Biotechnology : Measuring, Modelling, and Control / Karl Schugerl

Contents 1 Common Instruments for Process 12 Analysis and Control 5 6 Determination of Cell Concentration and Characterization of Cells 179 IV.. The measuring techniques are subdivided

Special Processes Volume 11 Environmental Processes Volume 12

Patents, Legislation, Information Sources, General Index

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British Library Cataloguing-in-Publication Data:

Biotechnology Second Edition

Biotechnology: Vol 4 Measuring, modelling and

control

Vol Ed Schiigerl, K

620.8

ISBN 3-527-28314-5

Die Deutsche Bibliothek - CIP-Einheitsaufnahme

Biotechnology : a multi volume comprehensive treatise / ed by

H.-J Rehm and G Reed In cooperation with A Piihler and P

Stadler - 2., completely rev ed - Weinheim; New York;

Basel; Cambridge: VCH

NE: Rehm, Hans J [Hrsg.]

2., completely rev ed

Vol 4 Measuring, modelling, and control / ed by K Schiigerl

- 1991

ISBN 3-527-28314-5 (Weinheim)

ISBN 1-56081-154-4 (New York)

NE: Schiigerl, Karl [Hrsg.]

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Preface

In recognition of the enormous advances in

biotechnology in recent years, we are pleased

to present this Second Edition of “Biotech-

nology” relatively soon after the introduction

of the First Edition of this multi-volume com-

prehensive treatise Since this series was ex-

tremely well accepted by the scientific commu-

nity, we have maintained the overall goal of

creating a number of volumes, each devoted to

a certain topic, which provide scientists in

academia, industry, and public institutions

with a well-balanced and comprehensive over-

view of this growing field We have fully re-

vised the Second Edition and expanded it from

ten to twelve volumes in order to take all re-

cent developments into account

These twelve volumes are organized into

three sections The first four volumes consider

the fundamentals of biotechnology from bio-

logical, biochemical, molecular biological, and

chemical engineering perspectives The next

four volumes are devoted to products of indus-

trial relevance Special attention is given here

to products derived from genetically engi-

neered microorganisms and mammalian cells

The last four volumes are dedicated to the de-

scription of special topics

The new “Biotechnology” is a reference

work, a comprehensive description of the

state-of-the-art, and a guide to the original

literature It is specifically directed to micro-

biologists, biochemists, molecular biologists,

bioengineers, chemical engineers, and food

and pharmaceutical chemists working in indus-

try, at universities or at public institutions

A carefully selected and distinguished Scien-

tific Advisory Board stands behind the series

Its members come from key institutions repre-

senting scientific input from about twenty

countries

The present volume, fourth in the series, re- flects the enormous impact of computer tech- nology on biotechnology, especially in the areas of measurement and control It describes monitoring of the biotechnological process with sophisticated analytical techniques, use of the resulting data by means of mathematical models, and computer-aided closed loop con- trol for improvement of the productivity of biotechnological processes While Volume 4

can be used independently, Volume 3 “Bio-

processing” is recommended as a companion volume

The volume editors and the authors of the individual chapters have been chosen for their recognized expertise and their contributions to the various fields of biotechnology Their will- ingness to impart this knowledge to their col- leagues forms the basis of “Biotechnology” and is gratefully acknowledged Moreover, this work could not have been brought to fru- ition without the foresight and the constant and diligent support of the publisher We are grateful to VCH for publishing “Biotechnolo- gy” with their customary excellence Special thanks are due Dr Hans-Joachim Kraus and Christa Schultz, without whose constant ef- forts the series could not be published Finally, the editors wish to thank the members of the Scientific Advisory Board for their encourage- ment, their helpful suggestions, and their con- structive criticism

G Reed

A Puhler

P Stadler

Scientific Advisory Board

August Kirchenstein Institute of Microbiology

Latvian Academy of Sciences

Biochemical Engineering Research Centre Indian Institute of Technology

Weizmann Microbial Chemistry Laboratory

Department of Chemistry

Manchester, UK

Prof Dr C L Cooney

Department of Chemical Engineering

Massachusetts Institute of Technology Alimentaire

Cambridge, MA, USA

Department of Applied Microbiology The Hebrew University

Prof Dr G Goma

Departement de Genie Biochimique et Institut National des Sciences Appliquees Toulouse, France

Institut fur Biotechnologie

Eidgenossische Technische Hochschule

Zurich, Switzerland

Prof Dr D A Hopwood

Department of Genetics John Innes Institute Norwich, UK

Prof Dr E H Houwink

Organon International bv Scientific Development Group Oss The Netherlands

Center for Molecular Bioscience and Biotechnology

Lehigh University Bethlehem, PA, USA

Department of Plant Sciences

University of Western Ontario

London, Ontario, Canada

Institute of Molecular and Cell Biology National University of Singapore Singapore

Prof Dr E.-L Winnacker

Institut fur Biochemie Universitat Munchen Miinchen, Germany

Prof Dr H Sahm

Institut fur Biotechnologie

Forschungszentrum Julich

Julich, Germany

Contents

1 Common Instruments for Process 12

Analysis and Control 5

6 Determination of Cell Concentration

and Characterization of Cells 179

IV Control and Automation

16 Control of Bioreactor Systems 509

S Shioya, K.-I Suga

19 Expert Systems for Biotechnology 625

A Halme, N Karim

Index 637

9 Cell Models 267

K -H Bellgardt

Contributors

Dr Graham F Andrews

E G & G

Idaho National Engineering Laboratory

Idaho Falls, ID 83415, USA

Prof Dr Aarne Halme

Automation Technology Laboratory Helsinki University of Technology Electrical Engineering Building Otakaari 5A

CH-8092 Zurich, Switzerland

Chapter 2

Prof Dr Karl-Heinz Bellgardt

Institut fur Technische Chemie

D-3000 Hannover 1, FRG

Chapters 9 and 12

Prof Dr Nazmul Karim

Department of Agricultural and

Fort Collins, CO 80523, USA

Chapter 19

Dr Irving J Dunn

Biological Reaction Engineering Group

Chemical Engineering Department

Eidgenossische Technische Hochschule (ETH)

Dr Sun Bok Lee

Pohang Institute of Technology

Pohang, Korea

Chapter 15

Prof Dr Henry C Lim

Biochemical Engineering Program

University of California

Irvine, CA 92717, USA

Chapter 16

Priv.-Doz Dr Andreas Liibbert

Institut fur Technische Chemie

Prof Dr Jose C Merchuk

Department of Chemical Engineering

Program of Biotechnology

Ben-Gurion University of the Negev

Beer Sheva, Israel

Chapter I 1

Prof Dr Axel Munack

Institut fur Biosystemtechnik Bundesforschungsanstalt fur Landwirtschaft Bundesallee 50

Prof Dr Matthias Reuss

Institut fur Bioverfahrenstechnik Universitat Stuttgart

Boblinger StraRe 72 D-7000 Stuttgart 1, FRG

Chapter I0

Prof Dr Dewey D Y Ryu

Department of Chemical Engineering University of California

Davis, CA 95616, USA

Chapter I5

Priv.-Doz Dr Thomas H Scheper

Institut fur Technische Chemie Universitat Hannover

CallinstraRe 3 D-3000 Hannover 1, FRG

Chapter 6

Prof Dr Karl Schiigerl

Institut fur Technische Chemie

Prof Dr Suteaki Shioya

Department of Fermentation Technology

Faculty of Engineering

Osaka University, Suita

Osaka 565, Japan

Prof Dr Gregory Stephanopoulos

Department of Chemical Engineering

Massachusetts Institute of Technology

Cambridge, MA 02139, USA

Chapter 7

Prof Dr Ken-ichi Suga

Department of Fermentation Technology Faculty of Engineering

Osaka University, Suita Osaka 565, Japan

Prof Dr Christian Wandrey

Institut fur Biotechnologie 2

Forschungszentrum Julich Postfach 1913

Chapter 12

Introduction

Hannover, Federal Republic of Germany

The fourth volume of the second edition of

“Biotechnology” presents a survey on an in-

creasingly important field of biotechnology:

monitoring of the biotechnological process

with sophisticated analysis techniques, use of

the resulting data by means of mathematical

models, and (computer-aided) closed loop con-

trol for improvement of the productivity of

biotechnological processes

The bottleneck in biotechnological process

control is the on-line measurement of con-

trolled process variables Except for tempera-

ture, impeller speed (for stirred tank reactors),

aeration rate (for aerobic microorganisms),

pH, po,, which are usually controlled process

variables, and the composition of the outlet

gas (0, and C 0 2 content), which sometimes is

a controlled process variable (respiration quo-

tient, RQ, C 0 2 production rate, O2 consump-

tion rate), no other process variables are usual-

ly measured on-line in commercial equip-

ment

However, manufacturers have recently

made great efforts to improve process analysis

and control In several laboratories, on-line

systems for the analysis of the chemical me-

dium composition are used to gain more infor-

mation about the process and to control the

concentrations of key components Further-

more, mathematical models have been devel-

oped in order to describe the production proc-

ess and to effect process optimization and con- trol Therefore, the Series Editors decided to add to Biotechnology a separate volume with

the title “Measuring, Modelling, and Con- trol”, and they asked the Volume Editor to or- ganize it

