controlled the flow rates. Fluorescence measurements. A microfluorometer developed by Beyeler et al. (1981) was used to measure the NADH concentration in the medium. A low-pressure mercury lamp was used as an excitation source for NADH fluorescence (366 nm). Calibration of the fluorescence intensity was carried out by quinine sulphate in 0.05 N sulphuric acid. The NADH concentration was measured by a modified lactate dehydrogenase method of Bergmeyer (1970). (For more details see Kuhlrnann, 1983.)

Results In Fig. 10, the concentrations of ethanol (E)*, glucose (S)*, nitrogen source (N), orthophosphate (PO:-), dissolved oxygen (0,) and dissolved CO, (C,) measured on line are shown during a batch cultivation. For the calculations of dissolved gases constant Bunsen coefficients were assumed because of their slight variation during cultivation (Table 3): when using their mean values (ao, = 0.025, aco2 = 0.64) the mean deviations were + 2.78% for ao, and - 1.0% for aCo,. The corresponding outlet gas composition and RQ values are shown in Fig. 11

180 E s 1gxr1 A

1 30

o- 20 9-

0, cFlm9d-I

8-

AA

76s- m

ioo

-10 -50

L-

-e

3-

-6 -30

t

4

-20

l-

-2

-10

,

-LO

20 tlhl

Fig. 10. Concentrations measured on line during the growth of Soccharomyces cereoisioe on D 3% nutrient medium in batch operation. E = ethanol; S = glucose (g 1-l); N = nitrogen, PO:= orthophosphate; 0, = dissolved oxygen; C, = dissolved CO, (mg 1-l).

t Ihl

Fig. 11. Respiratory quotient (RQ), during the growth of Sacchoromyces

QCOf

oxygen

consumption rate (Qo,) and CO, production rate (Qco,) cereuisiue on D 3% medium in batch operation X , RQ; 0, Q,,; q ,

181 TABLE

3

BUNSEN

COEFFICIENTS

FOR

Initial

Q2

OXYGEN

values

0.02469 0.6345

UCOl

sop AND

CARBON

Maximum

values

DIOXIDE

(ICON Final

0.02561 0.6570

values

0.02499 0.6366

and the data measured off line: cell mass concentration X, optical density OD, volume of the medium V, and the ratio K = X/OD in Fig. 12. The first exponential growth phase was completed after 9.25 h at a maximum ethanol concentration of 11.3 g 1-l. The second growth phase was completed after 19.85 h. In the first phase nonlimited growth on glucose, in the second phase nonlimited growth on ethanol prevailed. The concentrations of each of the components measured on-line can be used for process control. In Figs. 13 and 14, biological parameters are shown which were calculated from the process variables measured on-line. In Fig. 15, NADH fluorescence, the enzymatically determined NADH concentration and the ratio of fluorescence to cell mass concentration are shown. It can be recognized that fluorescence and NADH concentrations exhibited the same course. Furthermore, there is a good correlation between cell mass concentration and fluorescence intensity. A comparison of Figs. 13 and 14 with 15 indicates that the fluorescence signal was fairly closely related to the substrate '+ III 4.

t

x

i CellKkylMass

K

= Xl00

K .D

..20 ~%,6 ..19.6 ',19.4 -19.2 '49.0

'.0*4

0.2 0

1

2

3

4

5

6

7

6

9

10

11 12 13 14 15

16 17 16

19 20 t Ihl

Fig. 12. Growth of Sacchoromyces cereuisiue on D 3% medium in batch operation. Drq 1 CelI mass cell mass concentration (X) volume of liquid phase (0) and quotient (K). K = (dry concentration)/(optical density of cells).

182

Rx lg I-‘h-11 x

-f%

RE lg I-‘h-l I

1

5

10

15 I lhl

Fig. 13. Growth of .Sacc~urotnyces

rate R,, substrate consumption cereoisioe on D 3% medium

5

10

rate - R,, ethanol in batch operation.

15

rate R, during Rx: 0, - R,; 0, R,.

production X,

the growth

20 llhl

Fig. 14. Speoific growth rate (p) specific substrate consumption rate (Qs) specific ethanol production rate (v) during growth of Sacchoromyces cereuike on D 3% medium in batch operation. X, p; 0, Qs; n, Y.

183

consumption rate. A very significant signal variation occurred during the change of metabolism to another substrate, e.g. during the lag phase due to diauxy, especially during the change from aerobic to anaerobic metabolism (Fig. 16).

