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Doctoral Thesis

Continuous bioconversion of octane to octanoic acid

Author(s): Rothen, Simon Andreas

Publication Date: 1997

Permanent Link: https://doi.org/10.3929/ethz-a-001763396

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Continuous bioconversion

of octane to octanoic acid

Abhandlung zur Erlangung des Titels

DOKTOR DER NATURWISSENSCHAFTEN der

EIDGENOSSISCHEN TECHNISCHEN HOCHSCHULE ZURICH

vorgelegt von

Simon Andreas Rothen

Dipl. Natw. ETH

geboren am 23. September 1962

von Wahlern (BE)

Angenommen auf Antrag von:

Prof. Dr. Bernard Witholt, Referent

PD Dr. Bernhard Sonnleitner, Korreferent

PD Dr. Elimar Heinzle, Korreferent

Zurich, 1997

Table ofcontents

Table of contents

1 SUMMARY 1

2 ZUSAMMENFASSUNG 3

3 INTRODUCTION 5

3.1 Biotransformations 5

3.2 Product inhibition 6

3.3 Microorganisms and engineering sciences 7

3.4 Automatic bioprocess control 7

3.5 Escherichia coli 8

3.5.1 Escherichia coli K12 and B 8

3.5.2 Escherichia coli HB101 10

3.5.3 Escherichia coli HB 101 [pGEc47] 10

3.6 The project 'Kinetics and dynamics of biotransformations' 11

3.6.1 Previous work 11

3.6.2 Objectives and description of the process 12

3.6.3 Experimental set-up and strategies 13

3.6.4 Results presented in this thesis 14

4 CHARACTERIZATION OF ORGANISMS BY ON-LINE ANALYSES 15

4.1 Introduction 15

4.2 On-line biomass estimation: POSsmiLnTES and constraints 15

4.2.1 Optical density 15

4.2.2 Fluorescence 17

4.2.3 Carbon dioxide 18

4.3 the growth limiting substrate glucose 19

4.4 miniaturization: a step back to the future 22

5 DEVELOPMENT OF AN OPTIMAL DEFINED MEDIUM FOR HB10irPGEC471 25

5.1 Introduction 25

5.2 RESULTS 26

5.2.1 Approach to the development of an optimized medium 26

ii Table ofcontents

5.2.2 Growth of Escherichia coli HB101 and HB101 [pGEc47] in the absence

and presence of yeast extract 28

5.2.3 Metal and vitamin requirements of E. coli HBlOl [pGEc47] during growth on

medium smi 29

5.2.4 requirements of e. coli hb101[pgec47] for p, n, s, l-pro, l-leu 29

5.2.5 Determination of Dr 33

5.3 Discussion 38

6 OCTANOATE PRODUCTION AND PRODUCT INHIBITION 41

6.1 Introduction 41

6.2 Results 41

6.2.1 Basic effects of octane feed during continuous culture 41

6.2.2 the effect of shifts in octane-feed on the culture is reversible 46

6.2.3 production of octanoate at different dilution rates at low octane phase

RATIOS 47

6.2.4 INFLUENCE OF OCTANOATE ON HB101 [PGEC47] GROWING CONTINUOUSLY IN A

C-LIMTTED CULTURE 53

6.2.5 GROWTH OF HB101 [PGEC47] IN THE PRESENCE OF ACETATE 56

6.2.6 Growth of HB 101 [pGEc47] on defined medium with different octanoate

concentrations 57

6.3 Discussion 58

6.3.1 Growth of E. coli HB 101 [PGEC47] in the presence of octane 58

6.3.2 Influence of octanoate on growth of HB101 [PGEC47] 59

6.3.3 Simulation as a tool for planning, prediction and verification of experiments 59

7 PROCESS INTEGRATION FOR THE REMOVAL OF OCTANOATE 60

7.1 Introduction 60

7.2 Start-up procedure 60

7.2.1 Model simulation 62

7.2.2 performance of the system 63

7.3 redhtection of carbon fluxes 65

7.4 Octanoate production with attached membrane filter 66

7.4.1 influences on physiology of the cells 66

7.4.2 Demands on on-line analyses 69

Table of contents ui

8 CONCLUSIONS AND OUTLOOK 71

8.1 Growth and product characterization 71

8.2 hblol as suitable host for biotransformations 71

8.3 process integration 72

8.4 Alternative methods for on-line removal of octanoic acid 73

8.5 Alternatives to process integration 73

9 MATERIAL AND METHODS 74

9.1 Media 74

9.2 Microorganisms, storage, plates, inocula 94

93 Bioreactor, Cell recycle 75

9.3.1 Bioreactor 75

9.3.2 Cell recycle 75

9.3.3 Sterilization 77

9.4 Analyses 77

9.4.1 On-line analyses 77

9.4.2 Off-line analyses 78

9.4.3 Synchronization of the analytical subsystems 79

9.5 Modeling 79

10 SYMBOLS AND ABBREVIATIONS 81

11 REFERENCES 83

12 APPENDIX 91

12.1 Definitions 91

12.2 Derivatives of E. coli K12 and B 92

12.3 CGSC-DATABASE (COLI GENETIC STOCK CENTER, YALE UNIVERSITY, USA) 92

12.4 MATLAB M-FILE FOR DATA EVALUATION 97

12.5 PD3-CONTROLLERS 103

12.6 CONFIGURATION FR3 105

Summary 1

1 Summary

Escherichia coli HBlOl[pGEc47] is able to carry out the biotransformation of octane

to octanoic acid, but cannot oxidize octanoate further. The acid is excreted into the

cultivation liquid and can be determined in the supernatant. Analytical (on-line)tools such as in-situ probes, on-line FIA and on-line MS were used for acquisition of

precise and reliable data. The focus of the presented work lies on growthcharacterization of E. coli HBlOl and HB101[pGEc47], the development of an

optimized medium and the characterization and optimization of the

biotransformation.

Differences in growth behavior of E. coli strain HBlOl and strain HB101[pGEc47]could be related to yeast extract-enriched medium rather than plasmid content. An

optimal medium for growth of E. coli HBlOl[pGEc47] was designed based on the

individual yield coefficients for specific medium components (NHi+ 6 g g"1, P043~ 14

g g1, S042" 50 g g"1). The yield coefficient for L-leucine depended on the glucosecontent of the medium (20 g g"1 for 3 % glucose, 40 g g"1 for 1 % glucose) and the yieldcoefficient for L-proline depended on the cultivation mode (20 g g"1 for batch

cultivation, 44 g g"1 for continuous cultivation). Growth on defined medium after

medium optimization was as rapid as on complex medium (umax = 0.42-0.45 h"1). The

critical dilution rate (DR) above which undesired production of acetic acid occurs was

in the range of 0.23 - 0.26 h"1.

E. coli HBlOl[pGEc47] was grown on the optimized defined medium with glucoseas carbon source in batch and continuous culture. The biomass yield on glucosedecreased from 0.32 ± 0.02 g g1 in aqueous cultivations to 0.25 + 0.02 g g"1 in the

presence of octane. Maximal octanoate productivities of 0.6 g l"1 h"1 were the same as

found in cultivations on complex medium. The glucose based carbon recovery in

these experiments was 99 + 4 % (in extreme, between 90 and 105 %). An increase of

the octane feed from 1 % to 2 % (v/v) or more led to washout of cells. This effect was

reversible when the octane feed was decreased to its initial value of 1 %. Analysis of

experimental data by model simulation strongly suggested that washout was due to

inhibition by octanoate only. Pulses of octanoate to a continuous culture grown on

aqueous media were applied to analyze the inhibition further. Inhibition by acetate

was not significant but its presence in the medium reflected a physiological state

which made the cells more sensitive to octanoate inhibition. Model simulation with

linear inhibition kinetics could perfectly predict glucose consumption and the

resulting glucose concentration. The linear type of inhibition was confirmed by a

variety of batch experiments in the presence of different concentrations of octanoate.

The glucose based specific growth rate u decreased linearly with increasingconcentrations of octanoate and became zero at a threshold concentration pmax of 5.25

± 0.25 g l1.

The bioreactor was extended with a ceramic membrane filter to form a fullyautomated cell-recycle bioreactor system. The main advantage of this process

integration was the removal of the inhibiting octanoate via the permeate stream and

the possibility of decoupling the specific growth rate u from the dilution rate D,

2 Summary

which allowed a possible increase of the volumetric productivity. Model simulations

helped in designing an optimal start-up procedure of the cell-recycle bioreactor

system. Biomass concentrations of 40 g l"1 with non-induced cells and 35 g l"1 with

induced cultures at a dilution rate of 1 h"1 (with a growth rate of 0.1 h"1) could be

reached. The glucose dependent biomass yield of cultures grown in the cell-recyclebioreactor system in the presence of octane decreased to 0.13 g g"1 indicating that the

use of the cell-recycling led to a change of the metabolic pathway towards increased

production of metabolic overflow products. This finding could be confirmed bycalculation of carbon balances which showed significant deficits of up to 40 % carbon.

A maximal stable productivity of more than 1 g l"1 h1 could be established with the

cell-recycle bioreactor system. Therefore the use of process integration lead to a

doubling of the volumetric productivity compared to a system without cell-recycling.

Zusammenfassung 3

2 Zusammenfassung

Escherichia coli HBlOl[pGEc47] kann die Stoffumwandlung von Oktan zu

Oktansaure durchfuhren, ist jedoch nicht befahigt, die Oktansaure weiter zu

verwerten. Die Saure wird in die Kulturfliissigkeit ausgeschieden und kann dort

gemessen werden. Genaue und zuverlassige Daten wurden mit Hilfe von Sonden,

welche direkt im Bioreaktor (in-situ) angebracht waren, sowie on-line Analytik wie

FIA oder MS gewonnen. Im Mittelpunkt des Interesses standen in dieser Arbeit die

Charakterisierung des Wachstumsverhaltens von E. coli HBlOl und HBlOl[pGEc47],die Entwicklung eines optimalen Mediums sowie Charakterisierung und

Optimierung der Stoffumwandlung.Unterschiedliches Wachstumsverhalten der beiden Stamme HBlOl und

HB101[pGEc47] konnte auf unterschiedliche Mediumsbedingungen zuruckgefuhrtwerden und war unabhangig vom Vorhandensein des Plasmides. Aufgrund der

ermittelten Ausbeutekoeffizienten fur einzelne Mediumsbestandteile (NH4+ 6 g g"1,PO43" 14 g g1, SO42" 50 g g1) wurde ein optimales Wachstumsmedium fur E. coli

HB101[pGEc47] entwickelt. Der Ausbeutekoeffizient von L-Leucin war vom

Glukosegehalt des Mediums (20 g g1 bei 3 % Glukose, 40 g g1 bei 1 % Glukose),

derjenige von L-Prolin von der Kultivationsform (20 g g"1 bei Satzkultur, 44 g g"1 bei

kontinuierlicher Ziichtung) abhangig. Nach der Mediumsoptimierung wurde auf

definiertem Medium dieselbe spezifische Wachstumsgeschwindigkeit erreicht wie

auf Komplexmedium (umax = 0.42 - 0.45 h"1). Die kritische Verdiinnungsrate (Dr),oberhalb welcher die unerwunschte Nebenproduktbildung von Acetat auftritt,konnte im Bereich von 0.23 - 0.26 h1 festgelegt werden.

Das optimierte definierte Medium wurde verwendet, um E. coli HB101[pGEc47] in

Satz- und kontinuierlicher Kultur zu ziichten. Der Ausbeutekoeffizient von Biomasse

auf Glukose verringerte sich von 0.32 ± 0.02 g g"1 bei Ziichtungen auf rein wassrigemMedium auf 0.25 + 0.02 g g"1 in der Anwesenheit von Oktan. Die maximal erreichte

Produktivitat von 0.6 g l"1 h"1 war vergleichbar mit derjenigen, welche auf

Komplexmedium erreicht wurde. In den Experimenten auf definiertem Medium

betrug die Kohlenstoffwiederfindung 99 ± 4 % (bei Extremwerten von 90 und 105 %).Eine Erhohung der Oktanzufuhr von 1 % auf 2 % (v/v) oder mehr fuhrte zu einem

Auswaschen von Zellen. Dieser Effekt war reversibel. Mit Hilfe von

Modellsimulationen konnte die Biomasseabnahme auf einen Hemmungseffekt durch

die produzierte Oktansaure zuruckgefuhrt werden. Um diese Inhibierung weiter zu

untersuchen, wurde einer auf rein wassrigem Medium geziichteten kontinuierlichen

Kultur Pulse von Oktansaure zugesetzt. Glukoseaufnahme und resultierende

Glukosekonzentration in der Kulturfliissigkeit konnten mit einem Modell mit

linearer Inhibitionskinetik treffend vorausberechnet werden. Die lineare

Abhangigkeit zwischen Wachstum und Oktansaurekonzentration in der

Kulturfliissigkeit wurde durch eine Serie von Satzkulturen bestatigt. Die Glukose-

abhangige spezifische Wachstumsgeschwindigkeit u nahm mit zunehmender

Oktansaurekonzentration im Medium linear ab und erreichte bei einer

4 Zusammenfassung

Oktansaurekonzentration pmax von 5.25 + 0.25 g l"1 den Nullwert, bei welchem kein

Wachstum mehr beobachtet werden konnte.

Der Bioreaktor wurde durch hinzufugen eines Keramik-Membranfilters zu einem

vollautomatisierten ZeUruckfuhrsystem erweitert. Diese Prozessintegrierung machte

es moglich, dass die inhibierende Oktansaure durch den Permeatstrom aus^ dem

System entfernt werden konnte. Ausserdem ergab sichT so^^HeT Moglichkeit, die

Verdiinnungsrate unabhangig von der spezifischen Wachstumsgeschwindigkeitwahlen zu konnen, was eine mogliche Erhohung der volumetrischen Produktivitat

gestattete. Mit Hilfe von Modellsimulationen wurde eine optimale Anfahrstrategiedes Zellruckfuhrsystems entwickelt. Zelldichten von 40 g l"1 (bei uninduzierten

Kulturen) und von 35 g l"1 (bei induzierten Kulturen) konnten bei einer

Verdiinnungsrate von 1.0 h"1 und einer Wachstumsgeschwindigkeit von 0.1 h"1

erreicht werden. Der Ausbeutekoeffizient von Biomasse auf Zucker sank in

Anwesenheit von Oktan auf 0.13 g g"1. Dies entspricht beinahe der Halfte des Wertes,welcher ohne Zellriickfuhrung bestimmt wurde, was darauf schliessen liess, dass die

Zellruckfuhrung eine Veranderung des Stoffwechsels mit erhohter Ausscheidungvon Nebenprodukten bewirkte. Dies konnte indirekt durch das Berechnen von

Kohlenstoffbilanzen bestatigt werden, welche Verluste von bis zu 40 % Kohlenstoff

ergaben. Mit dem ZeUruckfuhrsystem konnte eine maximale stabile Produktivitat

von mehr als 1 g l"1 h"1 erreicht werden. Damit ermoglichte der Einsatz von

Prozessintegration eine Verdoppelung der volumetrischen Produktivitat verglichenmit einem System ohne Zellruckfuhrung.

Introduction 5

3 Introduction

Modern biotechnology is an interdisciplinary field influenced by several sciences

like microbiology, molecular biology, chemistry and the engineering sciences. It

covers the whole range from the genome to cultivation of microorganisms and

downstream-processing of products. Scientific investigations generally do not deal

with the whole range but rather concentrate on specific areas or questions. The

present study focused on the investigation of a biotransformation process performedby recombinant bacteria and its improvement by means of bioprocess engineering.

3.1 Biotransformations

Biotransformation is the enzymatic conversion of natural and chemicallysynthesized starting materials into products having specifically modified structures.

Biotransformations are carried out either with pure cultures of microorganisms or

with purified enzymes. The first processes which fulfilled these requirements were

the oxidation of alcohol to acetic acid by 'Mycoderma aceti' (Pasteur, 1862) and

'Bacterium aceti' (Brown, 1886), the oxidation of glucose to gluconic acid by'Mycoderma aceti' (Boutroux, 1880) and the oxidation of sorbitol to sorbose by'Bacterium xylinum' (Bertrand, 1896). Independent of whether the conversions are

carried out by whole cells or purified enzymes, biotransformations have

characteristics typical for enzymes. The catalytic activity is usually restricted to a

single reaction type (reaction specificity) and the substrate molecule is usuallyattacked at the same site (regiospecificity). Only one of the enantiomers of a racemic

substrate is attacked or only one of the possible enantiomers is formed

(stereospecificity). Furthermore, biotransformations are carried out under mild

reaction conditions. With these properties biotransformations are able to carry out

reaction steps that can hardly be accomplished by chemical methods. This is a

prerequisite if biotransformations are to compete with chemical alternatives.

The design of a biotransformation process involves several steps, the first of which

is to find a microorganism which catalyzes the reaction of interest. The screening for

microorganisms can be followed by mutagenesis to maximize the yield of the

product. This empirical approach to finding a suitable organism is now more and

more replaced by genetic engineering which allows the insertion of desired

functionalities into appropriate host organisms. An aqueous substrate (startingmaterial for the biotransformation) should be soluble in the medium, should be able

to pass the cell membrane and it should not be toxic to the microorganisms. The

biotransformation can be carried out either with growing cultures or with pre-

grown, resting cells. The separation of microbial growth and biotransformation has

several advantages. Each step can be separately optimized and a negative effect of

the starting material or the biotransformation product on growth can be excluded. A

third possibility is the biotransformation with immobilized cells. The immobilized

microorganisms normally show a higher stability than freely suspended cells. Highcell densities and high production rates are further possible advantages of

immobilized cells. Compared to immobilized enzymes, whole cell immobilization

6 Introduction

guarantees that the enzymes remain in their natural environment and artificial

coenzyme regeneration is not necessary, because it is done by the cells themselves.

Once a biotransformation process has been established, interest focuses on

optimization of the process to maximize, in the end, the profit. Optimization of the

environmental conditions involves improvement of the biomass to achieve a highlevel of enzymes and improvement of the yield of the biotransformation process.Both enzyme formation and enzyme activity are heavily influenced by medium

composition and cultivation conditions. Optimal physical and chemical conditions

have to be determined empirically or based on model simulations. Elimination of

side reactions in a biotransformation by applying conditions that suppress the

undesired enzyme activities or by genetic engineering is a further possibility of

improving the biotransformation.

The production of penicillin is an illustrative example of the establishment and

improvement of a biotransformation process. Penicillin was the first antibiotic that

could be produced biotechnologically in sufficient amounts to cover the

requirements. Alexander Fleming discovered the production of penicillin byPenicillium notatum in 1928. The need for penicillin during the second world war led

to the industrial production of this antibiotic. After an extensive screening,Penicillium chrysogenum was chosen as a production strain. Bioreactors for cultivation

of the microorganisms and tools for harvesting the product had to be constructed

and, finally, the isolation of the product also had to be solved. This process started in

1938 and, in 1944, enough penicillin could be produced to cover the needs of the US-

army hospitals. Penicillium chrysogenum was treated with IR, UV and mustard gas,and a total of 20 mutation steps led to the strain finally used for the production of

penicillin. The optimization of the bioprocess increased the amount of penicillinproduced per liter cultivation liquid from 60 mg in 1951 to more than 20 g in the

eighties. Apart from strain improvement, this more than 300 fold increase was also

due to medium optimization. Penicillium grew faster on glucose, but on lactose it

produced more penicillin. A mixture of glucose and lactose was chosen as a

compromise between microbial growth and penicillin production. Finally, corn steep

liquor turned out to be the cheapest and most effective basal production medium.

3.2 Product inhibition

The biotransformation product can influence the growth of the producingmicroorganisms. An example of this is the production of butanol and acetone,

another historic biotransformation process. Chaim Weitzman (later first president of

the state Israel) developed the bioconversion of starch to acetone and butanol byClostridium acetobutylicum in 1912. Again war, this time the first world war, was the

initiator of industrial acetone/butanol-production. Acetone was used in the first

world war for the production of ammunition. Both acetone and butanol inhibit

growth of Clostridium at low concentrations. The solution to producing still largeamounts of the products was to build huge vessels for cultivation of the organisms,thus increasing the reactor volume and diluting the products.

Introduction 7

3.3 Microorganisms and engineering sciences

The first industrial processes (production of lactic and citric acid, ethanol,

butanol/acetone) were carried out under non-sterile conditions. The presence of

(undesired) microorganisms not involved in the production was kept low only bymeans of cultivation conditions. This was possible as long as the product was not

degraded by these infections.

The production of penicillin, which can be degraded by several organisms, led to

the development of sterile processes guaranteed by complex bioprocess engineering.This allowed cultivating the organisms under optimal (rather than selective)conditions.

Processes with wild-type organisms isolated from nature mostly suffered from

low productivities. Strain improvement was therefore needed to increase the

productivity, to allow the use of cheaper raw material and/or to improve the

tolerance of the organisms to higher product concentrations. The development of

highly productive industrial strains by means of mutagenesis and selection has been

largely an empirical process. In contrast to this, genetic engineering allows the

targeted modification of organisms to introduce the desired capabilities.Modeling and simulation is an alternative and increasingly used method in order

to carry out an effective process design. This is only possible if basic physiologicalcharacteristics of the organism in use are known, which is not always the case for

industrial strains (Gram, 1996). Improved bioprocess engineering allows gatheringthe physiological knowledge needed for effective modeling. Modeling has increasingimportance for industry. Recent examples show that the use of model simulations

helped to choose a suitable operation mode or even totally replaced the empiricalprocess development (Hoeks et al, 1996; Rohner and Meyer, 1995).