This volume consists of four main parts:

“Modelling, design, and control” of down- stream processes are also considered because

of the great importance of downstream proc- essing However, because of their broad scope, they are too heterogeneous and not yet suffi- ciently developed for treatment in the same way as processes for growth and product for- mation

The instruments are subdivided into three groups:

0 common instruments for medium analy- sis,

0 instruments for gas analysis, and

0 biosensors

The last group of instruments is still being de-

veloped

Only instruments that are (or can be) used

for process control are considered in detail

However, modern off-line techniques (e.g.,

NMR) are also taken into account

The measuring techniques are subdivided

into four groups:

0 physical techniques for the characteriza-

tion of fluid dynamics,

0 chemical methods for the analysis of

broth composition,

0 physical methods for the determination

of cell concentration, and

0 physical/biochemical methods for the

characterization of the biological state

of the cells

Most of these are on-line techniques; others

can only be carried out in a quasi on-line

mode All of them are used to characterize the

reactor/medium/cell system

There are interesting new developments in

the characterization of such systems by mod-

ern mathematical methods, with optimization

of sampling to gain maximal possible informa-

tion These new techniques are also included in

0 aerobic wastewater treatment,

0 anaerobic wastewater treatment, and

0 models for recombinant microorgan-

isms

Models for animal and plant tissue cultures have not yet been included because no reliable kinetic data are available for mathematical modelling of these cultures

Modern control techniques are increasingly applied to closed loop control of bioreactor systems Therefore, different types of closed loop control techniques, including computer- aided control, are considered in detail Instrumental control is much more reliable than control by a human operator Further- more, long-range (many weeks or months) runs are only possible in the laboratories of re- search institutes and universities if automated equipment is used Thus, automation of bio- reactors is also taken into account

Chapter 18 on modelling, design, and con-

trol of downstream processing covers only the most important downstream processes Devel- opment in this field is still limited, so this chapter is not as extensive as the potential im- portance of downstream processing would warrant

Expert systems are being developed in dif- ferent aspects of technology, medicine, and the natural sciences Their use in biotechnolo-

gy is desirable, since they would permit the identification of equipment failures and their eventual elimination Furthermore, by means

of expert systems, large amounts of informa- tion from on-line and off-line measurements

as well as from the literature and from heuris- tic knowledge can be used with high efficiency The reviews consider only the most impor- tant techniques and omit some detail because

of limited space Further information can be gained through the reference notations

It is hoped that information given in this volume will help students, engineers, and scientists at universities, members of research institutes, and those in industry to increase their knowledge of this important and fast- growing field

Hannover, March 1991 K Schiigerl

I Instruments

1 Common Instruments for Process Analysis and Control

2.2 Dissolved Oxygen Partial Pressure, po, 7

2.3 Redox Potential, Eh, and Dissolved C 0 2 Partial Pressure, pcO2 9

4.4 Dissolved COz Partial Pressure, pCo2

3 Instruments for Determination of Physical System Properties 10

6 I Common Instruments f o r Process Analysis and Control

1 Introduction

Biological processes are influenced by sev-

eral control variables: temperature, pH, dis-

solved oxygen partial pressure p o 2 , as well as

by state variables such as redox potential, Eh,

and dissolved C 0 2 partial pressure, pco2,

which have a direct influence on cell metabo-

lism (FORAGE et al., 1985)

Other control variables (power input, aera-

tion rate) and state variables (liquid viscosity)

have an indirect effect on cell growth and

product formation They influence gas disper-

sion (bubble size, gas holdup, and specific in-

terfacial area) and the transport processes in

the broth The broth volume can be deter-

mined by means of the liquid level and the

holdup In continuous cultivation, the residence

time of the broth or its dilution rate, which

equals the specific growth rate of the cells, is

determined by the liquid throughput and broth

volume

With highly foaming broth the cells are

sometimes enriched in the foam by flotation

The diminution of the cell concentration in the

broth reduces cell growth and product forma-

tion rates Foam may be carried out of the

reactor by the air flow and then may clog the

gas analysis instruments and cause infection of

the broth Therefore, the foam detector be-

longs to the standard equipment of bioreac-

tors First, the use of these instruments for

process analysis, optimization, and control is

2.1 Temperature and pH

Cells have an optimum temperature and p H for growth and frequently another optimum for product formation Several authors have considered the calculation of the optimum temperature and p H profiles for product for- mation

FAN and WAN (1963) used the discrete maximum principle to calculate the optimum temperature and p H profiles for a continuous multistage enzymatic reactor to maximize product concentration

BOURDARD and FOULARD (1 973) consid- ered the optimization of yeast production in a batch process by means of optimum tempera- ture and p H profiles using the continuous maximum principle

SPITZER (1976) used a grid search method with subsequent steepest descent to maximize biomass productivity in a continuously oper- ated bioreactor by optimizing pH and sub- strate profiles

RAI and CONSTANTINIDES (1973) and CON-

STANTINIDES and RAI (1974) investigated the

production of gluconic acid with Pseudomo-

nus ovalis and of penicillin G by Penicillium

chrysogenum in batch operation and used the

continuous maximum principle to maximize the productivity by means of optimal tempera- ture and pH profiles

CONSTANTINIDES et al (1970) and KING et

al (1974) studied the production of penicillin

G by P chrysogenum in batch operation and

used the continuous maximum principle and/

or a specific optimal control to evaluate the optimum temperature profile for achieving maximum productivity ANDREYEVA and BIRYUKOV (1973) also investigated the batch production of penicillin G and used the contin- uous maximum principle to find the optimum

p H profile for maximum productivity

BLANCH and ROGERS (1972) maximized

profit in gramicidin S production by Bacillus

brevis by evaluating the optimum temperature

and pH as well as the number of stages using

the discrete maximum principle

Erythromycin biosynthesis in batch opera-

tion was maximized by CHERUY and DURAND

(1979) by evaluating optimal temperature and

pH profiles

On the other hand, the pH variation can be

used to control the production process PAN et

al (1972) reported on penicillin production

where carbohydrate and nitrogen source feed

rates were controlled by measurement of the

pH The nitrogen source was metabolized to

basic cations and the carbohydrate source to

CO, and organic acids The balance of the two

ingredients provided a basis for the pH con-

trol

ANDREYEVA and BIRYUKOV (1973) pro-

posed a model for this pH effect and its use

for calculating optimal fermentation condi-

tions CONSTANTINIDES (1979) reviewed these

publications SAN and STEPHANOPOULOS

(1984) also proposed a relationship between

the total rate of biomass growth and ammonia

addition to the reactor for pH control

ROSEN and SCHOGERL (1984) used the con-

sumption of sodium hydroxide solution at a

constant pH to calculate the cell mass produc-

tion rate of Chaetomium cellulolyticum and to

control the substrate feed by means of a mi-

croprocessor in a fed-batch biomass produc-

tion process SHIOYA (1988) developed an ad-

vanced pH control system for the measure-

ment of biological reaction rates

In many microbial or cell culture systems

the pH varies during growth Acid or base

must be added to the broth to keep the pH at

the optimal value In some enzymatic hydro-

lytic reactions acid or base must also be used

to keep the pH constant by neutralizing the

produced acid or base From the amount of

acid or base required for keeping the pH con-

stant, the growth rate or enzyme reaction rate

can be calculated

If ApH(k+ 1) and ApH(k) are the differ-

ences of pH from the set point at times k + 1

and k, F(k ) is the feeding rate of acid or base

for pH control, and R(k) is acid or base pro-

duction or consumption rate, then Eq (1) can

be used to calculate R(k):