I s =20 t $25 =8 20

l5

10

5-l 0

2

L

6

6

10

1 11

12

16

I 16

>

I 20 tlhl

Fig. 15. Comparison of enxymatically determined NADH concentration with fluorescence intensity and fluorescence intensity with dry cell mass concentration during growth of Socckrromyces cereuisiae on D 3% medium in batch operation. 0, enxymatically determined NADH concentration. 0, ratio of fluorescence to dry ceil mass concentration ((mV- 7.5)/X). Baseline signal of the fluorimeter = 7.5 mV, X = dry cell mass concentration. -, fluorescence intensity (mv) (lower curve); -, specific fluorescence intensity (mV/cell mass) (upper curve).

9

15

27

39

51 time

[min]

Fig. 16. Variation of the fluorescence intensity during the transition of the metabolism of Saccharomycts cereuisiae from aerobic to anaerobic metabolism. 1. oxygen supply stopped; 2, 3% Oa in the outlet gas; 3. 1% 0, in the outlet gas; 4, oxygen supply on.

184

The combination of the fluorescence signal with other process variables measured on line can be used for process control. This on-line analysis system is used to evaluate the data necessary for the development of a structured model for the growth of S. cereoisiae and ethanol production in batch and continuous cultures (Bellgardt, 1983). By means of this model simulations of cell growth and ethanol production were carried out and compared with literature data. A reduced version of this model was implemented on a minicomputer (Meyer, 1983) and the cell growth and ethanol production were predicted on line by means of a state estimator. The model, the simulations and the state estimator are described in separate papers. Symbols

concentration CO, concentration E ethanol concentration KF ratioX toOD dissolved oxygen concentration g:, specific 0, consumption rate QCol specific CO, production rate Rx growth rate Rs substrate utilization rate respiratory quotient substrate concentration I time T temperature V, medium volume X dry cell mass concentration specific growth rate P C

c

g l-‘, mol 1-l mg 1-l g 1-l g 1-l mg 1-l mm01 g-’ h-’ mm01 g-’ h-’ g l-’ h-’ g l-’ h-’ g 1-l h OC 1 g 1-l h-’

Acknowledgements

The authors gratefully acknowledge the financial support of the Stiftung Volkswagenwerk and thank Professor H. Diekmann, Institut fur Mikrobiologie, and Professor M. Thoma, Institut fti Regelungstechnik, Universitat Hannover, for their good cooperation. References Bellgardt, K.H: (1983) Modellbildung des Wachstums von Succhoromyces cereuisicre in Riihrkesselreaktoren. Bergmeyer, H.H. (1970) Methoden der enzymatischen Analyse, Vol. 2. Verlag Chemie, Weinheim.

185 Beyeler, W., Einsele, A. and Fiechter, A. (1981) On-line measurements of culture fluorescence: method and application. Eur. J. Appl. Microbial. Biotechnol. 13, 10-14. Buhler, H. and fngold, W. (1976) Measuring pH and oxygen in fermentors. Process B&hem. 11, 19-23. Fiechter, A., Fuhrmann, G.F. and Kappeli, 0. (1981) Regulation of glucose metabolism in growing yeast cells. Adv. Microbial Physiol. 22, 123-183. Gohla, W., Nielen, H.D. and Sorbe, G. (1979) Verfahren zur automatischen Schnellbestimmung von Phosphaten bei der Waschmittel-Herstellung und der Fertigproduktkontrolle. GIT Fachz. Lab. 23, 89-86. Knopfel, H.P. (1972) Zum Crabtree-Effect bei Succharomyces cereuisiae und Candida fropicalis. Diss. No. 4906, ETH Zurich. Kuhlmann, W. (1983) Untersuchungen zur on-line Proxesslenkung der satxweisen turd kontinuierlichen Kultivienmg von Soccharomyces cereoisiae. Diss. Universitlt Hannover. Lever, M. (1972) A new reaction for calorimetric determination of carbohydrates. Anal. Biochem. 47, 273-279, Von Meyenburg, K. (1969) Katabolit-Repression und Sprossungszyklen von Snccharomyces cereuisioe. Diss. No. 4279, ETH Zurich. Meyer, H.D. (1983) Prozessrechnereinsat zur Ubenvachung und Regelung ein- und mehrstufiger Bioreaktoren. Dissertation Universitlt Hannover. Pungor, E., Schaefer, E.J., Cooney, C.L. and Weaver, J.C. (1983) Direct monitoring of the liquid and gas phases during fermentation in a computer-mass spectrometer-fermenter system. Eur. J. Appl. Microbial. Biotechnol. 18, 135-140. Rieger, M. (1983) Untersuchungen zur Regulation von Glycolyse turd Atmung in Succharomyces cereuisiue. Diss. No. 7264, ETH Zurich. Schatxmarm, H. (1975) Anaerobes Wachstum von Succharomyces cereuisiae. Diss. No. 5504, ETH, Zurich.