3.4 Automatic bioprocess control

Better knowledge of microbial physiology and the ongoing process itself is

possible with improved process control and application of automatic control. In vivo

measurement and improved on-line analysis are prerequisites for better bioprocessmonitoring and control. Quality control and process safety are prominent demands

of industry, which can be satisfied by an effective process control. Automation of a

process improves its reproducibility and might lead to an improvement of productquality. An increase of the process safety is often accompanied by an increase of

process reproducibility. For effective process control, precise and accurate

knowledge of the state of the process is required. This knowledge can be acquired bymeasurement of a set of state variables. The necessary analytical data must be

obtained in situ, on-line and in real time and must cover a wide dynamic range

(Sonnleitner et al, 1991).A growing number of on-line analytical systems has been applied to bioprocesses.

On-line systems provide faster, more frequent and often more reliable data (van de

Merbel et al, 1996). The majority of the described systems is based on the flow

injection analysis (FIA) principle. The drawback of having only one data channel can

be avoided by using multi-channel FIA (van Putten et al, 1995; van der Pol et al,

1995). Other analytical methods or detection systems such as MS (Lauritsen and

8 Introduction

Gylling, 1995; Chauvatcharin et al, 1994; Oeggerli and Heinzle, 1994), HPLC (Chenand Horvath, 1995; von Zumbusch et al, 1994) or GC (Filippini et al, 1991) are also

useful for on-line monitoring of multiple substances. Increasing numbers of on-line

analyses applied in industry in Switzerland (e.g. CIBA, Nestle, Givaudan, personalcommunications) prove that on-line analyses can provide the robustness needed for

industrial application. Biosensors, however, although numerous, have to be

improved further in order to make them work in industry (Mattiasson, 1996).

3.5 Escherichia coli

Escherichia coli belongs to the family of Enterobacteriae (Procaryotes, class of

Eubacteriae, group of Proteobacteriae), so called because some representativeorganisms of this family, like E. coli, inhabit the human enteric tract. Escherichia coli

strains are nowadays the working horses of microbiologists and genetic engineers.This popularity grew over time. Bacterial physiologists isolated organisms from

environments that were easily accessible, not highly virulent and able to grow on

defined media. E. coli, being one of these organisms, was first isolated in 1885 byTheodor Escherich and became more and more important with work on

bacteriophages and phage genetics (e.g. Delbriick and Luria, 1942; Doermann, 1948;Arber and Dussoix, 1962; Arber, 1965a,b; Dussoix and Arber, 1965; Wood, 1966), but

especially with the work of Monod on growth physiology and enzymatic adaptation(Monod, 1949) as well as the discovery of conjugation and transduction in the late

1940s.

3.5.7 Escherichia coli K12 and B

Most of the E. coli laboratory strains originate from wild-type E. coli strain K12

and, to a lesser extent, strain B.

E. coli K12 was isolated from feces of a convalescent diphtheria patient in the fall

of 1922 at Stanford University (Bachmann, 1996; Kuhnert and Frey, 1996). Earlyderivatives of K12 were isolated after irradiation with X-rays in 1944. Since that time,

many thousands of K12 mutants have been isolated (Bachmann, 1996). One of these,Escherichia coli AB1157, originated from K12 after 3 X-ray treatments, 9 UV

treatments and 1 treatment with mustard gas (Bachmann, 1996, see table 12.1,

Appendix). E. coli K12 has recently been investigated regarding its safety. Very little

is known about the pathogenicity of the wild-type K12, but no case of disease, caused

by E. coli K12 derivatives, has ever been reported since 1944. The fact that no

virulence genes have been found so far in K12 derivatives confirms their non¬

pathogenic phenotype (Kuhnert and Frey, 1996). Because of this property, K12

derivatives are good strains for use in industrial production processes.E. coli B has been used chiefly for investigation of the radiation effects on bacteria.

It was originally isolated from water by Bronfenbrenner, the source of the water

being unknown (Barbara J. Bachmann, personal communication). The cultures

maintained in different labs took on different designations: 'B American' in Brenner's

lab, 'B' or 'BW by Mark Adams and Luria/Delbriick (Delbriick and Luria, 1942), 'B

Berkeley' in Berkeley, 'B' and 'S' in Hershey's lab.

Introduction

AC2517 F" X AC2515 F K12wtF+ x W2961f-= B/r lac" = HB5

/[d]

sulA1 \ (S. typhoss1 /

lac-14 \ carrying /

\ F-/ac from / F1 transfer

\£. coli)

/AC2516 F X W2961 F+= HB11 = AB266

sulA1 Mlac-14

po65Thr+

Str*F-lac

i

AC2601 F= HB16

[d]\thr-1 =[f][thr-hsd] from £ coli B (r+Bm+B)

NG

i

AB3045 Hfr X HB67F

=HB82 [f]recA13 his'

HvD132 Phr-hsd] (r+Bm+B)thi-1

lacZ4His+

rpstr8or33supE44po13

KLF4/ \ '

AB2463 F' X HB100 F

= HB77 [f][e] recA13

F104 Pro+ [thr-hsd] {r+Bm+B)

1

KLF4/HB1C(OF Segregat^ HB101 r

Pro+/Leu+: Pro /Leu":

[f]\ proA2,leuB6 [f]recA13 recA13

[hsdS20] (r"Bm"B) "ramC1" [hsdS20] (r"Bm"B) "ramC1"

F104 (at least [thr-hsd] from £. coli B)

Figure 3.1: Pedigree of E. co//HB101 based on the following sources:

AB266

AB2463

AC2515

AC2516

AC2517

HB16, HB77, HB100, HB101

Pedigrees:

Boyer (1964) and Boyer (1966)Howard-Flanders et al (1966a,b)Johnson etal (1964)

Boyer (1964) and Boyer (1966)

Boyer (1964)

Boyer and Roulland-Dussoix (1969)Bachmann (1996 and personal communication)

The derivations of E. coli AC2517, W2961 and AB2463 as well as detailed information

about mutations of AB1157, AB2463, HB11, HB16, HB101 and W2961 are described in

more detail in the Appendix (table 12.1).

[d] and [e] correspond to sets of mutations listed in table 12.1 of the Appendix.

10 Introduction

In Brenner's lab the E. coli B strains were derived from E. coli B (American

Wildtype), obtained from Dennis Kay of Oxford (around 1950). E. coli B from

Delbriick was used in the lab of Witkin's to select E. coli B/r on the basis of radiation

sensitivity (Mary Berlin, Coli Genetic Stock Center Yale, personal communication).

3.5.2 Escherichia coli HBlOl

HBlOl, the strain used in this thesis, arose from work done on restriction and

modification in E. coli (Boyer, 1964; Boyer and Roulland-Dussoix, 1969). HBlOl is

mainly a derivative of K12 but carries (mutated) restriction and modification genes

(r"B, m"B) from E. coli B. AB2463, a recA" derivative of AB1157 mentioned earlier,

served as host strain for KLF4/AB2463 (F') and was designed HB77 by Boyer (Boyerand Roullard-Dussoix, 1969). This F' was crossed with HB100 to yield a Pro"1" Leu4"

derivative (KLF4/HB100 F'). This derivative was allowed to segregate out the F', and

a screening for Pro" and Leu" gave HBlOl (figure 3.1).E. coli B wild-type spontaneously mutated to B/r and AC2517 (see Appendix,

table 12.1). Introduction of plasmid F-lac from a strain of Salmonella typhosa (AC2515)into AC2517 gave HBll. This was crossed with the K12 strain W2961 F+ (AB266),

yielding HB16 (AC2601), a K12 and B/r hybrid (see Appendix, CGSC database).

HB100 was then the product of a cross of HB16 with the K12 type HB82 (AB3045

Hfr).HBlOl is therefore a F" strain carrying 13 mutations (recA", Str R, Leu", Pro", thi",

ara", lac", gin", gal", X~, xylA", mtl", hsd" (r'Bm'B), according to CGSC database, see

Appendix).

3.5.3 Escherichia coli HBlOl(pGEc47)

Pseudomonads are able to proliferate on diverse organic compounds not normallyused by other bacteria. The genes for the degradation of these compounds are

encoded on plasmids. Alkanes are the main components of petroleum and petrol.The strain Pseudomonas putida contains the OCT-plasmid, which enables the

organisms to grow on medium chain length alkanes and fatty acids as sole carbon

source (Chakrabarty et al, 1973; Hansen and Olsen, 1978). P. oleovorans has been

shown to belong to the P. putida family also carrying the OCT plasmid (Stanier et al,

1966). The alkane oxidation genes of P. oleovorans were cloned into the broad host

range vector pLAFRI (Eggink et al, 1987). The plasmid pGEc47 contains the alkBAC

and the alkR locus. The genes of these two loci are organized as AlkBFGHJKL and

AlkST operons (Nieboer, 1996; Chen, 1996; Wubbolts, 1994). The two operons are

encoding for the alkane hydroxylase system (AlkBGT), the alcohol dehydrogenase(AlkJ), the aldehyde dehydrogenase (AlkH) and an acyl-CoA synthetase (AlkK),which is not used in E. coli. AlkS might be involved in binding to the Paik promoter

(Wubbolts, 1994). The functions of AlkF and AlkL are unknown. The plasmid

pGEc47 was introduced in E. coli HBlOl (Favre-Bulle et al, 1993), resulting in the

recombinant E. coli HBlOl[pGEc47].

Introduction 11

3.6 The project 'Kinetics and dynamics of biotransformations'

The results presented in this thesis were produced integrated in the project'Kinetics and dynamics of biotransformations' supported by the Swiss Priority

Program Biotechnology (SPP), module 2. The aim was to improve the volumetric

productivity of the bioconversion of alkane to alkanoate. High performancebioreactors were used to ensure satisfactory mass and energy transfer. In-situ sensors

and on-line analyses were applied for determination of the kinetics and for process

control.

3.6.1 Previous work

The biotransformation investigated in this thesis has been established earlier in the

group of Prof Witholt in Groningen (Favre-Bulle, 1992). The genes enablingPseudomonas oleovorans to oxidize alkanes have been cloned previously in various

Escherichia coli strains (Favre-Bulle et al, 1993). These strains are unable to grow on

medium-chain fatty acids because their P-oxidation system is repressed and not

induced by medium chain length fatty acids (Favre-Bulle and Witholt, 1992). The

cells must therefore be grown on a common carbon source such as glucose. Theyloose viability rapidly in the presence of octane when growth ceases (Favre-Bulle et

al, 1993). Induced growing cells, however, convert alkanes to the respective alkanol

or alkanoic acid depending on their recombinant gene set. In a two-liquid phase

system, alcohols preferentially partition to the apolar phase, whereas the acids

preferentially go to the aqueous phase.The strain chosen for this project was Escherichia coli HBlOl[pGEc47], because it

was found that the plasmid stability was excellent in this strain (Favre-Bulle et al,

1993, also reported by Lee and Chang, 1988). Octane was chosen as starting material.

It is known that, unlike many other organisms (Harrop et al, 1989; Hocknull and

Lilly, 1988), E. coli is able to grow in the presence of alkanes, especially in the

presence of octane (Favre-Bulle, 1992).The so far established biotransformation process was carried out on media

containing the complex additive yeast extract reaching a volumetric productivity of

0.5 g l1 h1 of octanoate in continuous cultivation (Favre-Bulle et al, 1993).

12 Introduction

Figure 3.2: The biotransformation of octane to octanoic acid carried out by recombinant E coli

HB101[pGEc47].Alk B,G,T: Alkane hydroxylase systemAlk J: Alcohol dehydrogenaseAlk H: Aldehyde dehydrogenase

3.6.2 Objectives and description of the process

The main objective of the project was to investigate and optimize the

biotransformation of octane to octanoic acid (figure 3.2) in a defined environment.

The project was of highly paradigmatic nature as most of the treated aspects were of

general interest. Increase of volumetric productivity, relaxation of inhibition as well

as process integration and product recovery are omnipresent challenges in industryand the project was intended to identify and overcome decisive industrial relevant

bottlenecks.

The biotransformation system consisted of four different phases, two liquid phases(octane as apolar, water as polar phase), one solid phase (microorganisms) and one

gaseous phase (air). The cells grew only in the polar phase but were affected by the

apolar phase. The substrate was converted from the apolar phase to form a productin the polar phase and the product inhibited the growth of the microorganisms.

The conversion represented a worst case situation. Instead of one liquid phase the

cells were growing in a two liquid phase system with an organic solvent forming the

second phase. And instead of forming an inhibitory product that was soluble in the

organic phase and therefore no longer affected the cells growing in the aqueous

phase, this reaction converted the substrate of the apolar phase into a water soluble,

inhibiting product.

Introduction 13

feeds apolar

,_i aqueous

!|

naFIA,GC,

HPLC,... &

rmr

exhaust (MS capillary)

^

Xin-situ sensors

MS (membrane)

monitoring and controlgas

waste

hydrophobicmembrane

hydrophilicmembrane

iZr-S

H^0

o

oO

product recovery& cell-recycle

Figure 3.3: Experimental setup of bioreactor with product recovery and monitoring and control.

The supply into the bioreactor consists of the aqueous medium and the octane (organic or

apolar phase). The bleed stream (waste) and the permeate fluxes of cell-recycle (for

product recovery) and analytical loop (for monitoring and control) formed the outflow of the

system.

Thus, instead of a reaction that converted an inhibitory substrate into a non-

inhibitory product thus relaxing the inhibition, this conversion led to an even more

inhibitory water soluble product. Knowledge on how to deal with such a systemshould add useful information to the understanding of and ability to develop and

control biotransformations in general.

3.6.3 Experimental set-up and strategies

Figure 3.3 describes the experimental set-up of the applied bioreactor system.Fresh medium (polar phase) and octane (apolar phase) are pumped into the reactor.

They are used to fill the reactor and, in continuous cultivations, to constantly supplythe culture with new medium and the biotransformation substrate. In continuous

mode, grown cells and used medium are continuously removed from the culture via

the bleed stream (waste) in order to maintain a constant liquid weight in the

bioreactor. Any cultivation must be started by inoculating a batch culture. A batch

culture was switched to continuous mode only after total consumption of the glucoseand reconsumption of the metabolic by-products. The signals of optical density (off¬line), glucose (on- or off-line), fluorescence, redox, p02 and exhaust gas were

evaluated for determination of a steady state. Off-line analysis of biomass and by¬products allowed the time-delayed verification of the steady state.

Several different types of experiments were performed: repetitive batch cultures

(here, 80-90 % of the culture was harvested, leaving 10-20 % of the bioreactor

contents which serves as inoculum for the subsequent batch culture after refilling the

bioreactor with fresh medium), pulses (singular addition of a defined amount of a

14 Introduction

substance within a very short time), shifts (stepwise change of a parameter to a new

value, which is maintained for some time) and ramps (continuous linear change of a

parameter with a defined velocity over a predefined time).

3.6.4 Results presented in this thesis

The kinetics of growth and the biotransformation were quantified in order to

design an optimal reaction system. The quantification of the bioprocess requiredmonitoring and control of temperature, pH, pressure, working volume and medium

fluxes to guarantee constant and reproducible growth conditions as well as analytical(on-line) tools for acquisition of precise and reliable data and for process control.

Chapter 4 shows examples where the use of on-line analyses applied to bioprocessesis demonstrated.

The characterization of the biosystem required the development of a chemicallydefined and quantitatively optimized medium. Appropriate operation conditions

needed to be found in order to reduce or eliminate the undesired production of

metabolic overflow products. Chapter 5 focuses on the development of an optimaldefined medium for growth of the recombinant E. coli HBlOl[pGEc47] and the

determination of the critical dilution rate DR above which undesired production of

metabolic overflow products occurs.

Acetate, the major metabolic overflow product as well as octanoic acid were

known to inhibit growth. The strength and the kinetics of the inhibition are

important parameters for establishing a successful biotransformation process. The

effect of acetate and octanoate on cell growth as well as the kinetics of octanoate

inhibition are described in chapter 6.

The use of cell retention and specific separation of a liquid phase by membrane

filtration is beneficial, if an inhibitory compound is accumulated in the liquid phasepassing the membrane because it allows removal of the accumulated substance

through the permeate stream. A cell-recycle bioreactor system with integratedmembrane filtration was therefore used to remove octanoate from the reaction

system. Chapter 7 describes start-up procedures and biotransformation experimentswith this cell-recycle bioreactor system.

Characterization oforganisms by on-line analyses 15

4 Characterization of organisms by on-line analyses

4.1 Introduction

Scientific investigation of a bioprocess requires representative analyses of the state

variables in the process. Most effort is normally spent on developing analyses for the

detection of the product(s) of the bioprocess. Two other key variables, namely the

biomass concentration and the concentration of the growth limiting carbon source,

may be even more important as they allow a sound characterization of the

bioprocess, which is the basis of a successful formation of the desired product.Continuous measurement of the glucose concentration allows verification of

carbon limited growth of the culture. Today's methods available for glucosemeasurements are good enough for the subsequent calculation of the specific glucoseuptake rate. The off-line determination of the biomass x, however, is still not

sufficiently accurate and frequent (Rothen et al, 1996).

4.2 On-line biomass estimation: possibilities and constraints

The biomass concentration is a key variable because it is used for instance for the

calculation of the specific growth rate (umax), the specific glucose consumption rate

(qs = rs / x) or product formation rates (qp). All mathematical models used to

describe microbial growth or product formation contain biomass as the most

important state variable. The observation of a constant biomass concentration is a

first criterion for assuming a population to be in steady state (Sonnleitner et al, 1992).Off-line analyses involve partially or entirely manual operations, a possible source

for human errors leading to variations in accuracy or precision during work (Fiechterand Sonnleitner, 1994). It is therefore of vital interest to supplement the classical off¬

line determination methods with on-line estimations of biomass.

4.2.1 Optical density

A direct comparison of seven sensors to estimate biomass in bacterial and yeastcultures was made by Nipkow et al (1990). Two of them, a probe measuring the

fluorescence of the culture (fluoro-sensor) and a probe for OD-measurements (OD-

sensor) were used also in this thesis. Locher et al (1992) showed a comparison of off¬

line biomass, OD-probe and fluoro-sensor in batch cultivations of Saccharomycescerevisiae.

The light reflection of the OD-signal is not only influenced by the size and the

number of the cells. The most prominent constraint for use of the OD-signal as

biomass estimation refers to changes of the coalescence of the culture. As long as the

foam production of the culture remains constant, the signal of the OD-probe is veryvaluable for biomass estimation. This is, however, the case only in very rare

situations.

16 Characterization oforganisms by on-line analyses

o

crro

^"

C\|

oo

-1

OD by aquasant

co2 ln(aqua/10)

6 9

time [h]

'aqua

15

Figure 4.1: Comparison of C02- and OD-signal in normal and semilogarithmic representation duringbatch growth of HB101[pGEc47].Medium SM3 containing 3 % glucose, preculture SM3-SK, 3 I bioreactor volume, 1 wm.

8c

CDUCO

2>o

5=

CM

oO

4 5 6

time [h]

10

Figure 4.2: Comparison of C02- and fluorescence-signal in normal and semilogarithmic representation

during batch growth of HB101[pGEc47].Medium SM2 containing 1 % glucose, 0.1 g I"1 L-leucine and 0.1 g I"1 L-proline, with 5 mg I"1

tetracycline, preculture LB, 3.5 I bioreactor volume, 1 wm.


24 48 72 96 120 144 168 192 216 240

time [h]

Figure 4.3: Shifts in dilution rate applied to a continuous culture of HB101[pGEc47].Medium SM1, with 5 mg I"1 tetracycline, bioreactor volume 3.8 I, 0.5 wm.

The data-points of the fluorescence signal were reduced using dynamic data reduction, the

biomass data were fitted using cubic spline interpolation (MATLAB spline-function).

In low density cultures the signal is, apart from gas bubbles, furthermore

influenced by light coming from outside of the bioreactor. A typical OD-signalduring batch growth therefore first decreases when the influence of entering lightdecreases with increasing cell density. The signal then increases over-exponentiallydue to the formation of biomass and increasing foam production towards the end of

the batch cultivation (figure 4.1). The use of this sensor in a two-liquid phasebiotransformation system as biomass signal is limited because of the high coalescence

activity of both the biotransformation substrate and product. The slightest change in

either octane or octanoate concentrations resulted in responses of the OD-signal in

the range of several % of the absolute signal level. It finally turned out that the OD-

probe could be best used as indicator of an upcoming infection as infections almost

always completely destroyed any foam production, which could be seen by a

reversed trend of the OD-signal long before a second organism could be detected

under the microscope.

4.2.2 Fluorescence

The fluoro-sensor is measuring the intracellular pool of NADH and NADPH. If

the mass fraction of this pool with respect to biomass does not change during a

cultivation and is sufficiently large, then biomass can be estimated from this signal(Sonnleitner et al, 1992).


Table 4.1: Maximal specific growth rates of two batch cultivations (taken randomly out of a total of 76

batches performed in this thesis) calculated from biomass measurements (gravimetrical cell

dry weight) and indirect estimation from carbon dioxide exhaust gas measurement.

Batch* Mm«(biomass) [h'1] Mm«(COj[h-1] AMm„ [h_1]

28

43

0.3086

0.4008

0.3118

0.4048

0.0032

0.0040

The use of the fluoro-sensor as indirect measurement of the specific growth rate,

however, is questionable. In not one single batch experiment measured with the

fluorescence probe was a semi-logarithmic plot of the signal linear (figure 4.2). The

specific size of the NAD(P)H pool seems to increase during batch cultivation time.

The signal of the fluorescence probe, however, can be used for the relative, non

quantitative biomass estimation in steady state situations. Figure 4.3 shows

fluorescence signal and biomass measurements during several steps in dilution rate

applied to a continuous culture of E. coli HB101[pGEc47]. Although no quantitativestatement is possible, the relative course of the biomass concentration is well

represented by the fluorescence signal. Finally, the most valuable pieces of

information are given by this sensor in regard to steady-state estimations. The signalof the fluoro-sensor was in most cases (during this work) the last one reaching steadystate. A macroscopic steady-state can therefore safely be assumed if the signal of the

fluorescence probe no longer shows any relevant deviations during several mean

residence times.