A pH(k + 1) = A pH(k) + aF(k) - bR(k) (1)

where a is a coefficient corresponding to the

pH deviation caused by adding a unit amount

of acid or base to the broth, and b is a coeffi- cient that corresponds to the pH deviation caused by the formation of a unit amount of cell mass or product

If the pH is kept constant, i.e.,

A pH(k + 1) = A pH(k) = 0, then the acid or base production or consumption rate R(k) can

be evaluated from

This principle was applied to

0 baker’s yeast production using a distur- bance predictive controller to determine the growth rate of the yeast,

0 determination of the reaction rate of N- acetyltyrosine ethyl ester (ATEE) with a-

chymotripsin to give ethanol and N-ace- tyltyrosine (AT) in a pH stat using a re- peated feedforwardlfeedback controller (repeated P F system),

0 determination of the overall production rate of (lactic) acid in hybridoma culture

Rhodotorula glutinis by means of the pH of

the medium The enzyme was excreted only in the pH range 4.5 to 6.5

Several microorganisms produce different

metabolites depending on the pH Thus, As-

pergillus niger produces citric acid in the pH

range from 2.5 to 3.5, whereas gluconic acid is produced at a higher pH and oxalic acid in the neutral pH range (SCHLEGEL, 1974)

Pressure, p o ,

Dissolved oxygen pressure or concentration

is a state variable widely used to calculate the biomass concentration by 0,-balancing and to

8 I Common Instruments for Process Analysis and Control

control the growth or production process of

aerobic microorganisms

Furthermore, Po,-electrodes are used as re-

search tools for determining oxygen transfer

rates (OTR) in pioreactors, biofilms, pellets,

and cells immobilized in beads

The use of oxygen balancing for real-time

estimation of the biomass concentration was

recommended first by HOSPODKA (1966) ZA-

BRISKIE and HUMPHREY (1978) worked out

this technique of observation in detail Using

the relationship between oxygen uptake rate

(OUR), the yield coefficient of the cell growth

with regard to the oxygen consumption,

Yx/02, the maintenance coefficient with regard

to the oxygen consumption, moZ/X, and the

where X, is the initial biomass concentration

Eq (4) forms the basis for estimating the

biomass concentration X(t) from the oxygen

uptake rate

The growth rate may be approximated using

Eqs (3) and (4):

With this method the biomass concentration

of Saccharomyces cerevisiae was estimated

Several other authors used this technique in

combination with the respiration coefficient,

RQ, to estimate the biomass (e.g., COONEY et

al., 1977; WANG et al., 1977; PERINGER and

BLACHERE, 1979; TAKAMATSU et al., 1981)

SQUIRES (1972) reported that po2 was used to

control the sugar addition to the broth of

Penicillium chrysogenum during penicillin pro-

duction The sugar feed was increased at a

high po2 value; at a low po2 it was reduced

Since a close relationship exists between oxy-

gen and substrate uptake rates, this control of the substrate feed is very popular

Under steady-state conditions, the oxygen uptake rate, OUR, and the oxygen transfer rate, OTR, are identical Knowing the driving force for the oxygen transfer, (0, - OZ), the volumetric mass transfer coefficient, KLa, can

be calculated:

0 TR

KLa =

(02 - 02) where 0, and 03 are the concentrations of the

dissolved oxygen in the bulk and at the inter- face (in equilibrium with the gas phase) By measuring the oxygen balance during cell culti- vation, the volumetric mass transfer coeffi- cient can be calculated in real time

In cell-free systems, KLa can be determined

by non-stationary or stationary measurements The non-stationary method is based on the re- lationship :

evaluated from Eq (7) However, the interre- lationships between sorption rate and driving force are in practice more complex Several re- lationships have been recommended for this calculation

A good review of these methods is given in a

‘Report of a Working Party on Mixing’ of the European Federation of Chemical Engineering (LINEK and VACEK, 1986) and in the review article of LINEK et al (1987)

Several papers consider the mass transfer of dissolved oxygen into biofilms, pellets, and cells immobilized in beads The dissolved oxygen concentration profiles are determined

by means of micro-oxygen electrodes (BUN-

GAY and HAROLD, 197 1 ; CHEN and BUNGAY, 1981; BUNGAY and CHEN, 1981; BUNGAY et al., 1969, 1983; WITTLER et al., 1986)

The combination of Eqs (10) and (11) gives

2.3 Redox Potential, Eh, and

Dissolved C 0 2 Partial Pressure,

Pcoz

The oxidation and reduction of a compound

is controlled by the redox potential of its envi-

ronment

The oxidation-reduction potential of a pair

of reversible, oxidizable-reducible compounds

is related to the equilibrium between the oxi-

dized (ox) and reduced forms (red) and the

number of electrons involved in the reaction

(ne-) (KJAERGAARD, 1977; KJAERGAARD

and JOERGENSEN, 1979; THOMPSON and GER-

SON, in KJAERGAARD, 1977):

The redox potential of this reaction is given by

the Nernst equation:

R T activity of ox

E h = E o + - In

n F activity of red

where Eh is the redox potential referred to the

normal hydrogen electrode,

Eo is the standard potential of the sys-

tem at 25 "C, when all activities of

any reactants are at unity,

R the gas constant,

T the absolute temperature,

n the number of electrons involved in

the reaction,

F the Faraday constant

(9)

JOERGENSEN (1941) introduced a concept ana-

logous to the pH, namely the rH, which is de-

fined as

where aH2 is the activity of hydrogen in the hy-

drogen-hydrogen ion redox system according

to Nernst For hydrogen

The redox potential is used in practice for microaerobic cultivations, i.e., at very low dis- solved oxygen concentrations, which cannot be measured by standard oxygen electrodes An

example is the production of exoenzymes by Bacillus amyloliquefaciens in continuous cul- ture at 0.5% oxygen saturation by means of

redox-potential control (MEMMERT and WAN- DREY, 1987)

In small stirred tank reactors, the dissolved

COz concentration in the broth can be calcu- lated from the gas composition by assuming an equilibrium between the phases In tower reac- tors and large commercial units, no equili- brium distribution of COz exists between the

phases; therefore, the direct measurement of

pco2 can be useful

The driving force, (Pco2-pEo2), can be evaluated from the calculated pEo, at the in- terface and the measuredpco2 in the bulk The

CO, production rate, CPR, can be determined

from the evolved gas stream and the gas com- position

The volumetric mass transfer coefficient of the C 0 2 desorption is given by

CPR (KLa)co2 =

(Pco2-PEo2) CPR can also be used for the calculation of the cell mass concentration and the specific growth rate, ,u The instantaneous specific growth rate of Penicillium chrysogenum was

calculated by Mou and COONEY (1983) by

measuring the CPR during the growth phase

By monitoring the O2 and/or COz concen-

trations in the outlet gas and its flow rate, O2

and/or CO, balances can be calculated and

used for state estimation of biochemical reac-

10 I Common Instruments for Process Analysis and Control

tors (e.g., STEPHANOPOULOS and SAN, 1982)

However, because this state estimation method

is based on measurements of the gas composi-

tion, it will be discussed in Chapter 2

3 Instruments for

Determination of Physical

System Properties

3.1 Temperature

Temperature is the most important control

variable for most biotechnological processes,

including sterilization as well as cell growth

and product formation In general, a precision

of f0.5 "C is necessary in the temperature

range from +20 to + 130 "C Only a few types

of the various industrial thermometers are

suitable because of this prerequisite (BUSING

and ARNOLD, 1980) Most popular are the Pt-

100 (100 ohm at 0 "C and 123.2 ohm at 60 "C)

resistance thermometers which are encased in a

protective steel tube fixed with a sealing com-

pound of high heat conductivity

According to DIN 43 760 (German Stand-

ard) the resistance of these instruments is guar-

anteed with the following precision: 1OOf 0.1

ohm at 0 "C, which corresponds to an error of

k 0.26 "C Therefore, these resistance ther-

mometers can be used without calibration

However, the resistances of all electrical con-

nections must be controlled These instruments

are steam-sterilizable at 121 "C Thermometers

with short response times for fluid dynamical

measurements are described in Chapter 4

3.2 Pressure

The absolute pressure is measured with re-

spect to zero pressure Gauge pressure is meas-

ured with respect to that of the atmosphere

The SI unit of the pressure is Newton per

square meter (N/m2) called Pascal (Pa)

(1 bar=0.1 M P a = 10' N/m2; 1 mbar = 100

P a = 100 N/m2.) Bar and millibar deviate with

less than 2% from the technical and physical atmosphere

Pressure measurements are necessary for the control of the sterilization and the state of the outlet gas filter as well as for the evaluation of the holdup and the partial pressures of the ga- seous components in the gas and liquid phases Membrane pressure gauges are commonly used

in biotechnology, because they are particularly suited to aseptic operations Numerous pres- sure gauges are used in the chemical industry (HIRTE, 1980; ANDREW and MILLER, 1979)

In biotechnology, the commonly employed pressure gauges are based on strain and/or capacitance measurements The capacitance pressure gauges can measure very small pres- sure differences; therefore, they are used for liquid level measurements For the construction

of the different pressure meters, see HIRTE (1980) and ANDREW and MILLER (1979)

3.3 Liquid Level and Holdup

Measurement of the liquid volume is impor- tant for filling bioreactors with nutrient solu- tions, for continuous and for fed-batch culti- vations It can be performed (OEDEKOVEN, 1980; ELFERS, 1964; ANDREW and RHEA, 1970) as follows:

0 by measuring the hydrostatic pressure difference between the bottom of the reactor, P b , and the head space, P h , by means of pressure gauges The pressure difference is proportional to the weight

of the liquid in the reactor:

where h is the liquid height above the

p the density of the broth, and

g the acceleration of gravity,

0 by measuring the total weight of the

bottom,

reactor by load cells The accuracy of the volume measurement is +0.2% for large reactors and + 1% for laboratory reactors

The measurement of the volume of an aer- ated broth is accomplished with a level con-

troller The common liquid level meters are

based on the variation of the capacitance C of

the sensor with the composition of the dielec-

tricum For plate condensers, the capacitance

is given by

A

C= E O E , -

d

where A is the area of the plates,

d the distance between the plates,

E o the absolute dielectric constant of

vacuum, and

E, the relative dielectric constant of the

aerated broth between the plates

The value of E, of the broth and of the air dif-

fer by a factor of about 80 In the case of non-

aerated broth the capacitance of the condens-

er, C, is given by

where Co is the capacity of the condenser

with air,

AC the capacity difference due to

broth per unit height, and

h the height of the liquid in the ca-

pacitor

The capacity of the condenser with aerated

broth is given by

where E is the gas holdup in the aerated broth

Analogous relationships hold true for cylindri-

cal condensers (OEDEKOVEN, 1980)

The accuracy of the level control amounts

to * 2-4% depending on the uniformity of the

liquid level In the case of large reactors, the

level variation can be extremely large There-

fore, only the level of the broth can be meas-

ured in the reactor, not that of the aerated

broth, which is measured outside of the reac-

tor, e.g., in a non-aerated section Also in the

case of foam formation, the measurement of

the aerated broth level by capacitance instru-

ments becomes difficult Under these condi-

tions floating bodies can be used as level con-

trollers

Since cultivation broths have adequate elec- trical conductivity, the liquid level can also be measured by inexpensive electrical conductivi-

ty probes Their application is restricted to aqueous broths In the presence of a second (organic) liquid phase, their application cannot

in Chapter 2 Special techniques for measure-

ments of local liquid velocities are treated in

Chapter 4

Of the large number of available instru- ments ( S C H R ~ D E R , 1980; ANDREW et al., 1979; ERICSON, 1979) only three types are im-

portant in biotechnological practice:

- floating body flowmeters,

- differential pressure flowmeters, and

- magnetic-inductive flowmeters

The floating body flowmeter or rotameter consists of a conical tube and a floating body with the upper diameter D,, mass M,, and den-

sity ps (Fig 1) In the upstreaming fluid, the lifting force, which is produced by the differ- ential pressure across the slot between the tube wall and the floating body, is balanced by the weight of the floating body minus its buoyan-

cy The position of the float is a function of the flow rate and the density of the fluid, p

The volumetric throughput q v is given by

The flow coefficient a is a function of the Rey-

nolds number and the diameter ratio Dk/D,,

where Dk is the diameter of the tube at the up-

per edge of the floating body

12 I Common Instruments for Process Analy> pis and Control

reading

.floating

mass M

body density e ,

Fig 1 Floating body flowmeter ( S C H R ~ D E R ,

1980)

Calibration of q v is necessary because of the

nonlinear relationship between the position of

the floating body and the throughput It can

be carried out with water or air and recalcu-

lated for the nutrient medium with known den-

sity by means of the a-Ru diagram, where

the Ruppel number

depends only on the instrument constants and

fluid properties, but not on the throughput

The accuracy of rotameters is between k 1

and k 3 % depending on the ratio qv/qV,max

( S C H R ~ D E R , 1980)

Differential pressure flowmeters consist of a

tube with a restriction (usually an orifice

plate) The pressures p 1 and p z upstream and

downstream of the orifice are measured The

p the density of the fluid,

D the tube diameter,

q the dynamic viscosity of the fluid,

and

w the mean flow rate of the fluid

the orifice-to-tube diameter ratio d / D for

smooth tubes

In practice, standardized orifices are used for which the flow coefficients are given in diagrams The accuracy of calibrated orifice

flowmeters is f0.5'70 of qv,max

According to the induction law of Faraday,

an electrically conductive liquid passing a mag- netic field induces a voltage between two elec- trodes positioned perpendicular to the direc- tion of the flow The voltage is proportional to the flow velocity:

where U is the induced voltage,

B the magnetic induction,

D the tube diameter, and

w the mean liquid velocity

The volumetric throughput q v is given by

Magnetic-inductive flowmeters are fairly ex- pensive However, they have important advan- tages:

- the voltage U is proportional to qv,

- they are independent of the density and viscosity of the fluid as well as of the velocity profile of the fluidin tubes,

- they d o not produce a pressure drop,

- they do not have moving parts,

- they can be used for suspensions,

- they can be steam-sterilized

Their accuracy is k 1% at qv,max, and

k 1.5% at 0.5 qv.max

The flow coefficient a is usually given as a

function of the orifice Reynolds number and

3.5 Power Input

In an agitated reactor, the power input, P,

can be calculated by Eq (23) by measuring the

torque on the shaft, MN, and the speed of ro-

tation, N

The torque is measured by torsion dynamome-

ters or strain gauges and the impeller speed by

an electronic tachometer In large-scale reac-

tors, the consumed electrical energy, as meas-

ured by the wattmeter, yields useful data on

power input, if the mechanical losses in gear,

seals, etc., are taken into account

In small laboratory reactors, the mechanical

losses are considerable in comparison with the

power input into the broth Therefore, power

input measurements are inaccurate and are not

recommended

In bubble columns, P can be calculated by

where MG is the gas mass flow,

R the gas constant,

T the absolute temperature,

pin the gas pressure at the column

However, since the second and third terms to-

gether make up only 0.2% of the overall pow-

er input, the power input due to the gas expan-

in the reactor is high, improvement of heat transfer is also important to keep the tempera- ture constant