4.2.3 Carbon dioxide

Park et al (1983) assumed a linear correlation between biomass and carbon dioxide

evolution rate and exploited this model for the estimation of cell concentration. Since

the maximal specific growth rate (umax) is directly correlated with the biomass

concentration, umax can be reliably estimated from exhaust gas analysis of the carbon

dioxide (table 4.1).The calculated differences are not significant because they are smaller by far than

the accuracy of biomass determination and C02-measurement. The estimation of the

specific growth rate from the semi-logarithmic plot of the C02-signal versus time

during an unlimited, exponentially growing culture is therefore identical to the

specific growth rate obtained from cell dry weight measurements with a precision of

± 0.01 h1 (confirmed by additional comparisons, data not shown).

Figure 4.4 shows an example of two batch cultivations with almost identical CO2-

curves during the exponential phase. Calculation of the specific growth rate from the

semi-logarithmic plot of the C02 signal gives a value of 0.4048 h"1 for batch 43 (seetable 4.1) and 0.4129 h1 for batch 23, with a difference of 0.0081 h1 between the two

signals, which is again not significant.


4.5

4

3.5

3

E 2-5

CM

°o

O 2

1.5

1

0.5

batch 43

batch 43

18

Figure 4.4: Batch growth of HB101[pGEc47] monitored via C02-production measured in the exhaust

gas.

Batch 23: medium SM2 containing 1 % glucose, 0.1 g I"1 L-leucine and 0.1 g I'1 L-proline,with 5 mg I"1 tetracycline, preculture LB, 3.51 bioreactor volume, 1 wm.

Batch 43: medium SM3 containing 3 % glucose, preculture SM3-SK, 3 I bioreactor volume,

1 wm.

4.3 The growth limiting substrate glucose

The growth limiting substrate plays a key role among the measurable substances.

In all of the experiments described, the growth limiting substrate was intended to be

glucose and the cultures were therefore expected to be carbon-limited.

Continuous control of the glucose concentration during batch growth is importantfor characterization of the growth behavior and medium control. It must be

guaranteed that the organisms can grow exponentially until depletion of glucose.

Figure 4.5 shows an example where exponential growth of the culture ceased at a

residual glucose concentration of almost 10 g l"1. The p02-signal sharply increased

and the course of the glucose signal changed from exponential decrease (followingthe p02-signal) to linear, indicating that a limitation other than glucose occurred. The

described phenomenon could be correlated to L-proline limitation (see chapter 5), an

amino acid for which E. coli HBlOl[pGEc47] is auxotrophic.The fact that glucose is the limiting substrate must be verified continuously. This

can be done by simply measuring the concentration in the cultivation fluid. If it is in

the range of Ks or below the detection limit of the analyzer it can be assumed that the

culture is glucose-limited. Double limitations, however, cannot be detected like this.


90 1 i 1 1 1 1 1 1 1 1 25

80 _^;""^^S^-. p02

70 -

".^

.3^T\. s^^"^20

60 -

V ., //"V [

15 *T

[%] 50 -

""a. S

CM "~-\ CD

oQ. 40

30 -

-

co

810 i,

20 -

'—**

--*.

5

10 glucose

Ql I I I I I I I , I I I l__l Q9 10 11 12 13 14

time [h]

Figure 4.5: Partial pressure of oxygen and glucose concentration during batch growth of

HB101[pGEc47] between 8.5 h and 14 h after inoculation.

Medium SM3 containing 3 % glucose, preculture SM3-SK, bioreactor volume 3.5 1,1 wm.The glucose concentration was monitored on-line with flow injection analysis at a samplingrate of 60 h'1.

The method of choice therefore is to apply pulses of the limiting substrate to a

continuous culture. Figure 4.6 shows a series of two glucose pulses (2.5 and 5 g l"1)applied to a continuous culture of E. coli HBlOl[pGEc47]. The C02-concentrations

during both pulses reached the same level, which was maintained until glucose was

fully taken up from the cultivation liquid. Obviously the glucose pulses transformed

the state of the culture from one limited by glucose to a state linuted by another

component. This was expected, as the medium used in this experiment was designedbased on individual yield coefficients for specific medium components to avoid

unwanted excess of medium components other than glucose (see chapter 5).The monitoring of the glucose concentration in the cultivation liquid is

furthermore of paramount importance for the quantification of the dynamics of the

metabolism of cells (Rothen et al, 1996). A good example for this are synchronizedyeast cultures.

The variation of qs, and most probably also s and Ks, may act as so called

attractors for initiation and maintenance of spontaneous synchronization (Hjortsoand Nielsen, 1993). Munch et al (1992b) postulated that oscillations of glucoseconcentration would occur during synchronized cultivation of Saccharomycescerevisiae.


2.5

time [h]

Figure 4.6: Two pulses of glucose (2.5 and 5.0 g I"1) applied to a continuous culture of HB101[pGEc47]running at a dilution rate of 0.1 h"1 on medium SM3 containing 3 % glucose.The glucose concentration was monitored on-line with flow injection analysis at a samplingrate of 60 h"1.

On-line measurements of byproducts such as acetate and ethanol are possible,unfortunately with relatively low time resolution (figure 4.7). Experimentalverification of the glucose concentration (figure 4.8) now confirmed the mechanistic

proposals of Miinch et al. (1992a,b). It is known, as a rule of thumb, that the

measurement frequency must be 10 times faster than the frequency of the dynamicsignal. In the case of glucose measurement of oscillating cultures a measuringfrequency of about 120 h"1 is required to fulfill this rule. On-line measurements byGC do by far not fulfill this requirement (measurement frequency of 10 h1).Although the measurement frequency of the FIA is still too low by a factor of two

(measurement frequency of 60 h"1) the short transient periods in the substrate

concentration could be trapped reproducibly (figure 4.8).It might well be that ethanol or acetate concentration in the culture liquid showed

the same dynamic response as the glucose concentration but, simply, they could not

be seen due to the insufficient measurement frequency of the GC. Note that the

duration of the transient glucose peak is only 5 min at most and the time difference

between on-line GC measurements is at least 6 min.


8 10

time [h]

12 14 16

Figure 4.7: Continuous culture of Saccharomyces cerevisiae H1022 (ATCC 32167) at a dilution rate of

D = 0.15h"1.Medium D (Hug et al, 1974) containing 3 % glucose, bioreactor volume 4 I, temperature 30

°C, pH 3.5,1 wm.

The GC measurements of ethanol (*) and acetate (x) during 5 oscillations of a synchronizedculture were made on-line with equipment described by Filippini et al (1991) at a

measurement frequency of 10 h"1.

4.4 Miniaturization: a step back to the future

On-line analytical systems require that the analyses be fast. Taking into account

the volume of a bioreactor — on the research level typically some liters — it soon

becomes clear that an analytical system for on-line monitoring has to require very

small sample volumes. Being fast and requiring small sample volume are the two

features that can easily be provided by miniaturized analytical systems.One way to create miniaturized analytical systems is the use of micro-chips. Chips

with different functionalities are stacked together to form the different sections of the

micro-analyzer. Usually there is a pumping section, a section for chemical reactions

and a detection region. A stack element for fluid handling can contain any channels

and holes to form proper connections or reaction loops. There are no fittings neededto connect the stackable elements, which minimizes the dead volume.

The combination of FIA and microsystem technology can result in multi substrate

analyzers requiring less than 10 ul of sample volumes. Furthermore, the up to 1000

fold reduction of the required reagent volume (compared to normal FIA as used in

this thesis) allows the use of enzyme solutions for (bio)analytics at reasonable costs.


CM

oo

TO

E,CDCOOO3

TO

1

26

22

18

10.

v* "V

I 14\.^ %W>^ V-^w**

0 4 6

time [h]

8 10 12

Figure 4.8: Continuous culture of Saccharomyces cerevisiae H1022 (ATCC 32167) at a dilution rate of

D = 0.15 h"1. Cultivation conditions as in figure 4.7.

The glucose concentration during 3 oscillations of a synchronized culture was measured

using on-line FIA at a frequency of 60 h'1

A stack was applied to a culture of Pseudomonas oleovorans which contained a 4-

glucose biosensor array (2 biosensors without and two with catalase membrane) and

two optical analogues which were sensitive for glucose as well (Busch et al, 1996).This resulted in a sensor capable of delivering glucose data on 6 individual channels.

The outputs of all these 6 sensors were compared with an external makro FIA of

normal size, the measurement of which was based on the GOD-POD-ABTS reaction

(Rothen et al, 1996).

Figure 4.9 shows the signals of the optical biosensor analogues compared to the

measurements of the makro FIA during two repetitive batch cultivations of

Pseudomonas oleovorans performed on 1 % glucose media. The data are in goodagreement which demonstrates that despite the reduction in size by a factor of more

than 50'000, the uFIA is capable of delivering data that are useful for bioprocessmonitoring.

Characterization oforganisms by on-line analyses

9: Glucose concentration of two repetitive batch cultures of Pseudomonas oleovorans Gpo1

(ATCC 29347).Defined medium bsm (10 g I"1 glucose, 1.18 g r1 NH4CI, 0.4 g I'1 (NH^HPO* 0.25 g r1

MgS04 7 H20, 1 ml MT-solution per I medium), bioreactor volume 4.0 I, 0.38 wm, 30 °C,

pH7.The measurements were made on-line at the same time attached to the same interface

(analytical loop with cross-flow filter) with normal size FIA (—•—) at a frequency of 60 h'1

and with a uFIA (two channels, x,+) at a frequency of 20 h"1.

Development ofan optimal defined mediumfor HB101[pGEc47] 25

5 Development of an optimal defined medium for HBlOl(pGEc47)

5.1 Introduction

Efficient production requires both a high concentration of biomass and a high

specific product formation rate (rP = qP x). Increasing the productivity is therefore

possible by increasing the specific productivity or the biomass concentration or both.

Maximum biomass concentrations are usually reached by addition of complexcomponents such as yeast extract, tryptone and casamino acids to culture media

(Olsson and Hahn-Hagerdal, 1995; Chung et al, 1992; Ye et al, 1992; Hasio et al, 1992).More recently, considerable efforts have been spent on cultivating E. coli to high cell

densities on defined media (Weuster-Botz et al, 1995; Korz et al, 1995; Riesenberg et

al, 1994). Minimal media are preferred because they are required for causal analyticalstudies of metabolism, kinetics and regulation (Fiechter and Sonnleitner, 1994). Theyallow the quantification of carbon compounds as well as calculation of specific rates.

In addition, the use of minimal media might allow savings compared to the use of

richer media due to lower medium costs and to savings in the removal of unknown

and unwanted complex compounds during downstream processing.Most of the media from literature were originally designed for use in shake flask

experiments. The buffering capacity of such media is usually enhanced by addingphosphate-salts in excess. This can lead to precipitations and even inhibitions at

higher medium concentrations (Riesenberg, 1991).The appearance of metabolic overflow products during aerobic growth in media

containing glucose might be problematic. The carbon flux is directed into an

undesirable by-product resulting in a lower biomass-yield. Acetate, the main

overflow-product of E. coli, inhibits growth at concentration greater than 10 g l"1 (Panet al, 1987). The production of this overflow product depends on the particular strain

and its cultivation conditions (Riesenberg et al, 1991). In continuous cultures the

production of by-products occurs above a "critical" dilution rate (DR). At this dilution

rate, the regulation of the glucose metabolism is switched to by-product formation.

In literature, DR is often referred to as critical growth rate u^ (Korz et al, 1995;

Riesenberg et al, 1991). Values of DR for E. coZi-strains are in the range between 0.17

h1 (Korz et al, 1995) and 0.35 h"1 on defined media (Riesenberg et al, 1991). An

effective biotransformation must be operated at dilution rates lower than DR.

26 Development ofan optimal defined mediumfor HB101[pGEc47]

ryL i i i i i i i i

0 3 6 9 12 15 18 21 24 27

time [h]

Figure 5.1: Batch growth of E. coli HB101 (x,+) and E. coli HB101[pGEc47] (o,«) on minimal medium

SM1 (+,•) and yeast extract supplemented medium SM1YE4 (x,o).Bioreactor volume 3.5 1,1 wm, media with addition of 5 mg I'1 tetracycline.One batch was performed with heat sterilized medium (*); the medium for all other batches

was filter sterilized (0.2 u).

5.2 Results

5.2.1 Approach to the development of an optimized medium

Fiechter and Sonnleitner (1994) described the principles of medium design.Evaluation of responses of a population to transient disturbances permits the

identification of limiting or inhibiting components. The first step is the identification

of limitations to obtain complete information about required medium components.This is done by testing the growth response of a culture to pulses of individual

components or groups of substances (pulse technique). A small (10-20 ml) volume of

dissolved substance is introduced into the bioreactor with a syringe or a pulse pumpwithin a short time frame (10-20 s, ideally as A-Dirac). Compounds which cause

increased growth (glucose and 02-uptake, cell mass increase) are assumed to be

limiting in the original medium and are added to the medium to eliminate the

limitation while undesired components are probably reduced or even eliminated. In

a second step the culture is then shifted to the new medium (shift technique). Pulse

and shift technique are repeated until all of the limitations are identified leading to a

qualitatively optimized medium.


211 1 r

19.2

-1 QI 1 1 I I I I I

0 2 4 6 8 10 12 14

time [h]

Figure 5.2: Effect of medium supplements on the oxygen concentration during exponential growth of E.

coli HB101 [pGEc47].A: medium SM1YE4 with 5 mg I"1 tetracycline, preculture grown on LB, B: medium SM1

with 5 mg I"1 tetracycline, preculture grown on LB, C: medium SM1 with 5 mg I"1

tetracycline, preculture grown on M9*. Bioreactor volume 3.5 1,1 wm.

Essential medium components can even exert an inhibitory effect at higherconcentrations. This is one of the reasons why the exact requirement of the organismfor the individual components should be known, i.e. the yield coefficients YX/s,i (gbiomass per g substratei) have to be determined for designing a quantitativelyoptimized medium. Therefore the organism is cultivated in continuous culture on a

medium with a limited amount of the compound of interest.

After reaching the steady state and measuring the biomass concentration, the

culture is either shifted up or down to a higher or lower amount of the same

component in the medium to establish another steady state. The yield can be

estimated from the slope of the linear part of the correlation between the biomass

and the substrate concentration.

Special attention was given to amino acid utilization as the investigated E. coli

strain is auxotrophic for leucine and proline.


Table 5.1: Growth comparison of E. coli strain HB101 and strain E. co//HB101[pGEc47]Yield coefficients Yx/gic [9 biomass per g glucose] and maximal specific growth rate u,

(h"1) determined from batch growth on different media.

Medium HB101 HB101[pGEc47]

YX/S Mmax Yx/S Mmax

SM1YE4 0.51 ±0.02 0.43 ± 0.02 0.50 ± 0.01 0.44 ± 0.01

LB precultureSM1 0.35 ±0.01 0.27 ±0.01 0.34 ± 0.01 0.27 ± 0.01

LB precultureSM1 0.33 ± 0.01 0.27 ± 0.01

M9* preculture

5.2.2 Growth of Escherichia coli HBlOl and HBlOl (pGEc47) in the absence

and presence of yeast extract

E. coli HBlOl and HBlOl[pGEc47] were grown in batch culture on media SMI and

SM1YE4. Figure 5.1 shows that there was no significant difference in the growthbehavior of HBlOl and HB101[pGEc47].

Addition of 0.4 % yeast extract increased the specific growth rate (umax) during

exponential growth on medium SMI from 0.27 + 0.01 h1 to 0.43 + 0.02 h1. The yieldcoefficient (YX/s) for the carbon source glucose was 0.34 ± 0.02 g g1 for both strains.

Addition of yeast extract to SMI resulted in a higher apparent yield, namely 0.51 ±

0.02 g g1. Based on the carbon balance (data not shown) this was due to the

additional carbon sources supplied with the addition of yeast extract. The

characteristic parameters are summarized in table 5.1.

The results of figure 5.1 and table 5.1 show that yeast extract had a significanteffect on the growth of both strains when added to minimal medium. The effects of

yeast extract were tested further by growing HBlOl[pGEc47] in media containingdifferent yeast extract concentrations (figure 5.2).

The growth of the microorganisms is reflected by their oxygen consumption

(figure 5.2). Culture A was grown on medium SMI with addition of 4 g l~x yeastextract. Cultures B and C were grown on minimal medium SMI, but a small amount

of about 0.2 g l1 yeast extract was introduced in culture B with the LB-preculture,while a minirnal-rnediurn preculture was used for inoculation of culture C.

The addition of yeast extract influenced the growth of E. coli HB101[pGEc47] in

two ways: it increased the growth rate of HB101[pGEc47] from 0.27 h"1 to 0.43 h"1 and

it also led to an alteration of the steadily decreasing oxygen signal; a period of

decrease was suddenly followed by an increase before it decreased again. These

increases were very sharp in the case of curve A, while the alterations of curve B

were more moderate and were absent in C.


"0 0.02 0.04„0.06 0.08

S04 [g I"1]0.25 0.5 ,0.75 1

NH4 [g r1]

0.2

L-leucine [gl"']0.3

Figure 5.3: Cell dry mass yields of E. co//'HB101[pGEc47] on sulfate, ammonia, L-proline, L-leucine.

E. coli HB101[pGEc47] was grown continuously at a dilution rate of 0.2 h'1 and 1.5 wm in

minimal medium (table 5.2), containing the limiting component at the concentration

indicated. The steady state concentration attained after 50 h is plotted; the yield coefficient

is represented by the slope of the line.

5.2.3 Metal and vitamin requirements of E. coli HB 101(pGEc47) during growthon medium SMI

E. coli HB101[pGEc47] was grown continuously at a dilution rate of 0.2 h1, which

was below the critical dilution rate DR (see 5.1.5). Pulse experiments were carried out

to determine whether additional trace metals and vitamins might enhance growth.The addition of trace elements (1 ml MT-solution per 3.8 1 reactor volume, 10 umol

NiS04, 10 umol A1C13, 10 umol (NH^Mo^ 4H20) and vitamins (3 mg Ca-

pantothenate, 6 mg meso-inositol, 0.17 mg pyridoxalhydrochloride, 3 ug biotin per3.8 1 reactor volume) had neither beneficial nor negative effects on the growth of

HBlOl[PGEc47].

5.2.4 Requirements of E. coli HBlOl(pGEc47) forP, N, S, L-Pro, L-Leu

HBlOl[pGEc47] was grown in continuous culture at a dilution rate of 0.2 h1 on

special media designed to establish nutrient limited growth (see table 5.2). The

limitation was verified by adding the limiting component.


Table 5.2: Composition of growth media used to establish nutrient-limited growthTrace elements and thiamin according to medium SM1, 5 mg I"1 tetracycline. Additional Mgin S04-Iimited cultures was added as MgCl2. Additional SO4 contained in trace elements

solution was considered when calculating the S04-yield.

Limitations of

Glc NH4 SO4 PO4 L-Leu L-Pro

Glucose. H20 [g I"1] 33 33 33 33 33 33 33 33 33 33

NH4CI [g r1] 6 3.0 1.5 6 6 6 6 6 6 6

MgS04 . 7H20 [g I-1] 0.72 0.72 0.72 .171 .078 0.72 0.72 0.72 0.72 0.72

H3PO4 (85%) [g I"1] 3.39 3.39 3.39 3.39 3.39 0.34 3.39 3.39 3.39 3.39

L-Leu [g r1] 0.6 0.6 0.6 0.6 0.6 0.6 0.3 0.15 0.6 0.6

L-Pro [g r1] 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.15 .075

Table 5.3: Yield coefficients for three different strains of E. coli

Yield coefficients (g biomass per g substrate) determined in continuous cultivations of E.

coli HB101[pGEc47] at a dilution rate of 0.2 h-1 and a medium content of 3% glucose.

E. coli £.co/i B/r1 E.coli X902

HB101[pGEc47]

Yx/nh4+ [9 g"1l 6±14 4 8

Yx/po43- [g g"1] 14±13 12 11

Yx/so42- [g g'1] 50 ±24 93 54

L-Leu [g g"1] 20 ±14

L-Pro [g g"1] 42 ±24

1Reilingetal(1985)

2 Yee and Blanch (1993)3 based on 2 independent measurements

based on 4 independent measurements4

A positive reaction (increase of biomass, decrease of glucose, increase of

respiratory activity) of the culture to the pulse indicated that the pulsed substance

was indeed the limiting component. The yield coefficient for the component in

question was determined by measuring the biomass concentration and the residual

concentration of the limiting component during steady state continuous growth.Steady state was considered to be established after at least 10 mean residence times,and verified by determining that the on-line signals for CO2, 02, p02, redox and

fluorescence had been constant for at least 2 mean residence times (10 to 14 h).The measured yields for N, P and S as well as for L-Leu and L-Pro are listed in

table 5.3. The yields were calculated under the assumption that all of these

components are essential, i.e. no growth is possible when the limiting component is

completely absent (figure 5.3).


10 12

time [h]

Figure 5.4: L-proline limitation during batch growth of E. coli HB101[pGEc47] on medium SM2

containing 3 % glucose, with addition of 5 mg I"1 tetracycline.Bioreactor volume 3.5 I, 1 wm, LB preculture.Batch A

, grown on SM2 containing 0.3 g I'1 L-leucine and 0.3 g I"1 L-proline, ran into a

limitation indicated by an abrupt decrease of the CCysignal at relative time 12.2 h. A pulseof 2.0 g L-proline (at relative time 15.7 h) in the following plateau-phase of the CCysignal

(indicated by *) positively demonstrated a proline-limitation. The second batch B was

performed on SM2 containing 0.3 g I"1 L-leucine and 0.9 g I"1 L-proline. The increased

amount of proline in the medium prevented a limitation and glucose was completelyconsumed by the organisms.

5.2.4.1 Amino acid utilization

The yields for L-proline and L-leucine listed in table 5.3 have been determined

during continuous cultivation at a glucose concentration of 30 g l"1 in the medium.

Additional experiments showed, however, that these yields varied under different

cultivation conditions.


0.41 S_\ 0.33

"""0 2 4 6 8 10 12 14

time [h]

Figure 5.5: Batch growth of E. co//HB101[pGEc47] on different amounts of L-leucine.

Medium SM2 containing 3 % glucose and 0.3 g I"1 L-proline, LB preculture.Growth is monitored via C02-production measured in the exhaust gas; the logarithmic plotof the C02-concentration results in slopes of 0.41 (for 0.3 g I"1 L-leucine) and 0.33 (for 0.9 g

I"1 L-leucine) respectively.

Batch cultures of E. coli HBlOl[pGEc47] in media with sufficient L-Pro based on

the yield determined in continuous cultivations always became limited prematurely.Addition of L-Pro relieved the limitation (figure 5.4). Accordingly, the yielddetermined for L-Pro differed when the organism was cultivated in batch (YX/Pr0 = 20

g g1) or continuous (YX/Pr0 = 42 g g1) mode.The yield for L-leucine decreased when E. coli HBlOl[pGEc47] was grown in

continuous cultivation on media containing increasing amounts of glucose: while at 1

% glucose Yx/Leu was 40 g g1, at 3 % glucose YX/Leu had decreased to 20 g g"1.Contrary to L-Pro no extra need was observed in batch cultures.

The effect of L-leucine on the growth of E. coli HBlOl[pGEc47] is demonstrated in

figure 5.5. Two batch cultures of E. coli HB101[pGEc47] on medium SM3 are shown,

one containing 0.3 and the other one 0.9 g l"1 L-Leu. The increase of L-Leu from 0.3 to

0.9 g l1 reduced the maximal specific growth rate from 0.41 h"1 to 0.33 h"1, as

estimated from the C02-signal.


1

co jt^E /g jf

-2- s*

"3" /^^

i i i i i i i i

2 4 6 8 10 12 14 16

time [min]

Figure 5.6: Batch growth of E. coli HB101[pGEc47] on media SM1YE4 (+) containing 5 mg I"

tetracycline and on the optimized medium SM3 (*).

5.2.4.2 Medium SM3

Based on the yields determined above a medium SM3 was formulated which

contains all required components in slight excess relative to glucose (table 5.4). This

medium permitted optimized glucose limited batch growth of E. coli HBlOl[pGEc47]at specific growth rates of 0.41 - 0.45 h"1 similar to these found with a complexmedium containing 0.4 % yeast extract (figure 5.6). The increase of the growth rate

achieved on medium SM3 compared to SMI must be related to the reduced amount

of the inhibiting L-Leu.

5.2.5 Determination of Dr

Metabolic overflow products decrease the biomass yield YX/s because of

redirection of carbon into undesired products which are excreted into the medium.

The main by-product of E. coli cultures is acetate which was reported to

significantly inhibit growth at concentrations above 10 g l"1 (Pan et al, 1987).Formation of metabolic overflow products occurs above a critical specific growth rate

(Ucrit)-


Table 5.4: Chemically defined minimal medium SM3 for glucose limited growth of E. coli strain HB101

[pGEc47] with reduced excess of other medium components.The concentrations are listed as g I"1 for a medium containing 1 % glucose. Tetracycline (10

mg I"1) and citric acid (2 g I'1) were added independent of the glucose content of the

medium.

Component concentration in requirement comment

medium [g I'1] according to

yield [g r ]

Glucose H20 11 11 = 10g I"1 water free;

C-source

NH4CI 2.0 1.7 N-source

MgSC-4 7H20 0.24 0.17 Mg- & S-source

H3PO4 (85 %) 1.13a 0.28 P-source

L-proline 0.1 [0.2]b 0.075 required growth factor

L-leucine 0.2 0.165 required growth factor

Thiamine 0.001 ?c required growth factor

CaCI2 2H20 0.00735 ?c trace element

FeS04 7H20 0.00556 ?c trace element

C0SO4 7H20 0.00281 ?c trace element

MnCI2 2H20 0.00162 ?c trace element

CuCI2 2H20 0.00017 ?c trace element

ZnS04 7H20 0.00029 ?c trace element

NaOH & KOH; according to . titrant mixture &

equimolar; 10 N pH-controller Na+- & K+-source

amount of phosphoric acid needed to guarantee supply of Na+ and K+ added with the

titrant mixture

the higher concentration is needed in batch growth to avoid Pro-limitation towards the

end of the culture but not in chemostat (according to calculated yield and pulse-tested)

yield not determined experimentally; neither limiting nor inhibitory at givenconcentration according to indifferent (= neither negative nor positive) pulse-responses


^ 3.5

co 3CO

co

1 2.5

0

„

0.35 -

3-

i-

© 0.25co 2^^ \4

-

ii —-

c

o 0.15 i--

**

_3^ y^^ \^ 5

0.05

i i i i ! I

12 16

time [h]

20 24 28 32

Figure 5.7: Continuous growth of E. co/;'HB101[pGEc47] at changing dilution rates.

Medium SM1 with 5 mg I"1 tetracycline, bioreactor volume 3.8 I, 0.5 wm.

The cultivation started at a dilution rate of D=0.05 h"1. At t=0, D was increased from 0.05 to

0.35 at a rate of 0.02 h"2. From 15 to 23.5 h D was kept constant at 0.35 h"1, after which D

was decreased at a rate of -0.05 h"2 until the initial dilution rate of 0.05 h"1 was reached

again. (*) represents the biomass concentration.

The two lines of the dilution rate represent the setpoint (straight line) and the actual outputof the controller signal (oscillating curve, the controller was badly tuned in the beginning of

the experiment).

Continuous cultivation of E. coli HBlOl[pGEc47] at dilution rates lower than DR =

Ucrit avoids the production of overflow products, thus resulting in almost zero acetate

production and maximal biomass yield.To determine the value of Dr, HBlOl[pGEc47] was grown in continuous culture at

different dilution rates. The dilution rate was increased and decreased continuouslyas described for yeast (Sonnleitner and Hahnemann, 1994). The dilution rate was

increased at a rate of 0.02 h~2 from 0.05 h"1 to 0.35 h1, followed by a constant period of

0.35 h"1 equivalent to 3 volume changes and, later, a decrease to 0.05 h"1 at a rate of

0.05 h2 (figure 5.7). Samples were taken for determination of biomass, glucose and

products formed.


,." 4

dilution rate [h~

Figure 5.8: Specific glucose consumption rate (qs) and biomass concentration at different dilution rates

Medium SM1 with 5 mg I'1 tetracycline, bioreactor volume 3.8 I, 0.5 wm.

The experimental setup is the same as for figure 7. The arrows indicate whether the dilution

rate was increased or decreased. A deviation of the data from the dotted line can be seen

between 0.23 h"1 (increasing ramp) and 0.26 h"1 (decreasing ramp).

Figures 5.7 and 5.8 show that the biomass concentration achieved was essentiallyconstant (3.3 ± 0.15 g l1) at dilution rates from 0.05 h"1 to 0.23 h1, after which it

decreased to 2.8 g l"1 at D = 0.35 h1. At dilution rates lower than 0.2 h1, the

concentration of acetate was always less than 0.5 g l"1. At higher dilution rates,

however, higher concentrations of acetate were excreted into the medium.

Figure 5.8 shows that the specific glucose consumption rate (qs) is a linear function

of dilution between 0.05 h"1 and 0.23 h"1 (for increasing dilution rates) and between

0.26 h'1 and 0.05 h1 (for decreasing dilution rates). YX/s was constant at 0.34 ± 0.01 g

g1 over this dilution rate range. When the dilution rate was increased above 0.23 h1,the yield decreased due to acetate production. Thus Dr, the dilution rate, at which

production of acetate begins, is approached from each side in the two different

experiments and is approximately 0.23 - 0.26 h"1.

A graphical representation of three on-line signals, namely the redox signal, the

fluorescence and the carbon dioxide production rate, versus time is presented in

figure 5.9.


-10

0.1 0.15 0.2 0.25 0.3 0.35 0.4

dilution rate [h ]

Figure 5.9: Redox, fluoresence and carbon dioxide production rate signals monitored at increasing and

decreasing dilution rates (ramps as shown in figure 5.7).Medium SM1 with 5 mg I"1 tetracycline, bioreactor volume 3.8 I, 0.5 wm.

(1) continuous growth at a constant dilution rate of 0.05 h"1

(2) continuously increasing dilution rate from 0.05 h"1 to 0.35 h"1 at a rate of 0.02 h"2


(4) continuously decreasing dilution rate from 0.35 h"1 to 0.05 h"1 at a rate of 0.05 h'2

a and b distinguish the signals into two parts according to the pattern of the signal


All three signals showed steady state (almost no deviation in Y-axis for 14 h) at the

starting point (1). During increase of the dilution rate (2), the effect of the badly tunedcontroller was visible in all three signals. The redox signal and the carbon dioxide

production rate reached the same level as was reached afterwards during the

decrease of the dilution rate (4). After the increase in dilution rate both signals againshowed steady state behavior (3). The fluorescence signal behaved differently. It

reached neither the same level during the increase of the dilution rate (2) as then

reached during the decrease (4) nor did it show steady state behavior at the top level


dilution rate (3). Instead, a decrease of the signal and thus of the NAD(P)H-poolcould be observed. The decreasing part of the dilution rate pattern is characterized

by a smooth increase of the fluorescence signal. The decrease of the dilution rate

forced a continuous increase of the NAD(P)H-pool.In contrast to this, redox and CPR showed two phases during the decrease of the

dilution rate. During the first phase (4a), representing the reconsumption of the

formerly produced metabolic overflow products, both signals remained at a constant

level. Then, in the second phase (4b), they suddenly left their constant course: the

redox signal started to increase earlier than the carbon dioxide production rate

started to decrease. The redox potential and the respiratory activity therefore

depended more on the metabolic activity of the cells than on the dilution rate. The

carbon dioxide production rate reached exactly the starting point (5), which was not

the case for the fluorescence and the redox signal.

5.3 Discussion

The growth of E. coli strains HBlOl and HB101[pGEc47] on defined and complexmedia revealed that the recombinant and host strains responded equally to different

growth conditions, showing that in the absence of induction, pGEc47 had no effect on

the growth of the recombinant.

The addition of 0.4 % yeast extract increased the growth rate of HBlOl[pGEc47] byabout 60 % compared to growth on SMI without yeast extract. The low residual

amount of yeast extract, which was added with a complex preculture (estimated to

be less than 0.2 g l1) did not increase the maximum specific growth rate but it did

allow exponential growth until depletion of the carbon source.

When HBlOl[pGEc47] was grown on SMI without yeast extract (figure 5.2, curve

C) the oxygen signal decreased smoothly. When the medium contained yeast extract,

the oxygen signal decreased more rapidly, reflecting the higher growth rate, and

showed reproducible discontinuities. This can be explained by polyauxic growth on

different carbon sources present in the yeast extract. When the first and most

favorably consumed carbon source is exhausted, the cells adapt their metabolism to

the utilization of the next favorable carbon source until this substrate is also

exhausted. The changes from one carbon source to the other result in an alteration in

the steady decrease of the oxygen signal.The results from the pulse experiments reveal that medium SMI contained all of

the essential minerals and vitamins required for optimal growth of E. coli

HB101[pGEc47]. The absence of negative responses indicated that none of the pulsedsubstances was supplied in inhibitory concentrations.

The determination of DR showed that above 0.23 h"1 the glucose yield decreased

due to production of metabolic overflow products. The requirements of E. coli

HB101[pGEc47] were therefore identified at a dilution rate of 0.2 h"1 which

guarantees optimal growth of the microorganisms.The optimization of the growth characteristics of E. coli HBlOl[pGEc47] was

performed with regard to its capability of converting octane to octanoic acid. An

optimal biotransformation requires maximal activity of the biocatalyst, which, at a

constant activity relative to biomass, implies a maximal biomass. The production of


acetate lowers the biomass yield and, thus, probably decreases the performance of

the biotransformation. As no acetate is formed at a dilution rate of 0.2 h1, the

medium optimization was carried out at this dilution rate, which is lower than DR.

The assumption that no growth is possible when the limiting component is

completely absent is true for the two amino acids and sulfate. The data obtained for

aminonia-limitation indicate that at low ammonia concentrations the amino acids

could serve as nitrogen source (positive interception on Y-axis). Another explanationmight be that the yield-coefficient for ammonia is not constant but decreases (from5.8 to 4.7 g g1) with increasing ammonia concentration. This was also reported byYee and Blanch (1993) and Thompson et al (1985); the latter correlated this behavior

with increased production and secretion of amino acids at higher ammonia

concentrations.

The optimization of the medium content of L-proline and L-leucine is of specialimportance as the two amino acids account for about one third of the total medium

costs. The requirements for these amino acids, relative to a constant glucose content

of 1 %, varied from 0.4 g H (Favre-Bulle et al, 1993) to 0.8 g L1 (Mak et al, 1992) and

1.0 g l1 (Seo and Bailey, 1985) for L-Pro, and from 0.25 g l1 (Seo and Bailey, 1985) upto 2.2 g l1 (Toray, 1989, Japanese patent JP-J01051078) for L-Leu.

Yee and Blanch (1993) reported an inhibitory effect for amino acids synthesizedfrom pyruvate. L-Leu is a member of the pyruvate family and, in fact, an inhibitoryeffect of L-leucine could be observed (figure 5.5). According to Gschaedler and

Boudrant (1994) this is due to inhibition of the activity of the LIV-II common amino

acid carrier. L-Pro, however, was reported to be used as growth accelerator for

another recombinant E. coli (Lee et al, 1995).Gschaedler and Boudrant (1994) found for E. coli HBlOl that the glucose

concentration influences the destination of the amino acids and their usage as energysource which might explain the dependency of the leucine yield on the glucoseconcentration.

A comparison of the media SMI and SM3 shows that no new components have

been added to medium SM3 with the exception of citrate, which is used as

complexing agent in order to prevent precipitation of P04 and Mg at highconcentrations (Vogel and Bonner, 1956). Citrate is not consumed by E. coli

(Riesenberg et al, 1990) and can thus be used to prevent precipitations during growthofHB101[pGEc47].

Table 5.5 compares the macro-elements and the amino acids contained in the two

media. In all cases, SM3 contains lower amounts of the medium components than

SMI does. Ammonia and phosphate were reduced only slightly, the latter because

Na+ and K+ were present only in the basic titrant (NaOH/KOH). The sulfate content

was reduced 25 fold. The concentrations of the amino acids L-proline and L-leucine

were reduced to a quarter and a half, respectively. As L-leucine exerts an inhibitoryeffect on the growth of E. coli HBlOl[pGEc47] the reduction of its concentration in the

medium might be the main reason for the increase of the growth rate. The same

growth rate of 0.43 h1 can be obtained with double the amount of L-leucine and

addition of 0.4% yeast extract. Yeast extract apparently decreases the inhibitory effect

of L-leucine.


Table 5.5: Comparison of medium SM1 and SM3

The concentrations of the macro elements and the amino acids are given in g per I and are

calculated for a medium containing 1 % glucose.

Component Medium SM1 Medium SM3

P04[gl_1] 1.20 0.96

S04 [g I*1] 2.33 0.09

NH4 [g I"1] 0.83 0.67

L-leucine [g I"1] 0.4 0.2

L-proline [g I"1] 0.4 0.1

The differences in DR for increasing and decreasing ramps suggest that, althoughit is assumed that u(t) was close to D(t) throughout the entire experiment, steadystates were not reached. This could also be demonstrated by the signals of CPR,

fluorescence and redox as shown in figure 5.9. The observed deviations of the signalsreflect different physiological states of the cells, characterized by different intra- and

extracellular metabolite concentrations. Increases or decreases of the dilution rate

have to be slow enough to allow the dynamic short- and medium-term behavior of a

culture to be neglected (Sonnleitner and Hahnemann, 1994). A shortage in substrate

feed will result in a more severe substrate limitation whereas an increase of substrate

feed can lead to an intracellular enzyme limitation (Sonnleitner et al, 1997).In conclusion, the medium optimization reduced or eliminated the needed

medium components. Yeast extract could be completely omitted and the

concentration of the required amino acids and the salts were decreased.

Octanoate production and product inhibition 41

6 Octanoate production and product inhibition

6.1 Introduction

The production of octanoate by high cell density cultures of E. coli HBlOl[pGEc47]grown in fed-batch cultivations with addition of 2 % yeast extract resulted in highervolumetric productivity during the first 5 h after induction (1.3 g l"1 h"1) compared to

previous batch experiments (Wubbolts et al, 1996; Favre-Bulle et al, 1991). The

accumulation of octanoate, however, led to a decrease of both biomass concentration

and specific productivity. To prevent such an accumulation, continuous cultivation

with increased medium concentration was chosen as operation mode. The resultinghigh biomass concentration and the advantage of a continuous culture, that,

compared to batch or fed batch cultivation, the product is not accumulated, should

allow high octanoate production rates, leading to higher product concentrations.

Special attention has to be given to product inhibition, which is well described in

the literature. Ethanol, 2,3-butanediol, formic acid, acetic acid, butyric acid, lactic acid

and free fatty acids (Borzani, 1995; Hamamci and Ryu, 1994; Kuhn et al, 1993; Misset

et al, 1993; van den Heuvel et al, 1988; Fond et al, 1985; Richter and Becker, 1985) are

a few examples of substances that result in product inhibition in E. coli as well as in

general. Some effort has also been spent on modeling the inhibition kinetics (Shuklaand Chaplin, 1993; Bhaskar and Rao-Bhamidimarri, 1991; Stefuca and Bales, 1990; Lee

and Rogers, 1983), using either linear or non-linear kinetics. This distinction is

important, as the resulting volumetric productivities are totally different. Volumetric

productivities with underlying linear inhibition kinetics show a unique globalmaximum at a distinct dilution rate, which makes it impossible to increase

volumetric productivities by simply increasing the dilution rate. Octanoic acid, the

product formed in the biotransformation presented here, is known to inhibit growthin complex media (Viegas and Sa-Correia, 1995; Favre-Bulle and Witholt, 1992).

The tolerance of microorganisms to starting material, overflow and

biotransformation products and an appropriate bioprocess design determine the

stability of a production process. Modeling such processes can be helpful in

complementing empirically developed biotransformation processes (Hoeks et al.,1996; Rohner and Meyer, 1995).

6.2 Results

6.2.1 Basic effects of octane feed during continuous culture

Induction behavior was studied as follows: HBlOl[pGEc47] was grown in

continuous culture at a dilution rate of 0.15 h"1 without addition of octane (figures6.1,6.2). Dilution rate and octane feed (% v/v) were changed according to the patternshown in figure 6.1.


Figure 6.1: Continuous growth of E. co//HB101[pGEc47] at changing dilution rate and octane feed.

The culture was grown on medium SM3 containing 3 % glucose in absence of octane at a

dilution rate of 0.15 h"1 to steady state. At relative time of 0.58 h, 100.17 h and 144.42 h the

octane feed was shifted from 0 to 0.84 %, 1 % and 2 % (v/v relative to the medium flux). At

100.17 h the octane shift was combined with a shift in dilution rate from 0.15 h"1 to 0.20 h"1.

On-line signals (C02 measured by infrared gas analyzer, p02, redox and optical density by

aquasant-probe) and experimental setup. The insert between the C02 and p02-signalshows a magnification of the p02-signal after initiation of the octane-feed.


24 48 72 96

time [h]

120 144 168 192


wWco

Eo

10

9

8

7

6

5

4

3

awesei* *-

4

3.5

C 3

S 2.5

25co

8CO

1.5

1

0.5

0U*^ *"

110

105

[%] 100

8c 9b(0

CO90

o

85

8024 48 72 96

time [h]

120 144 168

16

12 ^i

8 S8

4 o)

3

2.5

2

1.5

1

0.5

0

192

at

COoc

o


Figure 6.2 Continuous growth of E. co//HB101[pGEc47] at changing dilution rate and octane feed.

Off-line data (—x—, x and *) of the same experiment as presented in figure 6.1.

Biomass was determined by cell dry weight measurement, glucose with Yellow Springsglucose-analyzer and acetate and octanoate were determined by GC. The carbon balance

represents all carbon recovered including the GC-measurements. Accumulation terms

(dC/dt) were not considered for the calculation of the carbon balance. Calculations provedthat accumulation terms (dC/dt) did not add significantly to the carbon balance.


The first shift in octane feed at 35 min was applied to an uninduced culture.

Oxygen consumption increased 40 min after initiation of the octane feed (insert

picture figure 6.1), the time needed for induction of the alkane oxidation system. The

culture remained carbon limited but the biomass decreased (figure 6.2), indicatingthat the yield coefficient on glucose (YX/s) decreased significantly (from 0.3 g g"1 to

0.22 g g1) following induction.

Octanoate production began, reaching a concentration of 1 g l"1 within the first 12

h and increasing over the next 3 days to slightly above 1.5 g l"1. The combined

(second) shift in dilution rate from 0.15 h"1 to 0.2 h"1 and in octane feed from 0.84 % to

1% did not influence the octanoate concentration significantly. The effect on acetate

formation, however, was much more prominent.The concentration of acetate, at a level below 0.5 g l"1 before the shift, increased to

more than 2.5 g l"1 over the next two days (figure 6.2). During the first 24 h after this

second shift glucose showed up in the supernatant at up to 6 g l"1, but decreased

again during the next 24 h to levels close to carbon-limitation. The third shift in

octane-feed from 1 to 2% led to an increase in octanoate production and in formation

of acetate, a reduced glucose uptake and a corresponding decrease in biomass

concentration. The signal of the on-line OD-probe (Aquasant, CH) correlated well

with the biomass measurements, except that changes in foam-production and/or

changes in the coalescence of the culture fluid influenced the measurement (figure6.1, time 0.5 and 144 h). The glucose based carbon recovery in steady state was

excellent (97 - 102 %), but deviated significantly during transients, increasing to 106%

after the first shift and decreasing to 86% after the third shift (figure 6.2).