High viscosity can be caused by high sub- strate concentration (e.g., starch), high prod- uct concentration (e.g., xanthan), high cell concentration (e.g., penicillin), high solid con- tent (e.g., peanut flour), or by their combina- tion The most general description of the rheo- logical properties of fluids is given by the rela- tionship between the velocity gradient dv/dx and the stress, T, the so-called flow equation:

dv

- = f (7)

dx

as long as viscoelastic behavior is not present

or very slight This flow equation can be calcu- lated from the experimentally measured shear diagrams (shear rate versus shearing stress) It should be noted, however, that such a calcula- tion is not always possible In contrast to the shear diagram, the flow equation is indepen- dent of the experimental conditions (e.g., the type of viscosimeter) used for the determina- tion of the viscosity

There are many methods available to esti- mate the rheological behavior of fluids, but there are only a few that furnish true fluidity values These include the capillary, the falling sphere, the Couette, the Searle, and the tor- sional pendulum methods Until now, the eval- uation of the flow equation from the shear diagram has only been possible for the capil- lary, Couette, and Searle methods (MUSCHEL-

KNAUTZ and HECKENBACH, 1980)

The capillary viscosimeter cannot be em- ployed for cultivation broths because of ad-

14 1 Common Instruments for Process Analysis and Control

verse wall effects in the capillary As for the

falling sphere and torsional pendulum viscosi-

meters, the flow equation cannot be calculated

from the shear diagram (only partial solutions

are known)

The Couette and Searle viscosimeters can

only be used if the following conditions are

fulfilled: the annular slit between inner and

outer cylinders must be large enough to reduce

the wall effects, and measurements must be

made using different cylinder lengths to elimi-

nate the end effects In a Searle viscosimeter,

the speed of rotation is limited by the occur-

rence of Taylor instabilities

By measuring the torque MN on the shaft of

different types of stirrers at differing stirrer

speeds, N is suited for the evaluation of the

power input but not for the viscosity These

techniques, which are commonly used accord-

ing to the literature, are not suitable for the

evaluation of the shear diagram and the abso-

lute viscosity

Only the coaxial cylinder viscosimeters,

Couette with rotation outer cylinder and

Searle with rotation inner cylinder, are consid-

ered here, since they are the most popular

where w is the angular speed,

Mi the torque exerted on the inner cy-

linder, and

L the length of the inner cylinder

From Eqs (27) and (28) it follows that

The relationship between the angular velocity

of the rotating cylinder SZ and t is experimen- tally determined to obtain the shear diagram The relationship dv/dx=f(z) (flow equation) can be calculated from Eq (30) For this eval- uation, see MUSCHELKNAUTZ and HECKEN-

BACH (1980) and DINSDALE and MOORE (1962)

For Newtonian fluids, the following rela- tionship is valid:

dv

dx

T = - 9 -

where q is the dynamical viscosity

In practice, relative viscosities are frequent-

ly determined The shear stress is measured for different shear rates with fluids of known (oils) and unknown (broth) viscosities, and the relative viscosity of the broth can be calculated from the ratio of their shear stresses at the same shear velocity, if the broth has Newton- ian behavior

On-line determination of the broth viscosity

is sometimes useful for controlling a process The on-line techniques only yield relative vis- cosities The viscosity of the Aspergillus niger

broth was measured on-line by means of a tube viscosimeter by BLAKEBROUGH et al (1978) PERLEY et al (1979) used an on-line capillary technique for the measurement of the viscosity of the Hansenula polymorpha broth

LANGER and WERNER (1981) and NEUHAUS

et al (1983) developed an on-line slot-type vis- cosimeter and measured the viscosity of the

Penicillium chrysogenum broth KEMBLOWSKI

et al (1985) used an on-line impeller type vis- cosimeter to determine the viscosity of the

A ureobasidium pullulans broth

3.7 Foaminess

Integration of Eq (29) with s2 = R?/Ri = tilta

cially a combination of different surfactants with proteins, may cause stable foams in aer- ated bioreactors Foam control is necessary to

avoid the loss of broth, the clogging of the gas

analyzers, and infections caused by foam

carry-out

Foam can be suppressed by antifoam agents

(BEROVIC and CIMERMAN, 1979; SIE and

SCHOGERL, 1983; SCHUGERL, 1986; PRINS

and VAN'T RIET, 1987; VIESTURS et al., 1982)

or destroyed with mechanical foam breakers

(VIESTURS et al., 1982) Foam can be detected

by an electrical conductivity probe, capaci-

tance probe, heat conductivity probe, or light

scattering probe (HALL et al., 1973; VIESTURS

et al., 1982) Antifoam and mechanical foam

breakers are frequently combined, if the foam

is very stable

The presence of an antifoam agent in the

broth may influence cell growth and product

formation as well as downstream processing

Mechanical foam breaking may exert stress

and selection pressure on the cells

4 Instruments for

Determination of Chemical

System Properties

4.1 pH Value

The dissociation constant K, of the purest

water is very low (10-'5.74 at 25 "C) The con-

centration of water can be considered as con-

stant because of the low K, value Thus, only

the ion product K, is taken into account:

[ H + ] * [OH-] =K,= 1.008 at 25 "C (32a)

Forming the logarithm of Eq (32a)

log [H '1 + log [OH -1 = log K, (32b)

and by multiplication with - 1,

The pH can be measured with a galvanic cell

(chain) The potential E of the cell is given by

the Nernst equation:

R T

F

where Eo is the standard potential and

F the Faraday constant

In this definition the thermodynamic activities

of the ions were replaced by their concentra- tions since the activities cannot be measured The absolute potential cannot be measured either, only the potential difference U between the indicator electrode and a reference elec- trode

Silver-silver chloride electrodes are used in the galvanic chain for sterilizable electrodes Fig 2 shows the schematic assembly of a pH electrode (INGOLD I) In this figure E l is the

potential on the outer surface of the glass membrane, which depends on the pH value of

the sample solution E2 is the asymmetry (bias)

potential, i.e., the potential of the glass mem- brane with the same solutions on both sides

E3 is the potential on the inner surface of the

glass membrane, which is a function of the pH

value of the internal buffer solution E4 is the

potential of the internal Ag/AgCl lead-out electrode, dependent on the KCl concentration

in the internal buffer solution E5 is the poten-

tial of the reference AgCl/Ag electrode, which

reference

elect rulyt

internal buffer solution

Fig 2 Schematic assembly of a pH-electrode (Dr

W Ingold AG, Brochure I, with permission) For details see text

are obtained

16 I Common Instruments f o r Process Analysis and Control

depends on the KC1 concentration in the refer-

ence buffer solution, E6 is the diaphragm or

diffusion potential

Since El is the potential which we want to

measure, the individual potentials E2-E6

should be kept constant These are included in

the standard potential U", which has to be de-

termined by calibration

In modern pH electrodes, U" varies only in

a narrow range (e.g., Type U 402-K7 elec-

trodes of Ingold AG have a potential of

- 10.4k3.8 mV at pH 7.02 and 20 "C)

The potential difference between the indica-

tor and reference electrodes U is also given by

the Nernst equation:

-

2.3R T

F

where the Nernst potential UN = - -

59.2 mV at 25 "C However, in real pH elec-

trodes, the Nernst potential is not attained, but

only approached to 97.5% (in the case of new

electrodes) Furthermore, UN is reduced with

increasing age of the electrode The aging

causes sluggish response, increasing electrical

resistance, a smaller slope, and zero point (U")

drift During steam sterilization, a pressure

difference builds up on both sides of the glass

membrane Therefore, a counter pressure is

imposed to avoid the destruction of the elec-

trode Frequent steam sterilization has a con-

siderable aging effect Therefore, pH elec-

trodes must be recalibrated frequently with

buffer solutions

During in situ steam sterilization a consider-

able, irreversible signal drift of the pH electro-

des occurs Therefore, it is advisable to meas-

ure the pH value of the broth in the reactor

after each steam sterilization by an indepen-

dent method and correct the reading of the pH

meter

Since the potential U depends on the tem-

perature, pH-meters have a temperature com-

pensation, which is usually calculated by the

4.2 Dissolved Oxygen Partial

Pressure, po,

The dissolved oxygen concentration is also measured by electrochemical methods Two types of electrodes are in use:

- polarographic electrodes

- galvanic electrodes

In polarographic or amperometric elec- trodes the dissolved oxygen is reduced at the surface of the noble metal cathode in a neutral potassium chloride solution, provided it reaches 0.6-0.8V negative with respect to a suitable reference electrode (calomel or Ag/ AgC1) The current-voltage diagram is called the polarogram of the electrode (Fig 3)

1

Negative bias voltage Oxygen

Fig 3 Polarogram and calibration curve for a po,-

electrode (LEE and TSAO, 1979)

At the plateau of the polarogram, the reac- tion rate of oxygen at the cathode is limited by the diffusion of oxygen to the cathode Above this voltage the water is electrolyzed into oxy- gen and hydrogen In the plateau region (0.6-

0.8 V), the current is proportional to the par- tial pressure of the dissolved oxygen (Fig 3)

In this probe, the cathode, the anode, and the electrolyte are separated from the measur-

ing liquid by a membrane which is permeable

to gaseous oxygen In the electrolyte, the fol-

lowing reactions occur:

Since hydroxyl ions are constantly being sub-

stituted for the chloride ions as reaction pro-

ceeds, KCl or NaCl must be used as an electro-

lyte When the electrolyte becomes depleted of

C1-, it has to be replenished

The dissolved oxygen concentration is meas-

ured by the galvanic electrode which does not

require an external voltage source for the re-

duction of oxygen at the cathode Using a

basic metal such as zinc or lead as anode and a

nobler metal such as silver or gold as cathode,

the voltage is generated by the electric pair and

is sufficient for a spontaneous reduction of

oxygen at the cathode surface The reaction of

the silver-lead galvanic electrode is given by:

During the reduction of oxygen, the anode sur-

face is gradually oxidized Therefore, occa-

sional replacement of the anode is necessary

The polarographic or amperometric elec-

trode is in greater demand in biotechnological

practice than the galvanic electrode Fig 4

shows a schematic view of a steam-sterilizable

polarographic or amperometric oxygen elec-

trode

A constant voltage (ca 650 mV) is applied

between cathode (Pt) and anode (AgIAgCl) A

regular control of this voltage is necessary in

order to avoid incorrect measurements The

Fig 4

Electrolyte Anode Cathode Electrolyte film Gas permeable mernbr 'ane

Sterilizable po,-electrode (Dr W Ingold

AG, Brochure 11, with permission)

control is carried out by measuring the polaro- gram and adjusting the bias voltage to main- tain a voltage-independent current in the pla- teau region of the polarogram

The current ip,02 is proportional to po2 only

in the plateau region:

where K is a constant,

A the surface area of the cathode,

P the membrane permeability,

d the membrane thickness

The response time is proportional to d 2 / P

Therefore, thin membranes with high gas 02-

permeability are used Two membranes are used for the p o , electrodes for sterile opera- tion The inner membrane consists of a 25 pm teflon foil, the outer one of a 150 wm silicone membrane reinforced by thin steel mesh This type of electrode was developed by the Instru-

18 1 Common Instruments for Process AnalyJ :is and Control

mentation Laboratory Inc., Lexington, Mass.,

USA, and also produced by Dr W Ingold

AG, Urdorf, Switzerland (INGOLD 11) This

type of electrode has a fairly long response

time (45 to 90 s to attain 98% of the final sig-

nal)

During steam sterilization, the membrane

thickness and shape change irreversibly An

improved construction of BAUERMEISTER

(1981) enables the electrodes to endure many

(ca 20) sterilizations without any change in the

membranes

The temperature of the calibrations and

measurements must be controlled closely

(k 0.1 "C) because of the temperature sensitivi-

ty of the signal (temperature coefficient 3%/

"C) Since the electrode measures the partial

pressure of oxygen, the signal is independent

of the 02-solubility in the broth The calibra-

tion should be performed in the reactor under

the same fluid dynamic conditions (stirrer

speed) as those that prevail during cultivation

to avoid errors due to differences in diffusion

resistance at the surface of the membrane

The calibration is carried out with nitrogen-

and air-saturated broth by setting these values

at 0 and 100% The partial pressure of oxygen

is expressed as follows:

p o 2 = [PB-p(HzO)] x 0.2095 (37)

where pB is the temperature-corrected

(barometric) pressure in the

react or,

p(H20) the vapor pressure of the

broth at the temperature of

the calibration,

0.2095 the fraction of oxygen in at-

mospheric air

The sources of error in the measurement of

po2 are numerous: errors in reading of temper-

ature and pressure, drift due to membrane

fouling, change in membrane shape, variation

of bias voltage and electrical resistance as well

as capture of bubbles, etc With sufficient ac-

curacy of temperature and pressure measure-

ments, and with bias voltage in the plateau re-

gion, the precision of the measurements is on

the average f 5 % Below 5 % of the 02-satura-

tion, the error increases with decreasing po,

The dissolved oxygen concentration [O,] is calculated by the relationship:

and CY is the Bunsen coefficient

Bunsen coefficients CY of oxygen for some simple aqueous solutions and a few cultivation broths have been given by SCHUMPE (1985) For more details, see MELZNER and JAENICKE (1980), INGOLD 11, LEE and TSAO (1979), FRITZE (1980), BUEHLER and INGOLD (1976), and SCHINDLER and SCHINDLER (1983)

4.3 Redox Potential, Eh

The definition of the redox potential is given

by Eq (9) To determine E , the potential be- tween the redox electrode and a standard refer- ence electrode is measured The universal ref- erence reaction is the oxidation of hydrogen:

H 2 - + 2 H + + 2 e -

(39)

The standard potential Eo(H+/H2) is by defi- nition equal to zero at all temperatures The universal reference electrode is known as the Standard Hydrogen Electrode (SHE), which consists of a platinum-coated platinum foil that is immersed in a solution containing 1 mol

L - ' H + , and over which flows hydrogen gas

at a pressure of 1 bar The reference electrodes (Hg/calomel/sat KC1, or Ag/AgCl/KCl) used

in practice are referred to the SHE:

where Eh is the redox potential against the

The sterilizable redox meter consists of a Pt

electrode and an Ag/AgCl reference electrode

The electrodes are calibrated with redox buf-

fers in the range of Eh = + 200 mV to 600 mV

(INGOLD 111) Since the redox potentials have a

high temperature coefficient, knowing the

temperature of the broth is necessary for cal-

culating the correct standard potential for the

reference electrode For example, standard po-

tentials of Ingold reference electrodes are giv-

en in INGOLD I11 for different temperatures

Redox potentials occur in a range of - 1200

to + 1200 mV Measurement precision is + 5

mV A simple pH meter with a mV scale is an

adequate measuring instrument The redox po-

tential depends on the po, and pH in the

broth However, since both are measured in

bioreactors, these effects can be taken into ac-

count In aerobic cultivations the po, and re-

dox meters give nearly the same information at

a constant pH value In microaerobic and

anaerobic cultivations the redox potential gives

additional information about the state of the

broth components However, because of the

complex composition of the broth this infor-

mation is only qualitative

For more information on the redox poten-

tial, see MELZNER and JAENICKE (1980),

KJAERGAARD (1977), KJAERGAARD and

JOERGENSEN (1979), INGOLD 111, and FRITZE

(1980a, b)

4.4 Dissolved C 0 2 Partial Pressure,

Pco,

The presence of dissolved C 0 2 in the broth

influences cell growth and product formation

(Ho et al., 1987) Therefore, thepco2 can be

an important variable The pco, can be meas-

ured in-line using thepcoz meter of Dr W In-

gold AG (INGOLD IV) The instrument con-

sists of a pH meter and a hydrogen carbonate

solution, which is separated from the broth by

a gas-permeable membrane Fig 5 illustrates

the main features of the electrode

The dissolved Cot diffuses through the

membrane into the hydrogen carbonate solu-

tion The equilibrium of the reaction

CO,+H,O + HCO, + H +

Measu

Fig 5 Sterilizable pco,-electrode (Dr W Ingold

AG, Brochure IV, with permission)