6.2.2 The effect of shifts in octane-feed on the culture is reversible

The above results showed that the culture can be grown stably under carbon

limitation at a dilution rate of 0.2 h"1 and an octane feed of 1%, but not 2%. To

investigate whether or not the effect of an octane feed greater than 1% to a

continuous culture is reversible, a continuously growing culture of HBlOl[pGEc47]running at a dilution rate of 0.2 h"1 was shifted from 1% to 3% octane and back to 1%

according to the trajectory shown in figure 6.3.

Most of the signals presented in figures 6.3 and 6.4 reached their initial values

after the shift-down of the octane-feed. An exception was the dynamics of acetate

formation, which showed a different relaxation time. Instead of decreasing to its

initial value, the acetate concentration even increased without reaching a steady-statethroughout the duration of the experiment.

At 66.9 h, 73.1 h and 91.2 h respectively, 3 g casamino acids, 3 g yeast extract and

0.3 g methionine were pulsed (see negative spikes in redox signal, figure 6.3) in order

to determine whether additional nutrients might have beneficial effects. All three

pulses resulted in a slight increase of biomass (monitored on-line, see figure 6.3, and

off-line, data not shown, OD measurement) and of respiratory activity. Furthermore,

both acetate and octanoate formation were stimulated by the first two pulses (figure6.4).


Table 6.1: Production of octanoic acid at different dilution rates and at different octane-feeds.

E. coli HB101 [pGEc47] was grown in continuous culture (T 37 °C, pressure 1.02 bar, pH 7,

1 wm). Biomass, glucose, acetic acid and octanoic acid were measured at steady-stateconditions of different dilution rates and octane feeds. Productivity, biomass yield and

carbon recovery were calculated from the measured data. The last column shows the

fraction of the octane feed that was converted to octanoic acid.

D Octane Bio¬ Glucose Acetate YX/S Carbon Octano¬ Produc¬ Octane

mass recovery ate tivity[gi'V1]

converted

[h-1] [%] [g i"1] [g r1] [g i"1] [g g"1l [%] [g i"1] [%]

0.1 7.3 0.0 0.0 0.24 90 0.6 0.06 6.1

0.15 7.9 0.0 0.0 0.26 105 1.0 0.15 11.3

0.2 7.3 0.0 0.3/3.43 0.24 99 1.7 0.34 19.2

0.25 6.0 3.6 4.5 0.23 96 1.8 0.45 19.8

0.3 5.2 10.2 3.4 0.26 98 1.9 0.57 21.1

0.2 1 7.3 0.0 3.4 0.24 99 1.7 0.34 19.2

0.2 2 3.2 15.6 2.1 0.22 101 2.4 0.48 13.7

0.2 3 2.7 18.9 1.6 0.24 101 2.2 0.44 8.4

depending on whether the dilution rate of 0.2 h"1 was accessed from lower (first value)or higher (second value) dilution rate

6.2.2.1 Comparison of experimental data with model simulation

The data obtained were compared to the output of a simple Monod-type model

with linear inhibition kinetics (pmax = 5.5 g l"1). Data from on-line signals and model

output were almost identical. The biomass concentration (figure 6.4) correlated well

with the corresponding model output, however, the experimental data showed a

faster dynamic behavior. The cause for this dynamic difference is unknown and

therefore not included in the model. The measured glucose concentrations were

slightly lower than the simulated data, mainly due to the fact that the model did not

include by-product (acetate) formation. Overshoot and steady-state values of the

octanoate-concentration were well estimated by the model-simulation.

6.2.3 Production of octanoate at different dilution rates at low octane phaseratios

The production of octanoate and acetate was monitored at different dilution rates

and a (low) constant octane-feed of 1% (v/v). In pure aqueous systems, the dilution

rate of 0.2 h"1 is known to be close to the critical dilution rate for E. coli

HBlOl[pGEc47], above which the production of acetate occurs (see chapter 5.2.5).Depending on whether this dilution rate was accessed from lower or higher dilution

rates, acetate was produced or not. The octanoate concentration increased as D

increased from 0.1 h"1 to 0.2 h"1 and remained constant at higher dilution rates (figure6.5). The biomass concentration remained constant at low dilution rates.


Figure 6.3: Continuous growth of E. coli HB101 [pGEc47] during shift-up and down in octane feed.

The culture was grown on Medium SM3 containing 3 % glucose and 10 mg I'1 tetracycline,at a dilution rate of 0.2 h"1 with an octane-feed of 1 % (v/v relative to medium flux) to steadystate. At relative time 17.25 h the octane-feed was increased to 3 %. The octane feed was

kept constant for 94.4 h (18.9 mean residence times). At relative time 111.65 h the octane-

feed was decreased back to the initial 1%.

On-line signals and applied experimental trajectory.


12 24 36 48 60 72 84 96 108 120 132 144 156

time [h]


* 0

12 24 60 72 84 96 108 120 132 144 156

time [h]


Figure 6.4 Continuous growth of E. co//HB101[pGEc47] at shift-up and down in octane feed.

The culture was grown on Medium SM3 containing 3 % glucose and 10 mg I"1 tetracycline,at a dilution rate of 0.2 h"1 with an octane-feed of 1 % (v/v relative to medium flux) to steadystate. At relative time 17.25 h the octane-feed was increased to 3 %. The octane feed was

kept constant for 94.4 h (18.9 mean residence times). At relative time 111.65 h the octane-

feed was decreased back to the initial 1 %.

Off-line data (—x—, x and *) of the same experiment as presented in fig. 6.3 and

corresponding output of model simulation (—). Biomass, glucose and octanoate are

compared to model simulation with pmax = 5.5 g I"1.

Accumulation term (dC/dt) not considered for calculation of the carbon balance (see legendof fig. 6.2).


dilution rate [h~

Figure 6.5: Continuous growth of E. coli HB101[pGEc47] on medium SM3 containing 3 % glucose and

10 mg I"1 tetracycline, at changing dilution rates and constant octane feed of 1% (x-D-

diagram).The organisms were grown at different dilution rates. After steady-state was established for

a given dilution rate, biomass, glucose (always limited, data not shown), acetate and

octanoate were determined. The corresponding octanoate productivity was calculated. The

shadowed area reflects the estimated analytical error.

t


2 3 4 5 6 7 8

time [h]

Figure 6.6: Pulses of different concentrations of Na-octanoate to a continuous culture of E. coli

HB101[pGEc47].Cells were grown on medium SM3 containing 3 % glucose and 10 mg I"1 tetracycline, in the

absence of octane at a constant dilution rate of 0.2 h"1. Na-octanoate pulses of different

concentrations were applied to the culture.

On-line data (carbon dioxide C02 in exhaust, partial pressure of oxygen p02) of 4 different

pulses of Na-octanoate: 1 g I"1 (A), 2 g I"1 (B), 3 g I"1 (C), 5 g I"1 (D). The data are presentedas deviation from their corresponding steady-state values.

At dilution rates greater than 0.2 h"1, the biomass concentration decreased and

acetate and glucose (data not shown) showed up in the supernatant. The resultingproductivity is shown in figure 6.5.

Table 6.1 summarizes the steady state values obtained in the experimentsdescribed above.

6.2.4 Influence of octanoate on HB 101(pGEc47) growing continuously in a C-

limited culture

When pulses of 1 - 5 g l"1 Na-octanoate were injected in continuous cultures of

HBlOl[pGEc47] growing on glucose in the absence of octane, there were alwayschanges of CO2, p02, biomass and carbon-balance observed (figures 6.6, 6.7). All

concentrations pulsed led to an initial increase of the dissolved oxygen and to

varying changes of the C02-signal (figure 6.6).


11 1 1 1 1 rr 1 r

time [h]

Figure 6.7: Superimposition of off-line data of pulses of 4 different concentrations of Na-octanoate.

1 g I"1 (+), 2 g I"1 (o), 3 g I"1 (*), 5 g I"1 (x). Cells were grown on medium SM3 containing 3 %

glucose and 10 mg I"1 tetracycline, in the absence of octane at a constant dilution rate of

0.2 h'1. The data for biomass and carbon balance are presented as deviation from the data

point measured at time 0. The estimated analytical errors are the same as shown in figure6.5.

Accumulation term (dC/dt) not considered for calculation of the carbon balance (see legendof fig. 6.2).


Ql I I I I 1 I I I I I I 1 I

0 12 3 4 5 6

time [h]

.8: Pulse of 3 g I"1 Na-octanoate.

Glucose was determined on-line by FIA (•) and off-line with glucose-analyzer from YSI (*).Glucose and octanoate are compared to model simulation (pmax=4-1 9 I )• Octanoate and

acetate were measured by GC. Experimental conditions were identical as in figure 6.6.

Cells were grown on medium SM3 containing 3 % glucose and 10 mg I"1 tetracycline, in the

absence of octane at a constant dilution rate of 0.2 h'1.


Up to 50% of the carbon could not be recovered following the 5 g l"1 Na-octanoate

pulse. A surplus of carbon of up to 20% was calculated at lower pulsedconcentrations. The values of biomass, glucose and acetate showed a non-uniform

picture. For the pulses of 1 and 2 g l"1 Na-octanoate there was no appearance of

glucose determined. A slight decrease of cell mass and an increase of acetate resulted

for the 2 g l"1 Na-octanoate pulse. The glucose-concentration increased earlier and

faster after the 3 g l"1 Na-octanoate pulse, compared to the 5 g l"1 pulse. The acetate

concentration following the 3 g l"1 Na-octanoate pulse rose to 1.4 g l"1 within 1.5 h.

A similar acetate level was reached after 4.75 h, when the culture was pulsed with

5 g l1 Na-octanoate (figure 6.7).

6.2.4.1 Comparison of experimental data with model simulation

Glucose, acetate and octanoate data of the 3 g l"1 Na-octanoate (= 2.6 g l"1

octanoate) pulse are summarized in figure 6.8. The glucose concentration was

compared to simulations of the same model as used for the data shown in figures 6.3

and 6.4 with p^x = 4.1 g l"1. The lower value for pmax compared to the simulation

shown in figures 6.3 and 6.4 indicated that a stronger inhibition was encountered in

this experiment.

6.2.5 Growth of HB 101 (pGEc47) in the presence of acetate

To test the effect of acetate on HBlOl[pGEc47], pulse experiments were also

performed with Na-acetate (data not shown). These experiments quantitativelyconfirm indications given by Favre-Bulle and Witholt (1992) for E. coli W3110 grown

on complex medium, that acetate did not have a significant influence on the growthbehavior of E. coli HBlOl[pGEc47] in concentrations < 5 g l"1. The influence of acetate

is negligible even in combination with octanoate. When 5 g l'1 of Na-acetate were

pulsed in addition to octanoate, the differences of culture responses to cultures

where octanoate alone was added, were not significant.Acetate production is a good indicator for the state of the metabolism. The

organism tolerated more severe changes in the cultivation conditions when no

acetate was produced at the start of the experiment. This could be demonstrated

perfectly by the octanoate pulses. A pulse of 3 g l"1 of octanoate led to immediate

appearance of glucose in the supernatant when some acetate was present in the

supernatant at the beginning of the experiment. Without acetate present at the

beginning of the experiment, however, a much higher pulse of 5 g l"1 led to an even

slower appearance of glucose in the supernatant. This change in the dynamics of the

experiment was significant and could also be seen in other signals such as p02-


\l{S,p)= Umax^— (1 £-)

S + KS p max

S + Ks Ki.r + p

4 5 6

octanoate [g I" ]

10

Figure 6.9: Maximal specific growth rate and productivity as a function of the octanoate-concentration

in the medium.

18 independent batch cultivations in shake flasks (open circles) and bioreactor (filledcircles) were performed on medium SM3 with 3 % glucose (bioreactor) and medium SM3-

SK with 2 % glucose (shake flasks) containing different octanoate concentrations. The

maximal growth rate fits ten times better to the linear model than to the hyperbolic model.

The lower part of this figure shows theoretically achievable maximal octanoate

productivities. The values are the product of the X- and Y-component of the data-pointsshown in the upper part of the figure. The calculated productivity of the linear model results

in a global maximum.

6.2.6 Growth of HB 101(pGEc47) on defined medium with different octanoate

concentrations

The experiments presented in figures 6.1-6.2 and 6.3-6.4 showed that the growth of

HBlOl[pGEc47] was heavily influenced by the amount of octanoate present in the

cultivation fluid.

In order to determine optimal values for the production of octanoate, batch

cultures were carried out on defined medium containing different amounts of

octanoate (figure 6.9). The correlation between maximal specific growth rate and

octanoate concentration in the medium turned out to be linear. As a result, the

theoretically achievable productivity in a system without in-situ product removal

showed a global maximum of 0.6 g l"1 h"1 at an octanoate concentration of 2.5 - 3 g l"1.


6.3 Discussion

6.3.1 Growth of E. coli HBlOl(pGEc47) in the presence of octane

At low octane feeds, the octane was converted immediately to octanoate or blown

out of the bioreactor. The amount of octane converted at an octane-feed of 1%

increased with increasing dilution rate (table 6.1). At low dilution rates and a

constant airflow rate, relatively more octane was blown out of the reactor than at

high dilution rates. The amount of octane available for transformation was limited in

this situation. Air saturation (determined by on-line MS, data not shown) and

formation of a second phase could not be achieved in experiments with 1% octane

feed. A second phase, however, could be detected in experiments with 2% or more

octane feed. The relative amount of octane converted to octanoate decreased with

increasing octane feed (table 6.1). High phase ratios were therefore considered to be

counterproductive.The time difference between the start of octane feed and reaction of the p02-signal

was taken to be the minimal time needed for induction of the complete alkane

oxidation system.

Exposure of E. coli HB101[pGEc47] to octane decreased the biomass yield on

glucose in the order of 25%. The biomass yield remained remarkably constant at a

value of 0.25 + 0.02 g g1 independent of whether the culture was grown C-limited or

not. Carbon-limited growth could only be achieved at low dilution rates and low

octane-feeds.

The presence of octanoate in the medium decreased the maximal specific growthrate of the organisms. As a result, when the octane-feed was increased, the higherconcentration of the octanoate accumulated in the medium lowered the maximal

growth rate below the applied dilution rate and washout of cells occurred. If the

maximal specific productivity of 0.17 g g"1 h"1 for HBlOl[pGEc47] at a dilution rate of

0.2 h1, determined in this work (table 6.1) for defined medium and also reported byFavre-Bulle et al (1993) for complex medium, were to be fully exploited, a

concentration of more than 6 g l1 of octanoate could be formed with 7.5 g l"1 of

biomass for an expected productivity of 1.2 g octanoate formed per h and 1 of

aqueous phase. This value could not be reached however, because the inhibition of

octanoate led to wash-out of the cells at much lower octanoate concentrations.

Production of octanoate with high biomass concentration in a closed system will

therefore always result in an equilibrium of biomass, glucose and octanoate,

determined by the inhibitory potential of the octanoate at the established dilution

rate. The pulses of casamino acid, yeast extract and methionine (figures 6.3, 6.4) had

no significant influence on biomass and glucose concentration.

It must therefore be concluded that not primarily the octane, but the octanoate

inhibits the growth of the cells.


6.3.2 Influence of octanoate on growth of HBlOl (pGEc47)

The glucose based carbon balances in steady state situations were very close to

100% (see table 6.1). Deviations of the carbon balances, however, occurred in

situations, where the cells responded to dynamic changes of the cultivation

conditions during transient experiments. In cultivations with and without octane the

C-balances showed the same typical behavior and the deviations must therefore be

related to the appearance and disappearance of octanoate in the cultivation fluid. A

large increase of octanoate led to a significant deficit in the carbon balance. This

could be explained by substances produced and excreted by the organisms duringthese transients only. However, such compounds could not be detected by either GC

or HPLC measurements. A small increase in octanoate concentrations or a relaxation

of the octanoate inhibition by a decrease of the octane feed and, thus, a decrease of

the octanoate concentration, resulted in a surplus for the carbon balance. As shifts

and pulses were applied in steady state situations with carbon balances near 100%,

the increase of carbon could not be explained by substrates not detected in the

cultivation liquid. Solubility changes of carbon dioxide could be excluded for time

reasons. Even the possibility that octanoate might have triggered changes in the

composition of the cells, such as the differences in fatty acid composition of the

membranes seen on induction of alkane monooxygenase and in the presence of

alkanols (Chen et al, 1995; Nieboer et al., 1993), can hardly explain the deviations of

the carbon balance in dynamic situations.

The correlation between octanoate concentration and maximal growth rate is

linear, which means that the productivity must have a global maximum. This value

of 0.6 g l"1 h"1 cannot be exceeded unless the product is removed continuously from

the culture.

6.3.3 Simulation as a tool for planning, prediction and verification of

experiments

Simulation was used to analyze the experimental data with a model which

contains kinetics with product inhibition. The model was useful not only for

qualitative reflections of experiments, but also for quantitative purposes. Industry is

seeking simple, time inexpensive and robust models for improvement of

bioprocesses (Gram, 1996), and the generally excellent agreement between data and

model predictions demonstrates well how powerful even very simple models can be

for the experimentator as a planning, prediction and verification tool.

60 Process integrationfor the removal ofoctanoate

7 Process integration for the removal of octanoate

7.1 Introduction

The biotechnological formation of a product consists of a sequence of consecutive

steps. Process integration is the combination of two or more steps into one new step

leading to a reduction of total steps of the process. So far, process integration has

been used mostly for integrating bioconversions with one or more downstream

processing steps mainly with the goal to eliminate product inhibition (Mattiasson,

1996).Inhibition of growth and product formation by either biotransformation product

or by-product(s) can be reduced by applying cross flow filtration. Ceramic (metal

oxide) membranes, if wetted with aqueous medium, separate the aqueous (permeate)from both the solid and the apolar phase (retentate). The permeate withdrawn must

be replaced by fresh medium not containing the inhibitory compound. The latter is

thereby diluted and its inhibitory effect is relaxed.

Such a system has successfully been used for biotransformations with yeasts

(Rohner et al, 1992). It allows an uncoupling of the dilution rate from the specificgrowth rate. In a continuously operated system, the flow into the bioreactor (feed of

aqueous medium) is equal to the flow out of the bioreactor (the sum of bleed stream

and permeate). The dilution rate in a (pure aqueous) system with cell-recycling is

determined by the aqueous feed (D=Fin/V), whereas the growth rate is determined

by the bleed stream (flow of biosuspension including cells) out of the bioreactor

(u=Fout/V, see figure 10.1). The recircultation ratio R (R=Fp/Fjn) is used to calculate u

from D (u=D (1-R)). This means that the volumetric productivity can be increased byincrease of the dilution rate and the physiological parameter u can be tuned to

optimal values (e.g. to avoid acetate formation) by choosing an appropriaterecirculation rate. The compact loop bioreactor, as used in the previous experiments,was therefore extended by a tangential flow filter to form a fully automated cell-

recycle bioreactor system (see material and methods).

7.2 Start-up procedure

The cell-recycle bioreactor system was used to investigate the effects of cell-

recycling on the over-all performance of the process. The fact that dilution rates

greater than the specific growth rate were now achievable introduced a new element

in the operation of the biotransformation system: the start-up procedure.It was not possible to simply run the bioreactor system at the desired high dilution

rate. The approach to the desired production conditions had to be chosen carefully.

Any shift-up in dilution rate in a cell-recycling system leads temporarily also to an

increase of the actual growth rate (although the preset growth rate in steady state

(UsS) is set at a defined value) until the biomass concentration adapts to the increased

substrate supply.

Process integrationfor the removal ofoctanoate 61

cocoCD

Eo

Figure 7.1: Output of a simulation representing a shift from D=u=0.2 h"1 to D=0.4 h"1/R=0.5 (uss = 0.2

h"1) with a model parameter of umax=0.4 n~1-

£2COCOco

Eo

Figure 7.2: Output of a simulation representing a shift from D=u=0.2 h"1 to D=0.4 h"1/R=0.5 (uss = 0.2

h"1) with a model parameter of umax=0.3 h"1.


If the dilution rate is shifted up too frequently or the steps taken are too high, the

theoretically possible growth rate reaches the maximal specific growth rate. As the

organisms cannot grow faster than permitted by the maximal specific growth rate,

glucose can no longer be converted totally and shows up in the supernatant. In order

to avoid the undesired formation of metabolic overflow products, the growth rate

should therefore be kept even lower than the critical dilution rate DR. The design of a

successful start-up procedure therefore requires the definition of steps of dilution

rate and their durations avoiding the growth rate to exceed the value of the critical

dilution rate.

7.2.1 Model simulation

Model simulations with the same model as used in the previous chapter were

performed to find parameters to design an optimal start-up procedure. The steadystate growth rate (UgS) is reached when a steady-state has established after the

transient experiment. According to simulation, a shift-up in dilution rate from

D=u=0.2 h1 (R=0, no permeate flux) to D=0.4 hVR=0.5 (with ^=0.2 h1) fullyexploits the maximal specific growth rate of the organisms being 0.4 h"1 (figure 7.1).