Construction of a C0,-sensor: (1) 20 mL syringe, (2)

high-temperature coaxial cable, (3) cable screw con-

nection, (4) adjustment nut, ( 5 ) locking plug, (6) supply duct, (7) welding socket, (8) bore hole con- ductor, (9) draw tube, (10) pH-electrode, (11) refer-

ence electrode, (12) C0,-electrode, (13) membrane body, (14) calibration buffer, (15) glass membrane, (16) reinforced silicon membrane

is determined by the dissociation constant K

I H f l [HCO;I [CO2l

K =

according to Henry's law

where H is the Henry coefficient

Since the hydrogen carbonate concentration

in the electrolyte is high, it can be assumed to

be constant Thus, Eq (41) can be simplified

to

The potential of the inner pH electrode is a function of [H'I:

20 I Common Instruments f o r Process Analysis and Control

R T

F

E = Eo + 2.3 ~ log [H '1 (44)

where 2.3 R T/F=59.16 mV at 25 "C,

E is the measured potential, and

Eo is the standard potential

From relationships (43) and (44)

The response time is fairly long (one to sev-

eral minutes) and is influenced by the thickness

of the membrane and by the electrolyte solu-

tion as well as by the response time of the pH

electrode

The measuring range of the electrode is 1 to

1000 mbar COz The deviation is f 2 % , if the

electrode is calibrated with gas mixtures If the

inner pH electrode is calibrated by buffer solu-

tions, the deviation is f 10% To avoid errors

due to the complex temperature dependence of

the reading, the calibration should be carried

out at broth temperature

The electrode is sterilizable The steriliza-

tion is performed after the reduction of the

pressure of the p H electrode on the stainless-

steel reinforced plastic membrane The p H

electrode is calibrated by buffer solutions after

sterilization Then the electrode is filled with

the electrolyte and put into the measuring posi-

tion Fig 5 shows the electrode during calibra-

tion and measurement For more information,

see INGOLD IV

5 Performance

of Instruments

for Process Control

According to FLY" (1982) the relevance,

accuracy, and precision of the measured data

and the reliability, accuracy, precision, resolu-

tion, specificity, response, sensitivity, availa-

bility, and costs of the sensors/instruments are

important for their use in process control All

data which influence the productivity and the yield of the process and the quality of the product are relevant Accuracy of the meas- ured data is expressed as the difference be- tween the observed value of the variable and its true value, which is usually determined by calibration

The precision of the data relates to the probability that repeated measurements of the same system will produce the same values The distribution of the values around their mean is usually characterized by the variance and/or standard deviation, or, e.g., the 95% confi- dence interval

The most important property of a sensor is its reliability, which is made up of factors such

as failure rate, failure mode, ease of preventive maintenance, ease of breakdown maintenance, physical robustness, and its credibility in the mind of process operators (FLY", 1982) The latter plays a role only if the data are used by the operator and not by a n automated sys- tem

Based on information from three chemical works, LEES (1976) published data on the re- liability of the instruments important in the fermentation industry (Tab 1)

One can observe that at the time of investi- gation (1970-1975) the pH meter and the 0,

and COz analyzers were the least reliable in- struments During the last ten years, the relia- bility of these instruments has been improved considerably, provided an accurate flowmeter

is used for the 0, and CO, instruments

FLY" (1982) gave detailed results on the

performance of the instruments in a 1 m 3 pilot plant bioreactor (Tab 2)

One can see that the po2 measurement had

the lowest accuracy, the po2 and air flow con- trol the lowest precision, and the volume meas- urement the lowest resolution In the mean- time, the air-flow control should have attained

a much higher accuracy and precision, pro- vided the right instrument is used (e.g., mass flowmeter) In recent years, the accuracy of the po2 measurement has not been improved markedly, it can, however, be achieved by fre- quent calibration The accuracy and precision

of the po2 control is much better if one uses

three sensors and parameter-adaptive control

Tab 1 Instrument Reliability (LEES, 1976)

Tab 2 Performance of Measurement and Control Instrumentation in a 1 m 3 Pilot Plant Bioreactor

0.41 R

0.4 R

-7.0 R kO.1 R kO.l R

0.02 "C

0.08

0.001 0.01

0.05 mbar 9.95

0.004 psig 0.07

0.14 L 3.4

0.02 L/m 23.0

0.0005%

0.0005%

Notes: W , weekly; H, hourly; R, per run, which relates to the frequency with which the measuring instru- ments are recalibrated

22 1 Common Instruments f o r Process Analysis and Control

In this chapter, only H +-selective electrodes

are considered However, using ion-selective

membranes, in principle the concentration of

an arbitrary ion can be determined by means

of the Nernst equation:

IM the concentration of the measured

ion in the broth

The production of ion-selective carrier-mem-

brane-electrodes is very easy A standard pH-

electrode is combined with an ion-selective

membrane consisting of an ionophore in a

polymer matrix

The ionophore and the softener are usually

dissolved in a PVC solution, put on the sur-

face of the pH-electrode, and dried Fig 6

shows several constructions of such elec-

trodes

Ion-selective membranes may be prepared

by ionophore antibiotics (valinomycin, nonac-

tin, etc.) (SCHINDLER and SCHINDLER, 1983)

and synthetic carriers (crown ethers and cryp-

tates, cyclodextrins, cyclotriveratrylene, perhy-

drotriphenylene, etc.) (ATWOOD et al., 1984;

V ~ G T L E , 1975, 1981) Chiroselective transport

molecules are particularly interesting for the

more detailed analysis of broth components

(LEHN, 1988)

At present, reliability and selectivity of ion-

selective membranes are not always satisfacto-

ry However, host-guest-complex chemistry is

developing rapidly Therefore, it is expected

that some years from now reliable, ion-selec-

tive electrodes will be available

The combination of pH, po,-, pco,-, and

NH: -selective transducers with biochemical

receptors (enzymes, antibodies, lectins, etc.) is

considered in Chapter 3 (Biosensors)

(a) Glass membrane electrode (1) ion-selective glass membrane, (2) non-specific glass shaft, (3) Ag/

AgC1-lead-off electrode, (4) lead-off electrode (liq- uid), ( 5 ) cable

(b) Liquid membrane electrode with ion-exchanger reservoir (1) porous membrane, (2) ion-exchanger

reservoir, (3) lead-off electrolyte (liquid), (4) Ag/

(d) Coated wire-electrode (1) Pt-wire, (2) PVC ion-

exchanger membrane, (3) cable

(e) Disc electrode without O2 reaction barrier (1) carrier-PVC-membrane, (2) Pt-wire, (3) acryl/glass mantle, (4) PTFE-insulated, silver-coated Cu-wire,

( 5 ) PTFE or acryl glass

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2 Methods and Instruments

in Fermentation Gas Analysis

E LMAR HE I N Z LE IRVING J D U N N

Zurich, Switzerland

1 Introduction 30

2 Mass Balancing for Gas Analysis 31

2.1 Basic Gas Balance Equations 31

2.2 Inert Gas Balance to Calculate Flow Rates 33

2.3 Steady-State Gas Balance to Determine the Biological Reaction Rate 33

2.4 Determination of CPR with Accumulation of CO, in the Liquid Phase 34

2.5 Determination of KLa by Steady-State Gas Balancing with Well-Mixed Gas and

Liquid Phases 35

2.6 Determination of KLa by the Dynamic Method 35

2.7 Determination of Oxygen Uptake Rates by a Dynamic Method 36

2.8 Loop Reactors with External Aeration to Determine OUR 36

2.9 Methods to Measure Low Oxygen Uptake Rates 37

2.10 Oxygen Transfer in Large-Scale Bioreactors 38

3.1 Systematics of Elemental Balancing 41

3.2 Elemental Balancing for Monitoring a Poly-P-Hydroxybutyric Acid (PHB) Producing Culture 42

4.1 Objectives of On-Line Gas Analysis and Requirements for Accuracy and Reliability 45 4.2 Definition of Measurement Requirements 45