Indirect estimation from the carbon dioxide signal (see chapter 4) of the maximal

specific growth rate of E. coli HBlOl[pGEc47] grown in the cell-recycle bioreactor

system revealed that compared to the system without cell-recycle the maximal

specific growth rate of the organisms decreased from 0.4 h1 to 0.3 h1. The same

simulation of a shift from D=0.2 h"1 to D=0.4 h"1 with a UgS of 0.2 h"1 demonstrates that

the maximal specific growth rate of 0.3 h"1 is reached and glucose shows up in the

supernatant (figure 7.2). This finding was verified experimentally (data not shown).In order to keep the actual growth rate during start-up below the Dr, the steady

state growth rate u^ can be set to a value of zero as long as the actual resultinggrowth rate remains above the rate required to guarantee maintenance conditions

(figure 7.3).Even for an experienced scientist it is difficult to accurately estimate the time

course of the growth rate during start-up and transient experiments. Model

simulation supplements the researcher's estimation with objective data making it

easier to design a new experiment.The control of the growth rate could accordingly be achieved by simulating the

time-course of the planned experiment in advance and correcting the experimentalsetup to guarantee that the growth rate stays within the desired window.

It could be demonstrated in the previous chapter, that model simulation can be

efficiently used for verification of performed experiments. The example of the start¬

up procedure now demonstrated, that model simulation can also be useful (and save

time) for the planning and prediction of experiments.

Process integrationfor the removal of octanoate 63

0.4r

biomass30

20

cocoCO

Eo

10

24

time [h]

36 42 48.0

Figure 7.3: Simulation of a startup-procedure to reach a dilution rate of 1.0 h"1 with the cell-recyclebioreactor system.The continuous removal of liquid after batch growth was achieved by permeate only("perfusion": R=1, uss=0 n"1) until the dilution rate of 1.0 h'1 was set.

The following pattern was applied: 10 h batch growth, 4 h D=0.15 h"1/R=1, 4 h D=0.25

h-1/R=1, 4 h D=0.375 h-1/R=1, 12 h D=0.5 h'1/R=1 (uss=0 h'1), 14 h D=1.0 h'1/R=0.9

(Mbs=0.1 h"1).

7.2.2 Performance of the system

The start-up procedure evaluated by model simulation was applied to cultures of

HB101[pGEc47] without and with addition of octane during start-up (non-inducedand induced cultures). The start-up with non-induced cells resulted in a biomass of

40 g l"1 at a dilution rate of 1 h"1 (R=0.9), whereas with induced cells a biomass

concentration of 35 g I"1 could be reached at the same dilution and recirculation rate.

Figure 7.4 shows model simulation and biomass measurements during a start-upexperiment according to the previously determined shift-up pattern. The simulation

output of the growth rate shows that it hardly exceeded a growth rate of 0.1 h"1 but

decreased continuously over time. The deviation of the biomass data from the

corresponding model output after 30 h (data not shown) is related to the fact, that

after 30 h, carbon limited growth could no longer be maintained (at D=0.75 h"VR=l)and glucose was detected in the supernatant. The model output of the growth rate at

that time dropped below 0.06 h"1.

64 Process integration for the removal ofoctanoate

0.12

COCO

CO

£o

time [h]

Figure 7.4: Start-up of a cell-recycle experiment compared to the output of the similar model simulation.

HB101[pGEc47] was grown on medium SM3 containing 1 % glucose. Bioreactor volume

2.6 I, indirect measured loop volume 1.4 I, total volume of cell-recycle bioreactor system 4.0

I, airflow rate 4.0 I min"1. The following start-up pattern was applied: 1 h transition from

batch to continuous culture after total consumption of glucose (D=u=0 h"1), 2 h D=u=0.15, 3

h D=0.15/uss=0, 8.75 h D=0.25/uss=0, 4.75 h D=0.375/uss=0, 3.5 h D=0.5/uss=0, 4.5 h

D=0.625/uss=0, 2.5 h D=0.75/uss=0 (all units h"1). The glucose concentration of the

cultivation liquid was lower than 10 mg I"1 (the detection limit of the glucose analyzer).

As during shift-up in dilution rate the growth rate of the organisms cannot be

controlled, special attention had to be given to maintain the growth rate in a well

defined window between a minimal growth rate required for maintenance

metabolism and the upper limit, above which undesired production of metabolic

overflow products would occur. It could be shown previously, that the maintenance

requirements of uninduced cells are very low (see figure 5.8). The fact that induced

cells grown at growth rates lower than 0.06 h"1 could no longer maintain carbon

limited growth indicates that the maintenance requirements of induced cells

cultivated in the cell-recycle bioreactor system are significantly higher.The perfect agreement of biomass data and corresponding model simulation in

figure 7.4 could only be reached by fitting of one model parameter. A significantlylower yield of 0.13 g biomass per g glucose had to be chosen to bring the biomass

data in accordance with the model simulation. Biomass yields of induced cultures

were generally 0.25 + 0.02 g g1 (see table 6.1) in systems without cell-recycle.Hjorleifsdottir et al (1991) postulated that cells operated at high cell densities in a

cell-recycle bioreactor system suffer from starvation. The cells change their

metabolism to produce proteins that help them survive the starvation.


Table 6.2: Carbon balances calculated for different continuous cultivations of HB101 [pGEc47].K31 and K32 were performed without, K74 to K76 with cell-recycling (R = 0.5).The amount of carbon determined in biomass (bleed), C02 (exhaust), glucose (bleed and

permeate) and acetate (bleed and permeate) were calculated as fractions of total carbon

fed with the glucose in the medium. The sum of these fractions is the total recovered

carbon.

Fractions Continuous cultivations

K31 K32 K74 K75 K76

biomass [%] 26.6 29.7 15.8 12.0 27.8

co2 [%] 71.6 66.2 39.7 35.4 29.5

glucosetrfeed [%] 0.3 0 0 0 0

glucosepermeate [%] 0 0 0.1 0.1 0.2

acetatebieed [%] 1.1 3.6 0.1 1.3 0.8

acetatepermeate [%] 0 0 1.1 12.9 3.3

Carbon recovered [%] 99.6 99.5 56.8 61.7 61.6

From oxygen limited growth of E. coli cells it is known that the cells immediatelychange their physiological behavior and maintain the new physiological state even

when the oxygen supply is increased again to sufficient levels (unpublished data). It

is very likely that the same mechanism is responsible for the discovered low biomass

yield.The filtration capacity of the applied ceramic filter, allowing permeate fluxes of up

to 10 1 h"1 (i.e. 50 1 m~2 h1), was never limiting the overall performance of the cell-

recycle bioreactor system, which could be operated for more than one month (withbiomass concentrations between 15 and 35 g l"1) without having to back-flush the

membrane filter. The biological limits (growth rate) as well as the technical

boundaries (oxygen supply, recirculation velocity in the loop) could therefore be

fully exploited according to the experimental needs.

7.3 Redirection of carbon fluxes

It has previously been demonstrated that cells operating in cell-recycle bioreactor

systems show signs of stress and change their metabolic pathway towards increased

production of metabolic overflow products (Hjorleifsdottir et al, 1991). Although no

metabolic overflow products other than acetate could be detected by GC the fact that

they were really produced is clearly demonstrated by the calculated carbon balances

(table 6.2).5 continuous cultures, performed with octane-induced cells, were compared

regarding the contribution of biomass, glucose, carbon dioxide and acetate to the

recovery of total carbon fed into the bioreactor by the glucose contained in the

medium. The numbers represent the mean values of 2 to 3 successive samples taken

at steady state. The cultures K31 and K32 were performed without cell-recycle, the

cultures K74 to K76 with cell-recycle. Without attached cell-recycle, almost 100 % of

the carbon fed by glucose could be recovered, whereas in cultures operated with cell-

recycle, a deficit of carbon of about 40 % was detected. The carbon in the carbon

dioxide is always at least twice the carbon contained in the biomass (in extreme cases,


between 2.2 and 3.0) except in culture K76, where the carbon fractions of biomass and

CO2 were almost identical. The contribution of acetate to the total carbon is, due to

the high permeate flows, up to ten times greater in the permeate than of acetate in

the bleed stream. A (non-detected) substance, although maybe only present at low

concentrations in the medium, might therefore significantly contribute to the carbon-

balance due to losses through the permeate.The importance of avoiding undesired metabolic overflow products to form an

efficient biotransformation process could be demonstrated with the carbon balances.

In experiments without cell-recycle the carbon supplied with the growth substrate

could be recovered quantitatively. In the cell-recycle bioreactor system, however,

already very low concentrations of a compound can lead to significant losses of

carbon through the permeate flux, if the system is tuned to high dilution and low

growth rates leading to high permeate fluxes.

7.4 Octanoate production with attached membrane filter

The production of octanoate with the cell-recycle bioreactor system was

investigated with media containing different amounts of glucose. The amount of

glucose in the medium determined the biomass concentration reached at various

dilution rates.

7.4.1 Influences on physiology of the cells

Figure 7.5 shows startup and initiation of octanoate production with

HB101[pGEc47] growing on 1% glucose medium. In the first phase the dilution rate

was shifted to 0.5 h1 with a constant steady state growth rate of 0.05 h1 resulting in

actual growth rates that never decreased below 0.1 h"1 (evaluated using model

simulation, data not shown) in order to avoid complications due to maintenance

metabolism.

After reaching a dilution rate of 0.5 h1, the growth rate was kept constant at 0.1 h"1

throughout the rest of the experiment. During increase of the dilution rate, the octane

feed was kept constant at a low level of 0.75 % (v/v) of the aqueous medium feed.

Increase of the octane feed at 24 h (see MS signal m/z 43, figure 7.5) started the

increase of the octanoate production which reached a maximal concentration of

about 1.35 g I"1 after 38 h. As indicated by the C02-signal, octanoate became

inhibiting leading to washout of biomass (biomass data not shown). In none of the

cell-recycle experiments performed on 1% glucose medium, octanoate concentrations

of 1.5 g l1 could ever be exceeded.


Figure 7.5: Continuous culture of HB101[pGEc47] growing on SM3 containing 1 % glucose.Bioreactor volume of 2.6 I and loop volume of 1.4 I give a total volume of the cell-recyclebioreactor system of 4.0 I, airflow rate 4.0 I min'1. The carbon dioxide was measured in the

exhaust gas. Medflux and permflux represent the calculated values of medium and

permeate flow. Octanoate was measured off-line by GC, m/z43 represents the relative MS

signal of mass fraction 43, a fragment of the octane molecule measured in exhaust gas.

The following shifts in dilution and growth rate were applied: from D=0.15 h'1/R=0.83 to

D=0.25 h-1/R=0.9, at time 0.5 h, to D=0.375 fr1/R=0.93 at time 4.5 h (all uss=0.025 h'1), to

D=0.5/R=0.8 (uss=0.1 h"1) at time 8.5 h, octane was shifted from 3 ml h"1 to 4.5 ml h"1 at

time 24.25 h, to 6 ml h'1 at time 27.25 h, to 7.5 ml IT1 at time 29.25 h, to 9 ml h_1 at time

31.25 h, and to 10.5 ml h"1 at time 33.25 h.

In more concentrated glucose media, maximal values of up to 2.5 g l1 of octanoate

could be reached (figures 7.6, 7.7). Figure 7.6 shows a cultivation on 2 % glucosemedium at a dilution rate of 0.5 h"1 and a growth rate of 0.1 h1, reaching a volumetric

productivity of more than 1 g H h"1. In all similar experiments, a significant decrease

of the carbon dioxide in the exhaust gas was observed.

With medium containing 3% glucose, high biomass concentrations of 30 g l"1 and

more were reached at dilution rates greater than 0.5 h1. The oxygen supply within

the loop became critical under high biomass conditions. The oxygen content of the

culture in the loop could not be monitored because no oxygen probe could be

mounted directly at the outlet of the loop.


CM

OO

3CD

COo

Io

12

time [h]

Figure 7.6: Continuous culture of HB101[pGEc47], growing on SM3 containing 2 % glucose, at a

constant dilution rate of 0.5 h'1 and a growth rate of 0.1 h"1.

Bioreactor volume 2.6 I, loop volume 1.4 I, total volume 4.0 I, airflow rate 4.0 I min"1. An

octane shift from 6 ml h"1 to 9 ml h"1 was applied at time 1.6 h. The octanoate concentration

was measured off-line by GC.

CN

OO

<D

COoc

%o

2

1.5

1

0.5

0*

.^^^ilV^ **5

•#

* *..„^ * %

12

time [h]

16 20 24

Figure 7.7: Continuous culture of HB101[pGEc47], growing on SM3 containing 3 % glucose, at a

constant dilution rate of 0.5 h'1 and a growth rate of 0.15 h"1.

Bioreactor volume 2.6 I, loop volume 1.4 I, total volume 4.0 I, airflow rate 4.5 I min"1. The

following octane-shifts were applied: from 0 to 6 ml h"1 at time 0 h, to 12 ml h"1 at time 4.5 h,to 18 ml h'1 at time 6.5 h and to 0 ml h"1 at 24 h. The octanoate concentration was

measured off-line by GC (•) and on-line by FIA (*) using HPLC measuring principle.


c

E

1

CDC

CO

o

CD

COOcCO

CJo

12 15 18

time [h]

21 24 27 30

Figure 7.8: On-line octanoate measurements of a continuous culture of HB101[pGEc47] growing on

medium SM3 at a constant dilution rate of 0.33 h"1 and a growth rate of 0.15 h"1.

Bioreactor volume 2.6 I, loop volume 1.4 I, total volume 4.0 I, airflow rate 4.5 I min"1. The

following octane-shifts were applied: from 0 to 6 ml h'1 at time 0 h, to 9 ml h"1 at time 1.3 h

and toO ml h"1 at 25.1 h.

However, two oxygen electrodes in the bioreactor, one mounted near the outlet of

the cell-recycle and the other one mounted at the opposite side, showed differences

of up to 20 % in the dissolved oxygen content of the culture. It had to be expectedthat oxygen limitation occurred towards the end of the loop. In order to avoid

oxygen limitation, the accumulation of biomass was limited by applyingrecirculation rates of 0.7 or lower at maximal dilution rates of 0.5 h"1. This resulted in

biomass concentrations of about 20 g l"1 using the 3%-glucose medium.

HB101[pGEc47] showed different sensitivities to octanoate inhibition when grownon media containing 1 or 3 % glucose with corresponding amounts of salts and trace

elements. It could previously be observed that cells grown on media with addition of

yeast extract were less sensitive towards octanoate inhibition (unpublished data).It is possible therefore, that the ion content of the medium influences the

sensitivity of E. coli HBlOl[pGEc47] to octanoate inhibition. This topic was not

further investigated in this thesis.

7.4.2 Demands on on-line analyses

Figure 7.7 shows octanoate concentration measured by GC off-line and HPLC on¬

line of a culture of HB101[pGEc47] grown on 3% glucose medium.


The decrease of the C02-signal indicates again that the inhibitory influence of the

octanoic acid could not be avoided under the conditions selected for this experiment(D=0.5 h"1, u=0.15 h1). In fact, the actual octanoate concentration monitored on-line

by HPLC turned out to be insufficient for accurate supervision of the process (see

comparison in figure 7.7). It was never possible to maintain the octanoate productionat a low enough value (2 g l1 for 2 and 3% glucose media) to prevent the onset of

inhibition. Time courses of octanoate as shown in figure 7.8 (on-line HPLC) were

always observed.

In a biotransformation process yielding an inhibitory product, reliable and

accurate on-line measurements of the product are absolutely necessary. The applieddetection of octanoic acid by on-line FIA based on the HPLC principle could not

fulfill these requirements. Standard measurements showed insufficient precisionwith deviations of up to 20% from the actual standard concentration. Even more

important was the fact, that significant differences between GC and FIA-HPLC

measurements were observed. The value measured by FIA was often more than one

g l1 lower than the value measured by GC. The accuracy of the applied measurement

technique therefore turned out to be insufficient for successful and stable operationof the biotransformation.

Comparison between GC and HPLC off-line measurements using the same HPLC-

column as inserted in the on-line HPLC system showed no significant differences

(data not shown). Chemicals and method for the off-line HPLC measurements were

identical with the on-line analyses, as well as sample pretreatment and amount of

injection. The only difference was the measurement frequency. The injections of the

on-line system were twice as frequent and much more measurements were

performed. A possible explanation for the differences between GC and on-line HPLC

data is therefore, that the HPLC-column was saturated and the injection frequency of

the on-line system was too high to allow a proper and complete elution.

Conclusions and Outlook 71

8 Conclusions and Outlook

8.1 Growth and product characterization

The growth rate of E. coli HBlOl[pGEc47] grown on the optimized and chemicallydefined medium (0.41 - 0.45 h"1) is similar to that found with a complex medium

containing yeast extract. With the optimized chemically defined medium and

information on DR the biotransformation system could be optimized further. A

bioprocess for the production of octanoate should be operated at a specific growthrate u of 0.2 h"1 in order to prevent redirection of carbon source to the overflow

product acetate.

A maximal volumetric productivity of 0.57 g l"1 h"1 could be reached growingHB101[pGEc47] in continuous culture at a dilution rate of 0.3 h'1. Almost the same

productivity (0.50 g l"1 h"1) was also reached by Favre-Bulle et al (1993) growingHBlOl[pGEc47] continuously at a dilution rate of 0.32 h1, on a different, yeast extract

enriched medium. Both cultures were no longer glucose limited due to inhibition byoctanoate. The highest volumetric productivities under glucose limited conditions in

this work and reported by Favre-Bulle et al (1993) were 0.34 g l"1 h1 and 0.44 g l1 h'1

at dilution rates of 0.2 h"1 and 0.24 g l"1 h"1, respectively.Inhibition of growth by acetate turned out to be negligible in the concentrations

produced physiologically by HB101[pGEc47] at a growth rate of u < 0.2 h1. The

inhibitory effect of octanoic acid on defined medium was more severe than observed

on medium with addition of yeast extract. A linear correlation of productconcentration and achievable growth rate could be determined. Since the productconcentration of 3 g l1 cannot be exceeded due to biological causes, this results in a

theoretical maximal productivity of only some 0.6 g l"1 h"1, which cannot be exceeded

unless octanoate is removed continuously from the culture.

8.2 HBlOl as suitable host for biotransformations

E. coli HBlOl[pGEc47] has been chosen as host organism for this study because of

its excellent plasmid stability. The test system needed a strain that maintained the

plasmid. Among the available organisms, HBlOl[pGEc47] showed the best plasmidstability (Favre-Bulle et al, 1993), which justified the use of HB101[pGEc47] in this

work.

In the long term, HBlOl is less suitable as production strain. The first aspect is the

pathogenic safety of this organism. K12 and derivates are assumed to be safe (see

Introduction), but only little is known about the pathology of E. coli B (Barbara J.

Bachmann, personal communication). HBlOl contains genes from both K12 and B

and has, in addition, a Salmonella typhosa-strain in its pedigree (see figure 3.1).Because of the background of a pathogenic SaZmoneZZa-strain, it is recommended that

HBlOl should be handled carefully in the laboratory (Barbara J. Bachmann, personalcommunication).

72 Conclusions and Outlook

0 1 2 3 0 0.1 0.2 0.3

% glucose in medium L-proline [g I]

Figure 8.1: Dependency of amino acid requirement by HB101[pGEc47] on cultivation conditions.

A: the required leucine concentration depended on the glucose content of the medium.

B: less proline is required for the same biomass concentration in continuous (Yconti=44

g g"1) than in batch (Ybatch=20 g g"1) culture.

A second drawback is that this organism is auxotrophic for two amino acids and

one vitamin and, even more important, it could be shown during this thesis, that the

requirements of the two amino acids are not constant but change depending on the

cultivation mode (L-Pro) and the glucose-content of the medium (L-Leu, figure 8.1).Furthermore, leucine, which had to be added to the medium, is known to inhibit

growth. The amount of L-Leu in the medium therefore determines the achievable

maximal specific growth rate.

A really simple defined medium is a prominent demand even for scientific

research and this can be best achieved if non-auxotrophic organisms, therefore not

requiring any additional medium components, are chosen as host organisms. For use

of HBlOl on a larger scale, the auxotrophies have to be eliminated. Alternatively, a

better strain with fewer requirements than HBlOl and which is generally regarded as

safe (GRAS) could be used.

8.3 Process integration

Process integration, realized with a membrane filter, allowed a doubling of the

volumetric productivity. The development of an even more productive system was

hampered by irregularity of the total cultivation volume and lack of precise and

accurate on-line data of the inhibiting product.Better volume control requires reliable control of the foam behavior of the culture.

In the bioreactor the mass of culture liquid could be kept constant independent of the

volume by gravimetrical measurements. This was not possible for the volume added

by the tubings of the recirculation loop. Changes in foaming therefore increased or

decreased the amount of cells in the loop and, consequently, also of the total system.These changes influenced the growth rate, which could therefore no longer be

controlled with the accuracy needed. Classical antifoam agents are not a possiblesolution because of the risk of clogging the membrane. Addition of octanol in low

amounts with the octane feed, however, might be successful as octanol is known to

act as an antifoam agent. Gravimetrical control of the loop content as a further

solution could not be implemented due to lack of appropriate material.

Conclusions and Outlook 73

Accuracy and precision of the on-line HPLC analyses turned out to be insufficient.

HPLC was the method of choice because no sample pre-treatment was needed and

the permeate sample from the cross-flow filter could be injected directly. The

measurement frequency was too high for the used column and an increase of the

measuring time was not practical. Measurements by off-line GC allowed accurate

measurements within 10 min with a temperature profile designed to measure both

the short and medium chain length acids like acetate and octanoate. A tuning of the

temperature profile to octanoate measurements only (with a similar internal

standard such as nonanoate) allows a further increase of the measurement frequency.The need for sample pretreatment (acidifying of the sample, addition of solubilitymediator such as ethanol, addition of internal standard) makes this system more

complex compared to HPLC analyses. Nevertheless, such a GC-system is available

on the market and was used in house with a maximal measurement frequency of

more than 8.5 h"1 (Manfred Zinn, personal communication).

8.4 Alternative methods for on-line removal of octanoic acid

The use of a cell-recycle bioreactor system is a first and easily achievable option for

the integrated removal of an inhibitory product. The advantage, that high dilution

rates can be obtained resulting in high volumetric productivities by keeping the

growth rate below umax, must be paid with an increased demand for medium.

Selective removal of the inhibitory product rather than just diluting it out would be a

possible alternative to the applied method. Methods successfully applied to the

production of acids are for instance affinity adsorption (Nilsson et al, 1994),

crystallization (Alba, 1988) as well as anion exchange and precipitation (Zihao and

Kefeng, 1995). Adsorption can easily be applied after phase separation, thus

combining membrane filtration with extraction methods (Yang and Tsao, 1995).

8.5 Alternatives to process integration

Apart from process integration there are other possibilities to improvebiotransformation processes, which are not treated in this thesis. These

improvements are aimed mostly at the biological component of the process. By¬product formation can be prevented at the molecular level (Dedhia et al, 1992). End-

product inhibition might be successfully overcome by selecting strains that are less

sensitive or even resistant to product inhibition (Nelms et al, 1992; Sahm, 1991). The

use of different types of strains with different membrane properties might also lead

to a host organism that is less sensitive to the product formed. The application of

immobilized cells (Kuhn et al, 1991) or even enzyme systems may offer further

alternatives for improvement of tolerance towards product inhibition.

74 Material and Methods


9.1 Media

The LB medium contained: 10 g l"1 tryptone, 10 g l"1 NaCl and 5 g l1 Yeast Extract

(YE).LB plates contained 1.5 % Agar in addition.

The MT-solution contained: 2.78 g l"1 FeS04 7H20,1.98 g H MnCl2 4H20, 2.81 g l"1

C0SO4 7H20,1.47 g l1 CaCl2 2H20, 0.17 g l1 CuCl2 2H20, 0.29 g l1 ZnS04 7H20 in 1

N HC1 (Lageveen et al, 1988).The M9* medium, based on the original M9 medium (Miller, 1972), contained: 1 %

glucose, 12.8 g l1 Na2HP04 7H20,3 g l1 KH2P04,0.5 g l1 NaCl, 1.0 g l1 Nr^Cl, 0.24 gl"1 MgSO^ 1 ml per 1 MT-solution, 0.4 g l"1 L-leucine, 0.4 g l"1 L-proline, 1.0 mg l1

thiamine (Favre-Bulle and Witholt, 1992).SMI medium is derived from that used by Favre-Bulle et al (1993) for continuous

cultivation of HB101[pGEc47]. It contained: 1 % glucose, 0.75 g l"1 KH2P04, 1.0 g l"1

K2HP04 3H20, 0.5 g l1 Na2HP04 2H20, 3.0 g l1 (NH4)2S04, 0.02 g l1 NH4C1, 0.4 g l1

MgS04 7H20,1 ml per 1 MT-solution, 0.4 g l"1 L-leucine, 0.4 g l"1 L-proline, 1.0 mg l1

thiamine. Tetracycline was added only when mentioned in figure legends.SM1YE4 medium is SMI supplemented with 4 g l"1 yeast extract.

Medium SM2 contained per % glucose: 2.0 g l"1 NH^Cl, 0.24 g l"1 MgS04 7H20,1.13

g l"1 H3P04 (85 %), 1.0 mg l1 thiamine, 1 ml per 1 MT solution. Tetracycline was

added only when mentioned in figure legends, proline and leucine were added in

different amounts and are mentioned in figure legends.Medium SM3 contained per % glucose: 2.0 g l"1 NTLtCl, 0.24 g l1 MgS04 7H20,1.13

g l1 H3PO4 (85 %), 0.1 g H L-proline, 0.2 g l1 L-leucine, 1.0 mg l1 thiamine, 7.35 mg l1

CaCl2 2H20, 5.56 mg l1 FeS04 7H20, 2.81 mg l1 CoS04 7H20, 1.62 mg l1 MnCl2

2HzO, 0.17 mg l1 CuCl2 2H20, 0.29 mg l1 ZnS04 7H20. Citric acid (2 g l1) and

tetracycline (5-10 mg l"1, only when mentioned in figure legends) were added

independent of the glucose content of the medium.

The following components were added sequentially and dissolved completely in

approximately 80 % of the final volume of deionized H20 before addition of the next

component: glucose, phosphoric acid, citric acid, salts, amino acids, vitamin, trace

elements (as a 60-fold concentrated solution in 0.1 M HC1), titrant of equimolar ION

NaOH/KOH to give pH 5 and deionized H20 to make up the final volume. The

medium was filter sterilized (0.2 um) in order to avoid the uncontrolled formation of

by-products during thermal sterilization.

Medium SM3-SK was used as shaking flask medium. It was identical to medium

SM3 but was entirely prepared from stock solutions and contained additional

phosphate as a buffer. The final stock-solution contained the following: 66 ml of 30%

glucose H20,10 ml of 20% citric acid H20, 20 ml of 20% NH4CI, 2 ml of 36% MgS047H20, 20 ml of 2% L-leucine and L-proline, 2 ml of 0.1% thiamine, 2 ml of trace

elements solution, 3 ml of 10M equimolar NaOH/KOH, 875 ml 0.1 M

Material and Methods 75

KH2P04/Na2HP04 2H20. The trace element solution consisted of the trace elements

of medium SM3 for 1% glucose in a 1000 fold concentration dissolved in 0.1 M HC1.

All stock solutions were sterilized by autoclaving except those containing amino

acids, vitamin and trace elements. These solutions were freshly prepared and filter

sterilized (0.2 um). Defined inocula were prepared by adding 100 ml SM3-SK

(containing 2 % glucose) to a shaking flask with 150 ml of sterile water to give 0.8 %

final glucose concentration.

9.2 Microorganisms, storage, plates, inocula

The organisms used in this study were Escherichia coli strains HBlOl and

HBlOl[pGEc47] (Favre-Bulle et al, 1993). The plasmid pGEc47 contains the alkane

oxidation genes of Pseudomonas oleovorans cloned into the broad host range vector

pLAFR I (Eggink et al, 1987).The microorganisms were stored at -70 °C in 15% (v/v) glycerol stock cultures.

Inocula were prepared from a single colony of an LB plate, which was made from the

stock culture. The plate was incubated for 24 h at 37 °C and stored for no longer than

3 d at 4 °C sealed with Parafilm.

Precultures were grown on LB (complex inoculum), M9* or SM3-SK (defined

inoculum) medium at 37 °C for less than 20 h. Bioreactor cultivations were carried

out in medium M9*, SMI, SM1YE4 and SM3. Addition of amino acids (L-leucine and

L-proline) and vitamin (thiamine) was needed due to auxotrophies of HBlOl. In

some cultivations of HBlOl[pGEc47] tetracycline was added as selective pressure

(mentioned in figure legend).

9.3 Bioreactor, Cell recycle

9.3.1 Bioreactor

The cultivations were performed in a computer controlled 4 1 high performancecompact loop bioreactor (COLOR, Sonnleitner and Fiechter, 1988). A mechanical

foam separator replaced the use of antifoam agents. The automation of the

bioprocesses consisted of a hierarchical and highly modular structure (Sonnleitner et

al, 1991; Locher et al, 1991). The medium flux was calculated and controlled

gravimetrically from the time dependent decrease of the signal of a balance on which

an intermediate vessel was placed.The following cultivation conditions were kept constant in the bioreactor unless

otherwise mentioned: working volume 3.0 ± 0.011, temperature 37 + 0.02 °C, pH 7 +

0.02, air flow rate 1 + 0.05 wm, pressure 1.02 ± 0.005 bar, motor speed of stirrer and

foam separator 2000 ± 10 rpm. During steady state continuous cultivation the

dilution rate was kept constant with an accuracy of better than + 3 %.


Figure 9.2: Semi schematic diagram of the cell-recycle bioreactor system.1 compact loop bioreactor, 2 recirculation pump (type L 16-1, Socsil Inter SA, CH), 3

ceramic membrane filter 0.2 um (type 1 P-19-40, Membralox, F), 4 analogue valve (typeAFP, Saunders, USA), 5 digital valves (Valv AG, CH), 6 pressure probes (piezoresistive,Kistler, CH), 7 flow measurement (magneto-inductive, Foxboro, USA), 8 temperature probe

(Pt-100, Degussa AG, CH), 9 permeate pump (Watson-Marlow, UK), 10 harvesting pump

(Watson-Marlow, UK), 11 intermediate vessel (Pyrex 5 I), 12 balance (type PG 4001,

Mettler, CH), 13 connectors (25 mm ID) to bioreactor, 14 Try-clamp connectors, 15 flexible

pressure tubes, 16 pipe stainless steel (26 mm ID), 17 pipe stainless steel (8 mm ID), 18

puncture mug for permeate periphery, 19 silicone tubes, 20 air filter, 21 bioreactor balance,

22 motor for stirrer, 23 stirrer, 24 foam separator, 25 motor for foam separator

9.3.2 Cell recycle

A semi-schematic drawing of the cell-recycle bioreactor system is presented in

figure 9.2. The cell-recycle was connected to the bioreactor with flexible pressure

tubes. This allowed the weight control of the bleed stream by using the bioreactor

balance. The loop was constructed with pipes of stainless steel (inner diameter 26 and

8 nun). The flow back into the bioreactor via a tangential entry was immediatelydispersed by the mechanical foam separator.

The total volume of the loop was 3.4 1. The actual volume of the suspension in the

loop differed depending on the foam formation and was determined to be 1.4 ± 0.11

under continuous cultivation conditions, independent of glucose concentration in the


medium and biomass concentration. The volume of the suspension in the loop could

be significantly higher in cases where there was only little foam formation (e.g.beginning of batch culture).

The system was operated with a bioreactor volume of 2.6 1 (gravimetricallycontrolled) and a loop volume of 1.4 1 (no monitoring and no control), resulting in a

total volume of the cell-recycle bioreactor system of 4 1. The setpoint of the

recirculation pump was set to 3 m3 h"1. The permeate pressure was controlled byvariation of the pump speed or the restriction valve. An intermediate vessel on a

balance was used for the gravimetrical determination of the permeate flux.

The intermediate vessel was harvested periodically by a harvesting pump. An

additional connection to external air through an air filter avoided an unwanted

hydrostatic emptying of the vessel if the harvesting pump did not properly close the

pump tubing.Automation of the cell-recycle was integrated in the process control system of the

bioreactor and allowed fully autonomous operation of the cell-recycle bioreactor

system. For more detailed information see Rohner et al (1992) and Rohner (1990).

9.3.3 Sterilization

Bioreactor and cell-recycle bioreactor system were sterilized for 30 min at 121 °C

with acidified water. Heat up of the bioreactor lasted 20 min, of the cell-recyclebioreactor system 60 min (due to additional energy losses of the steel tubings of the

cell-recycle). Peripheral tubings for feed, bleed, permeate, air, pH control and

analytical loop were autoclaved at 121 °C for 30 min. 300 1 and 90 1 storage vessels

(mixed) were sterilized with water at 121 °C for 60 min. 20 1 medium bottles as well

as intermediate bottles for feed and permeate were autoclaved with only little water

at 121 °C for 30 min. Base (10 M 1:1 NaOH, KOH) and acid (4 M H3P04) were freshlyprepared in autoclaved bottles and added to the storage bottles. The bottles

containing base and acid were autoclaved periodically with content to prevent

growth. The medium was prepared in the final concentration and filter sterilized (0.2u). Octane, as delivered by the manufacturer, was fed to the bioreactor through a 0.2

um membrane filter.

9.4 Analyses

9.4.1 On-line analyses

The bioreactor was equipped with sensors for temperature (Pt-100, Degussa AG,

CH), pressure (piezoresistive, Kistler, CH), pH , redox, pC02 and p02 (all Mettler-

Toledo, CH).The exhaust gas of the bioreactor was analyzed with a paramagnetic 02 (Servomex

oxygen analyzer 540 A, Sybron Taylor, UK) and an infrared C(52 analyzer (Binos I,Leubold Heraeus, D).A quadrupole mass spectrometer (Leybold Heraeus, D) was used for the detection

of the partial pressure of gases. The MS was used to measure the relative octane

content in the exhaust gas by monitoring m/z 43 and 57, the latter being less


sensitive but also less influenced by CO2, which has its molecular peak at m/z 44

with ca. 70 % yield.A FIA system (Rothen et al, 1996) was used for the on-line detection of glucose

concentration in the bioreactor. Samples of 20 ul were injected into a carrier stream

(0.1 M phosphate buffer, pH 7.0). The carrier stream transported the sample over an

enzyme column. The enzyme (Glucose oxidase, Grade I from Aspergillus niger, 250 U

mg1, Boehringer Mannheim, FRG) was immobilized on controlled-pore-glass(Aminopropyl-CPG-550 A, Fluka Chemie AG, Buchs, CH), pretreated with 0.5 %

glutaraldehyde (Fluka Chemie AG). After the enzyme column, a reagent-streamconsisting of 1.5 mM ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid),

Sigma Chemical Co., St. Louis, USA) and 5760 U of peroxidase (Type VI from

horseradish, 288 U mg1, Sigma Chemical Co.) in 0.1 M phosphate buffer (pH 7.0) wasadded. The detection of the dye formed was carried out at 660 nm.

The same FIA equipment was used as on-line HPLC analyzer. The enzyme column

was replaced by a Micro Guard Cartridge packed with Aminex Resin as a precolumnand a Fast Acid Analysis Column (both columns Bio-Rad, USA). The analyses were

performed at room temperature. The octanoate was separated by using 80 % (v/v)0.01 N H2S04 / 20 % (v/v) acetonitril at a flow rate of 0.7 ml min1. The elution was

monitored at 210 nm.

Cell-free permeate (sample) for both on-line FIA and HPLC was prepared bycross-flow filtration with a filter described by Miinch et al (1992a).

9.4.2 Off-line analyses

The biomass dry weight concentration was determined gravimetrically using 0.2

um membrane filters (Zetapor, Cuno AMF Inc., USA). 1 to 5 ml of culture liquid wasfiltered and washed with 1 to 5 ml of deionized water. Before and after filtration the

filters were dried for 24 h at 105 °C, cooled down under vacuum in a desiccator and

finally weighed on an analytical balance AE260 (Mettler, CH).For determination of glucose and metabolites culture liquid was centrifuged about

20 s after sampling in an Eppendorf centrifuge 5414 for 5 to 10 min (depending on

cell density). Part of the supernatant was used to determine the glucose content with

a Glucose Analyzer 23 AM (Yellow Springs, USA). The remaining supernatant was

stored at -20 °C for further analysis.Acetate, propionate and octanoate were determined on GC and HPLC. The gas

chromatograph HP 5890 series II plus (Hewlett Packard, USA) was equipped with a

25m/0.25mm Permabond Carbovax 20M (Machery-Nagel, D) column. The internal

standard contained 5 g l"1 butyrate, 1 g l"1 Na-azide, 1:1000 H3P04 cone (85 %), in 1:1

ethanol/water. Temperature program: 100 °C to 140 °C (10 °C min"1), to 180 °C (20 °C

min1) and to 220 °C (10 °C min"1), injector 240 °C, detector 280 °C.

The HPLC measurements were performed on a HP Ti series 1050 (HewlettPackard, USA) equipped with a Micro Guard Cartridge packed with Aminex Resin

as a precolumn and a Fast Acid Analysis Column (both columns Bio-Rad, USA). The

analyses were performed at room temperature. The octanoate was separated byusing 80 % (v/v) 0.01 N H2S04 / 20 % (v/v) acetonitril at a flow rate of 0.7 ml min1.

The elution was monitored at 210 nm.


9.4.3 Synchronization of the analytical subsystems

The supervisory computer controlling the bioreactor was responsible for data

acquisition from the on-line probes and the on-line MS. On-line GC and on-line FIA

were controlled by two distinct and different PC units, each using its own individual

system time. For each experiment, therefore, supervisory computer, on-line GC and

on-line FIA had to be synchronized.In the case of the FIA, the injection time is known but the time needed to transport

the sample to the injection loop can be estimated only as 30 + 5 s. Limitations in the

manual handling during experiments, e.g. the time required to apply a pulse, created

a further uncertainty of approximately 30 s.

The various signals generated by the individual analytical subsystems thus could

be synchronized to within < 1 min.

9.5 Modeling

For data analysis (see appendix for master m-file), data reduction and simulation

the software MATLAB/SIMULINK (MathWorks Inc, Natik, USA) was used. The

advantage of this software is the rapid handling of large data matrices and its

flexibility regarding user written additions (m-files). Simulation was used routinelyto predict and evaluate experiments. The model used for the simulations is basicallya Monod-type model, extended with inhibition kinetics. The model contains a globalbalance of octane without characterization of the transfer mechanism from the

organic into the aqueous phase or to the cells. Evaporation losses of octane from the

reactor were compensated in the model by decreasing the value of o0, the feedingconcentration of octane, accordingly.

The following mass balance equations were used in the model:

(1)dx Fout

dt=^x- v'x

(2)ds Fm Fout + Fp

,

-

,,

-so + qs-x-dt V

yV

(3)do Fo Fout

-=v.oo+ qo.x-y

-a

(note that o refers to octane i

(4)dp Fout + Fp

,

- qp-x-p

dtH

Vy


The kinetics of the model was described by the following equations:

(5) qs = qs «». 1--S + Ks V P maxy

The term describing the inhibition kinetics (l-p/pmax) was chosen according to

the experiment presented in figure 6.9.

(6) H = -qs -Yx i s

(7)o

qu-qumn-o + Ko

(8) qp = —qo -Yp i o

Note that P/O refers to product per octane (not oxygen). The description of

the product formation is very simple. It is assumed that octane is converted to

ocanoate according to their stoichiometric yield.

Parameters:

qsmax = -1-333 [gg^h1]

qomax = -0.15[gg1h1]

Yx/s = 0.25[gg"1]

YP/0 = 1.25 [g g1]Mw(octanoate) 144.2

I Mw(octane) 114.2«1.25

Ks = 0.001 [gl1]

Ko = 0.0001 [g l1]

Pmax = 4.1 - 5.5 g l1 (see individual figures)

Formula, Symbols and Abbreviations 81

10 Formula, Symbols and Abbreviations

Fin f&)X

/^S Fout.

(feed) w y W (bleed)

k >v

FP

<oo (permeate)Figure 10.1: Schematic representation of the different liquid flows in and out of the bioreactor.

Fin: flow rate of medium feed in the bioreactor

Fout: flow rate of bleed stream (waste) out of bioreactor

FP: flow rate of permeateR: recirculation ratio

The following equations describe the system in steady state:

F,n = Fout + FP and R = FP / Fin, therefore: Fout = Fm (1-R)Balance equations for the biomass X:

dx/dt = u x - (Fout / V) x, therefore in steady state: u = Fou1 / V

dx/dt = u x - D (1-R) x, therefore in steady state: u = D (1-R)

specific growth rate [h1]maximal specific growth rate [h1]

specific growth rate at steady state, = D (1-R)

designation of genes for the alkane oxidation

signal of OD-probe from Aquasantcell dry weight (= biomass concentraton) [g l"1]carbon dioxide concentration in exhaust [%]carbon dioxide production rate (for formula see appendix)cell-recycle bioreactor systemdilution rate [h"1] (= Fm/V)

change of octane concentration with time (differential)change of product (octanoate) concentration with time (differential)change of substrate (glucose) concentration with time (differential)change of biomass concentration with time (differential)European Federation of Biotechnologyflow injection analysissignal of fluorescence probeflow rate of medium feed in the bioreactor [1 h1]flow rate of octane feed [1 h1]

Pmax

alk

aqua

CDW

co2CPR

CRBS

D

do/dt

dp/dtds/dt

dx/dt

EFB

FIA

fluoro

Fin

Fo

82 Formula, Symbols and Abbreviations

Fout flow rate of bleed stream (waste) out of bioreactor [1 h"1]

(= biosuspension including cells)

Fp flow rate of permeate [1 h"1]GC gas chromatograph(y)GOD glucose oxidase

HBlOl designation of an E. coli strain

HPLC high performance liquid chromatograph(y)K^p inhibition constant of growth by product [g l"1]

Ko saturation constant for octane [g l"1]

Ks saturation constant for glucose [g l"1]L-Leu L-leucine

L-Pro L-prolineIn natural logarithmm/z mass to charge ratio

MS mass spectrometero2 oxygen concentration in exhaust [%]OD optical densityOECD Organization for Economic Co-operation and Developmento octane concentration [g l"1]

o0 octane concentration in octane feed [g l"1]

(= virtual value because octane feed was separate and pure)

p product (octanoate) concentration [g l"1]PC personal computerpGEc47 designation of plasmid containing the Alk-genes

Pmax maximal product concentration [g l"1]

p02 partial pressure of oxygen [%]POD peroxidaseqo specific octane conversion rate [g g"1 h"1]

CjOmax maximal specific octane conversion rate [g g"1 h1]

qp specific product (octanoate) formation rate [g g"1 h"1]

qs specific substrate (glucose) consumption rate [g g"1 h1]

qSmax maximal specific substrate consumption rate [g g"1 h1]R recirculation ratio (R = FP/Fm = 1 - Fout/Fm = 1 - u/D)s substrate (glucose) concentration [g l1]

So substrate concentration in medium [g l"1]

vR working volume of bioreactor, controlled by weight [kg]vL volume of cell-recycle loop (no monitoring and control) [1]

V total volume of cell-recycle bioreactor system [1]

X biomass concentration [g l"1]

Yp/o yield of product per octane [g g1]Yx/s yield of biomass per substrate [g g"1!