4.3 Errors Caused by Simplification of Balancing 46

3 Application of Gas Analysis Results to Elemental Balancing Methods 41

4 Error Analysis for Gas Balancing 44

4.3.1 Simplifications Concerning Pressure, Temperature, Humidity, and Gas Flow 4.3.2 Errors Caused by Steady-State Assumption 47

4.4 Erroneous Estimation of Reaction Rates Caused by Measurement Errors 49

4.4.1 Errors in the Measurement of Gas Flow 49

4.4.2 Statistical Error Propagation 49

4.4.3 Errors in Oxygen Gas Analysis 50

4.4.4 Instantaneous Error Analysis for the Elemental Balancing Example PHB

4.4.5 Dynamic Error Analysis for Reaction Rates 54

5.1 System without Removal of Condensable Volatiles 56

Rates 46

51

5 Sample Pretreatment and Multiplexing 56

5.2 Application of Paramagnetic and Infrared Analyzers to the Measurement of Oxygen

and Carbon Dioxide 56

5.3 Special Valve Manifolds for Mass Spectrometers 57

6.1 Positive Displacement Devices 58

6 2 Rotameters 59

6.3 Thermal Mass Flow Monitors (MFM) 59

7 Instruments for Analysis of Gas Composition 60

7.1 Paramagnetic Oxygen Analyzers 60

List of Symbols and Abbreviations 29

List of Symbols and Abbreviations

C concentration (kg m-3)

CPR carbon dioxide production rate (mol s - ')

CTR carbon dioxide transfer rate (mol s - ')

G gas flow rate (m3 s-')

H Henry coefficient (L bar mol-')

I ion current (A)

K equilibrium constant

K L a mass transfer coefficient (s-')

L liquid flow rate (m3 s - ' )

m/z mass-to-charge ratio

M total mass flow (kg s - ' )

n number of moles

N molar gas flow rate (mol SKI)

OUR oxygen uptake rate (mol s-')

OTR oxygen transfer rate (mol s-')

p pressure (bar)

PHB poly-P-hydroxybutyric acid

Q specific reaction rate (mol kg -' s - ')

r reaction rate (mol L -' s - ')

R gas constant (=0.08314bar L mol-' K - I )

6, 6 molar flux (=specific reaction rate) (mol k g-ls- ')

Subscripts and Superscripts

E electrode

G gas phase

i index for component

inert inert gas

L liquid phase

re1 relative

X biomass

0 input into the reactor

1 output from the reactor

* refers to gas-liquid equilibrium

' relative value ( - )

biomass concentration (g L - I )

gas phase molar fraction ( - )

1 Introduction

It is evident from Chapter 1 of this volume

of “Biotechnology” that on-line fermentation

analysis is of increasing importance because

precise control of environmental variables is

necessary to optimize process yield and selec-

tivity Most biological products are not vola-

tile and are either dissolved in the fermentation

fluid, precipitated, or enclosed within the cell

membrane boundary These products are

usually difficult or presently impossible to

measure on-line in a process environment

This is also true for the biocatalyst itself (cell

or enzyme)

In industrial processes each sensor causes

risks of infection, whether located in the sterile

region or connected to the process with a liq-

uid sampling device This risk does not exist if

measurements are made in the effluent gas

stream outside the sterile region

On-line gas analysis is of general interest be-

cause almost any biological process using liv-

ing organisms involves consumption and pro-

duction of gases and volatile compounds Es-

pecially oxygen consumption and carbon

dioxide production occur in any aerobic fer-

mentation process Measurement of these reac-

tion rates gives direct information about the

culture activity Oxygen consumption rate

usually is directly proportional to the heat evo-

lution of any aerobic process (COONEY et al.,

1969)

Historically, one of the first instruments for

gas analysis was the Orsat apparatus (HERON

and WILSON, 1959) In this apparatus CO, and

O2 are subsequently absorbed in sodium hy-

droxide and pyrogallol solutions, and volume

changes are detected Inert gases are deter-

mined by difference CO, production was one

of the first biological activities to be quantified

in yeast alcohol production Traditionally, the

measurements were made using volumetric

methods

Under normal conditions, where the ideal

gas law is valid, gas volume, pressure, and mo-

lar amount are directly linked with each other

This makes barometric and volumetric meas-

urements very useful In microbiology the

Warburg apparatus is still a very popular

method of measuring gas reaction rates Oxy-

gen and CO, production can be measured si- multaneously by first measuring pressure change and subsequently absorbing CO, in an alkaline solution, then making a final pressure measurement Volumetric and barometric methods were also further developed to give on-line readings of gas composition (VANA, 1982)

Historically, the results of on-line gas analy- sis have almost exclusively been used to moni- tor fermentations Since more reliable analyti- cal instruments and on-line data acquisition and computing hard- and software have been developed, it is now possible to use gas analy- sis data together with other measurements to quantitatively characterize fermentation kinet- ics Cheap and reliable process computer sys- tems, together with increasingly powerful and easy to use software, have dramatically im- proved capabilities

Gas analysis usually involves measurement

of gas flow rates and gas composition Setting

up appropriate mass balances allows evalua- tion of actual production and consumption rates Today, gas flow rates can be measured with mass flow meters which directly give an electric signal This facilitates automatic data

evaluation using computers A whole series of instruments to measure gas composition on- line has been developed The instruments in- clude paramagnetic oxygen analyzers, infrared absorption photometers, gas chromatographs (GC), mass spectrometers (MS), flame ioniza- tion detectors (FID), amperometric and poten- tiometric sensors, and semiconductor devices Generally speaking, excluding pH measure- ment, gas analysis is the most widespread and most reliable on-line analysis in industrial fer- mentation processes It has been applied to the on-line analysis of bacterial, fungal, and high-

er cell culture systems Its potential in animal and plant cell culture has not yet been fully ex- ploited This is clearly seen by the fact that in a recent review of on-line analysis of animal cell culture the possibility of oxygen uptake rate measurements has not even been mentioned (MERTEN et al., 1986)

Mass Balancing for Gas Analysis 31

ficient; V, and VL (L3), gas and liquid volume;

r (MT-'L-3), reaction rate

The above equations have been written to apply to any component (oxygen, carbon dioxide, ethanol, etc.) They include accumu- lation, convective flow, inter-phase transfer, and reaction terms Usually there is only one biological reaction term, but a special excep- tion is the case of COz dissociation to yield bi- carbonate In a batch reactor the liquid flow terms are L 1 =Lo = 0 In a fed-batch culture

L o # L 1 , and in a continuous culture Lo= L,>O

Here Ct, is the liquid phase concentration

in equilibrium with CG,, and it is calculated by Henry's law

C G I R T = C t , H (3)

2 Mass Balancing

for Gas Analysis

2.1 Basic Gas Balance Equations

Balances which consider gas transfer and

gas reaction rates are necessary to characterize

the aeration efficiency and to follow biological

activity The same equations can be applied to

any component Well-mixed phases, whose

concentrations can be assumed to be uniform,

can be described simply, while situations with

spatial variation require more complex mod-

els The following general gas balance equa-

tions can be written for a well-mixed (tank

geometry) system (Fig 1):

are: Ho, = 856.9 L bar/mol; Hco, = 34.01

L bar/mol; HN,= 1484 L bar/mol The solu-

bility of pure gases in water can also be ex- pressed in liters of gas per liter of water At 30°C the values are: Nz, 0.0134; 02, 0.0261;

v, - dCG1 - GoCt.-jo-GICG,-

dt

(1)

-K,a(Ct, CLJ VL

Pressure and Temperature Effects

+ KLa (Ct , - C L , ) v L + z r VL (2)

The variables and their dimensions are as fol-

lows:

concentrations; G and L (L3T-'), gas and liq-

uid flow rates; KLa (T-l), mass transfer coef-

It is often convenient to write gas balances

in terms of partial pressures instead of concen- trations Using the ideal gas law

where R = 0.08205 atm L/mol K = 0.08314

(bar L/mol K) = 8314 (Pa L/mol K) or its equi-

valent for a flowing system,

where N is moledtime and G is volume/time

Thus, useful expressions are:

Fig 1 Gas transfer in a stirred tank reactor with ni = cGi v = ~ ~