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(1995) On-line control of an immobilized hybridoma culture with multi-channel

flow injection analysis. J Biotechnol 43,229-242

van Putten A B, Spitzenberger F, Kretzmer G, Hitzmann B, 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

Viegas C A, Sa-Correia I (1995) Toxicity of octanoic acid in Saccharomyces cerevisiae at

temperatures between 8.5 and 30 °C. Enz Microb Technol 17,826-831

Vogel HJ, Bonner DM (1956) Acetylornithinase of E. coli: partial purification and

some properties. J Biol Chem 218,97-106

von Zumbusch P, Brunner G, Meyer-Jens T, Maerkl H (1994) On-line monitoring of

organic substances with high-pressure liquid chromatography (HPLC). ApplMicrobiol Biotechnol 42,140-146

Weuster-Botz D, Pramatarova V, Spassov G, Wandrey C (1995) Use of a genetic

algorithm in the development of a synthetic growth medium for Arthrobacter

simplex with high hydrocortisone delta 1-dehydrogenase activity. J Chem

Technol Biotechnol 64,386-392

Wood W B (1966) Host specificity of DNA produced by Eschericha coli: Bacterial

mutations affecting the restriction and modification of DNA. J Mol Biol 16,118-

133

Wubbolts M (1994) Xylene and alkane mono-oxygenases from Pseudomonas putida.Genetics, regulated expression and utilization in the synthesis of optically active

synthons. PhD thesis, University of Groningen, Groningen, The Netherlands

90 References

Wubbolts M, Favre-Bulle O, Witholt B (1996) Biosynthesis of synthons in two-liquid

phase media. Biotechnol Bioeng, 52,301-308

Yang X, Tsao G (1995) Enhanced acetone-butanol fermentation using repeated fed-

batch operation coupled with cell recycle by membrane and simultaneous

removal of inhibitory products by adsorption. Biotechnol Bioeng 47,444-450

Ye Q, Kang F, Lu S, Tao J, Zhang S, Yu J (1992) Continuous and high cell densitycultivation of Escherichia coli W3110(pE901) harboring human Interferon alphaA gene. Biochem Eng 2001,221-224

Yee L, Blanch HW (1993) Defined media optimization for growth of recombinant

Escherichia coli X90. Biotechnol Bioeng 41,221-230

Zihao W, Kefeng Z (1995) Kinetics and mass transfer for lactic acid recovered with

anion exchange method in fermentation solution. Biotechnol Bioeng 47,1-7

Appendix 91

12 Appendix

12.1 Definitions

12.1.1 Definition Biotechnology

• The integrated use of biochemistry, microbiology and engineering sciences in

order to achieve technological (industrial) application of the capabilities of

microorganisms, cultured tissue cells and parts thereof (European Federation

of Biotechnology, EFB, 1981)• The application of scientific and engineering principles to the processing of

materials by biological agents to provide goods and services (Organization for

Economic Co-operation and Development, OECD, 1982)• The integration of natural sciences and engineering sciences in order to

achieve the application of organisms, cells, parts thereof and molecular

analogues for products and services. (EFB, 1989)

12.1.2 Definition Biochemical (bioprocess) Engineering Science

• Biochemical Engineering Science represents the fundamental research into all

aspects of the interactions between engineering and other disciplinesnecessary to underpin the development of industrial scale biologically-basedprocesses (Lilly, 1996)

92 Appendix

12.2 Derivatives of E. coli K12 and B

Table 12.1: Some derivatives of E. coli K12 and B

uv

K12wtF+x^v_ 679 F+ x"ray

» 679-680 F X-ray Y10F" EMB-Lac^ Y53F"

thr-1 thr-1

leuB6

rfbDI

thr-1

leuB6

supE44[a]'

thr-1

leuB6

lacYI

rfbDI supE44thi-1 rfbDI

N-must uv uv UVI thi-1

Y53F EMB-Mal^ W1 F EMB-Ga^ W894F EMB-Ara*- W904 F- EMB-Xyl^ W922F-

[a]

UV

[a]

malT1(),r)

[a]galK2malT1(Kr)

uv

[a]ara-14

galK2malT1(Kr)

[a]ara-14

galK2xyl-5

malTipj)

W922F EMB-Mtl^ W945F" EMB-M't W291AF- UV->

W2915 F Str-sel^W2961 F

[a]'

[a] [b] [b] f [b]ara-14 ara-14 mal* proA2 proA2galK2

[b]'galK2 r [d] { rpsL20

xyl-5 xyl-5 rac' uv\\

r

malT1(kr) mtl-1

, mgl-51\ I rac'

malT1(kr) \ AB1103 F

[b]proA2hisG4(Oc)kdgK51r

rac'

AB1103F

[b]

UV AB1115F" Str-sel AB1133F- T6-sel-

AB1157 F"

[c]

NG>AB2463F

r ic]r m [c]proA2 proA2 rpsL3^ rpsL31 rpsL31hisG4(Oc) hisG4(Oc) tsx33 [e] < tsx33

kdgK51 [c] I kdgK51 qsr qsfr argE3(Oc) [ recA13

rac X'

I rac'

BwtF+ spont (Witkin) B/r F" spont (Boyer)>

AC2517FsulA1 = B/r lac"

sulAI

lac-14

12.3 CGSC-database (coli genetic stock center, Yale University, USA)

Information about different E. coli strains can be found in the World Wide Web on

the home page of the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu). The

following are pieces of information directly extracted from the Web-site without any

changes (except addition of URLs to make them visible):

Appendix 93

CGSC: E.coli Genetic Stock Center

The CGSC maintains a database of E.coli genetic information, including genotypesand reference information for the strains in the CGSC collection, gene names,

properties, and linkage map, gene product information, and information on specificmutations. The public version of the database includes this information and is

accessible in the forms shown below. The CGSC DB_WebServer (first option below)

provides a fill-in-the-blank form that results in direct querying of the database. The

direct login to our Sybase APT forms frontend (the third option) provides somewhatmore powerful, but less convenient, query capabilities. The Stock Center and the

database development are supported by the National Science Foundation. Requestsfor strains or additional information, as well as questions about the contents or use

of the database or guest logins to the aptforms interface, can be addressed to MaryBerlin ([email protected]).

Access to CGSC Information

• CGSC DB-Webserver (http:/ /cgsc.biology.yale.edu/cgsc.htrnl)• Current CGSC Working Map

(http:/cgi-bin/sybgw/cgsc/Map?!Name=CGSC(Mary Berlyn))• Map Diagrams (Postscript Files) (mapdiags.html)• Alternate access: CGSC Gopher (gopher://cgsc.biology.yale.edU/l)• A lternate access: guest logins to CGSC Sybase database

(telnet:/ /cgsc.biology.yale.edu)Contact us for password info.

• Genera Web-Sybase Gateway:(http://info.gdb.org/~letovsky/genera/genera.html)This is a general-purpose bridge between the Web and Sybase databases.

Check it out.

A query for some strains mentioned in the introduction produces the followingresults:

Strain AB1157

• ID#: 4509

• Strain Designation

• Designation Source Person Choice

AB115 7 Adelberg E.A. 1

• CGSC#: 1157

• Sex: F-

• No. of Muts Carried: 2 0

• Mutations

• Mutation Mapcode

94 Appendix

thr-1 0.00

ara-14 1.40

leuB6 1.74

DE(gpt-proA)62 5.70

lacYI 8.00

tsx-33 9.30

qsr'-O 12.60

glnV44(AS) 15.05

galK2 17.01

LAM- 17.40

rac-0 30.30

hisG4(Oc) 45.03

rfbDI 45.43

mgl-51 46.34

rpsL31(strR) 73.00

kdgK51 78.00

xylA5 80.00

mtl-1 80.70

argE3(Oc) 90.00

thi-1 90.30

• References

• DeWitt 1962. Genetics 47:577

Strain AB2463

• ID#: 9260



F-Trim 3

AB2463 Howard-Flanders P 1

• CGSC#: 2463

• Sex: F-

• No. of Muts Carried: 2 0

• Mutations

• Mutation

thr-1

ara-14

leuB6

Mapcode0.00

1.40

1.74

DE(gpt-proA)62 5.70

lacYI 8.00

tsx-33 9.30

qsr'-0 12.60

glnV44(AS) 15.05

galK2 17.01

LAM- 17.40

rac-0 30.30

hisG4(Oc) 45.03

rfbDI 45.43

recA13 60.77

rpsL31(strR) 73.00

kdgK51 78.00

xylA5 80.00

mtl-1 80.70

argE3(Oc) 90.00

thi-1 90.30

• References

Appendix 95

• Howard-Flanders, P. Boyce, R.P. Theriot, L. 1966. Three loci in

Escherichia coli K-12 that control the excision of pyrimidine dimers

and certain other mutagen products from DNA. Genetics 53:1119-1136

Howard-Flanders, P. Theriot, L. 1966. Mutants of Escherichia coli K-

12 defective in DNA repair and in genetic recombination. Genetics

53:1137-1150

Strain HBll

• ID#: 13606



HBll Boyer H. 1

• CGSC#: 2516

• Sex: F'

• Episome: F42

• No. of Muts Carried: 1

• Mutations

• Mutation Mapcodelac-14 1000.00

• Comments:

• This is the Jacob-Adelberg F-lac$~{+}$ in {\it E. coli} B/r lac$~{-

}$•

• In Lyophil Only

• References

• Boyer, H. 1964. J.Bacteriol. 88:1652

Boyer, H. 1966. J.Bacteriol. 91:1767

Jacob, F. Adelberg, E.A. 1959. Compt.Rend.Acad.Sci.Paris 249:189

Strain HB16

• ID#: 8212


• Designation Source Person

HB16 Boyer H.

• CGSCtt: 2 601

• Sex: F-


• Mutations

• Mutation Mapcodeara-14 1.40

leuB6 1.74

DE(gpt-proA)62 5.70

lacYI 8.00

galK2 17.01

rpsL2 0(strR) 73.00

thi-1 90.30

hsd([]) 1000.00

Choice

1

96 Appendix

• Comments:

• This is a K-12 and B/r hybrid. K-12 with B restriction and

modification (r+B,m+B).

• Not a pure K-12 strain.

Strain HBlOl

• ID#: 26752


• Designation Choice

HBlOl 1

• CGSC#: 652 4

• Sex: F-


• Mutations

Mutation Mapcodeara-14 1.40

leuB6 1.74

DE(gpt-proA)62 5.70

lacYI 8.00

glnV44(AS) 15.05

galK2 17.01

LAM- 17.40

recA13 60.77

rpsL20(strR) 73.00

xylA5 80.00

mtl-1 80.70

thi-1 90.30

hsdS20([]) 98.67

Strain W2961

• ID#: 9130



W2 961 Lederberg J. 1

• CGSC#: 266

• Sex: F-


• Mutations

• Mutation Mapcodethr-1 0.00

ara-14 1.40

leuB6 1.74

DE(gpt-proA)62 5.70

lacYI 8.00

glnV44(AS) 15.05

galK2 17.01

LAM- 17.40

rac-0 30.30

Appendix 97

rfbDI 45.43

mgl-51 46.34

rpsL20(strR) 73.00

xylA5 80.00

mtl-1 80.70

thi-1 90.30

12.4 Matlab m-file for data evaluation

Experimental raw-data obtained from the process control system Alert50 were

stored on the VAX-workstation. The raw-data were later transferred via FTP from

the VAX-workstation to the PC. The following code is written in the MATLAB

scripting language and can be used for evaluation of the raw-data on the PC. The m-

file needs as input from the workspace several process parameters and measured

values (written in round brackets), calculates then CPR, OUR, RQ, qC02, q02 and

the carbon balance and finally exports the calculated values (written in square

brackets) afterwards to the workspace.

function

[messdata,C02,02,OUR,CPR,RQ,q02,qC02,C,Ctot,OURex,CPRex,C02ex,Vex,Fin,Pout,

Fout,Cglc_m,Cglc_out,Cglc_per,Cbm_out,Cbc_out,Cbc_per,Cco2_out,Cac_out,

Cac_per]

cbil_l(time,o2,co2,weight,air,press,medflux,permflux,timebm,bm,timeglc, glc,

timegc,acetat) ;

%

% m-file zum auswerten von on- und offline daten unter berucksichtigung

% von permeatfluss!

% kann folgende berechnungen ausfuhren:

%

% 1) CPR/OUR/RQ

% 2) qC02/q02

% 3) kla

% 4) C-bilanz, mit beruecksichtigung von bicarbonat

%

% synchronisation von off- und on-line signalen muss vorherher durchgefuhrt

% werden!

% 'time' ist korrigierte zeit fur alle on-line signale

% fur 'bm' und 'glc' mussen ebenfalls zeitvektoren 'timebm' und

% 'timeglc' ubergeben werden

% alle zeitvektoren mussen in [min] vorliegen

% fuer C-bilanz werden aus medflux und permflux die fluesse berechnet

%

% beschreibung der gebrauchten abkurzungen%

% GASBILANZ

%

% MAFRin : MassAirFlowRate in zuluft (direkt aus zuluftsignal) [Nl/h]

% MAFRout : MAFR in abluft (aus inertgasbilanz) [Nl/h]

% 02in : 02 in zuluft (20.946 %) [%]

0=d%02/dt=>0=do/dt=>0=o%

sein:%

erfulltbedingungenfolgendemussenkann,werdenberechnetkladamit%

%

klavonBERECHNUNG%

%

[Mol/Mol]CPR/OUR=RQ%

%

[mMol/gh]CPR/(V*X)=qC02%

[mMol/gh]OUR/(V*X)=q02%

%

[mMol/h]1000/22.4*C02m)*MAFRin-C02out*(MAFRout=

CPR%

[mMol/h]1000/22.4*02out)*MAFRout-02in*(MAFRin=

OUR%

[Nl/h]

1000/22.4*C02m)*MAFRin-C02out*(MAFRout=1000/22.4*02out)*MAFRout-02in*(MAFRin=

%

C02out)-(l-02out/(l-02m-C02in)*MAFRin=MAFRout%

cC02-c02-1=C(inert)%

(inert)Cout*MAFRout=(inert)Cm*MAFRin%

GASBILANZ%

[-]

[-]

[l/h]

[1/h]

[l/h]

[g/l]

[g/l]

[g/l]

[g/h]

[g/h]

[g/h]

[g/h]

[g/h]

[g/h]

[1]

[1]

[1]

[bar]

[%]

[mg/lbar]

[mMol/1]

[mMol/1]

[1/h]

[mMol/lh]

[Mol/Mol]

[mMol/gh]

[mMol/gh]

[mMol/h]

[mMol/h]

[%]

[%]

[%]

o.48)(annahmebiomasse

(0.4)glucose

rate

ablaufimentration

ablaufimentration

zulaufimentration

abluftinC02in

ablaufinbiomassein

ablaufinglucosein

zulaufinglucosein

ablaufim

zulauflm

inCanteil

inCanteil

permeatfluss

abflussrate

zuflussrate

biomassekonz

substratkonz

substratkonz

kohlenstoff

kohlenstoff

kohlenstoff

kohlenstoff

kohlenstoff

kohlenstoff

zellruckfuhrsystemtotalvolumen

cell-recycle-volumen

reaktor-arbeitsvolumen

reaktor-mnendruck

flussigkeitderinsauerstoff-partialdruck

30°C)bei(36.502furhenry-konstante

aktuellflussigphasein02

gleichgewichtbeiflussigphasein02

stoffubergangskoeffizientvolumetrischer

OxygenTransferRate

respirationskoeffizient

C02-produktionsratespezifische

sauerstoffaufnahmeratespezifische

CarbondioxideProductionRate

OxygenUptakeRate

gasanalysesignal)(ausabluftinC02

gasanalysesignal)(ausabluftin02

%)(0.033zuluftinC02

%

Abm%

Aglc%

Pout%

Fout%

Fin%

X%

S

SO%

Cco2_out%

Cbm_out%

Cglc_out%

Cglc_m%

Cout%

Cm%

%

C-BILANZ%

%

V%

VL%

VR%

P

p02%

Ho2%

o

osat%

kla%

OTR%

RQ%

qC02%

q02%

CPR%

OUR%

C02out%

02out%

C02in%

%

%

Appendix98

Appendix 99

%

% OTR = OUR/V * 32

% osat : O2out/100 * Ho2 *p

% o = pO2/100 * O2m/100 * H

%

%

%

%

%

kla = OTR/(osat-o)

C-BILANZ

Cm = Caus

% Cm = Cglc_m

% Cout = Cglc_out + Cglc_per +

% Cglc_m = Cglc_out + Cglc_per +

% Cglc_m = SO * Fm * Aglc

% Cglc_out = S * Fout * Aglc

% Cglc_per = S * Pout * Aglc

% Cbm_out = X * Fout * Abm

% Cco2_out = CPR * 44/1000

Cbm_out + Cco2_out

Cbm_out + Cco2_out

[mg/lh]

[mg/1]

[mg/1]

[1/h]

[g/h]

[g/h]

[g/h]

[g/h]

[g/h]

% BEGINN BERECHNUNGEN

% bei aufruf ohne paramter wird meldung ausgegeben, mit welchen

% parametern das m-file gestartet werden muss:

if nargm==0,

disp('usage:')

disp(' [messdata,C02 , 02,OUR,CPR,RQ,q02,qC02,C,Ctot,OURex,CPRex,C02ex,Vex,

Fin,Pout,Fout,Cglc_m,Cglc_out,Cglc_per,Cbm_out,Cbc_out,Cbc_per,

Cco2_out,Cac_out,Cac_per]=cbil_l(time2,o2,co2,weight,air,press,mflux,

pflux, timecbil,bm_cbil,timecbil,glc_cbil,timecbil,acetat_cbil);')

break;

end % if

% fuer analyzer.m wird eine matrix (messdata) kreiert, die nur positive

% C02-werte enthaelt:

nco2 = fmd(co2>0) ;

messdata ( :, 1) =time ( nco2 ) /60 ;

messdata ( :, 2) =co2 (nco2) ;

% kalibrationsdaten aus' co2 ' und ' o2 ' herausflitem,

% resultierende 'C02' und '02' wieder auf ursprungliche Grosse erweitern,

% mdem der letzte messwert emgefroren wird (achtung: ergibt stufen bei

% an- und absteigenden C02 und 02 signalen):

O2=o2;

io2=find(o2<=0);

if o2(1)<=0,

n=l;

while o2 (n) <0,

ii=n+ l;

end; %while

02 (l)=o2 (n) ;

io2=io2(2:size(io2,1) ) ;

end; %if

for i=l:size(io2,1),

% umbenennung m interne variable

% zeiger auf kalibrationswerte von 'o2'

% falls gleich zu begmn von' o2 '

em

% kalibrationswert steht, muss erster pos.

% wert mnerhalb von 'o2' gesucht und

% dieser an erster Stelle von '02'

% geschrieben werden

% erster wert festlegen

% zeiger um ems kurzen

% an alle stellen, an denen em negativer

100 Appendix

02(io2(1))=02(io2(1)-1); % wert steht wird der letzte vorangegangene

end; % for % pos. wert geschrieben

C02=co2; % identisch fur C02

ico2 = fmd(co2<= 0) ;

if co2 (1)<=0,

n = l;

while co2 (n)<0,

ii=n+ l ;

end;

C02 (l)=co2 (n) ;

ico2=ico2(2:size(ico2,1));

end;

for 1=1:size(ico2,1),

C02(ico2(l))=C02(ico2(i)-l);

end;

% definition der konstanten:

02m = 20.946/100;

C02in = 0.033/100;

Ho2 = 36.5;

SO = 10; % [g/l]

Aglc = 0.4;

Abm = 0.48;

Aac = 0.4;

Apr = 0.486;

Abe = 0.2;

% zuordnung von reaktorsignalen:

% folgende on-line signale mussen vorhanden sem:

% o2, co2, press, weight, air

02out = 02/100; % umrechnung [%] in [-]

C02out = CO2/100; % umrechnung [%] in [-]

VR = weight; % mhalt des bioreaktors

VL =1.4; % volumen der schlaufe

V = weight + VL; % totalvolumen

p = press; % reaktordruck

MAFRin = air*60; % zuluft

% berechnung von fm und pout

% medflux und permflux mussen vorhanden sein

fm = medflux/1000; % umrechnung von ml/h in 1/h

pout = permflux/1000;

% zuordnung von ausgewerteten off-line daten:

% biomasse und substrat als bm und gle-vektoren:

% zeitbasis von on- und off-line Signalen mussen ubereinstimmen!

% zuordnung der vorhandenen GC-daten zu internen vektoren

if exist ('acetat'), Ac=acetat; end

if exist ('propionat'), Pr=propionat; end

% berechnung der C-bilanz

disp('GASBILANZ wird berechnet...')

% MAFRin * Cm (inert) = MAFRout * Cout (inert)

Appendix 101

% C(inert) = 1 - c02 - cC02

MAFRout = MAFRin * (l-02m-C02in) ./ (1-O2out-C02out) ;

OUR = (MAFRin * 02in - MAFRout .* 02out) * 1000/22.4;

CPR = (MAFRout .* C02out - MAFRin * C02m) * 1000/22.4;

RQ = CPR./OUR;

disp('C02, 02 (ohne kalibrationswerte), CPR, OUR, RQ berechnet!');

% uberprufen, ob biomasse-daten vorhanden, sonst wird ausfuhrung

% abgebrochen:

if -exist('bm'),

disp('ACHTUNG: keine biomassedaten vorhanden, ausfuhrung wird

abgebrochen!!')

break;

end % if

% falls biomasse vorhanden, zuweisung zu mternem vektor

X = bm;

% OUR, CPR, V zur zeit der probenahmen berechnen

disp('q02 und qC02 wird berechnet...')

for i=l:size(timebm,1),

n=find(time<timebm(i) +1 & time>timebm(i)-1);

OURex(l,:)=mean(OUR(n));

CPRex (l, : ) =mean(CPR(n) ) ;

C02ex(i, :) =mean(C02 (n) ) ;

Vex(i,:) =mean(V(n) ) ;

% berechnen von q02 und qC02 zur zeitpunkt der biomasseprobenahme:

q02(l,:) = OURex(l)/(V(l)*X(i));

qC02(i,:) = CPRex(l)/(V(i)*X(i));

end % for

disp('q02 und qC02 berechnet!')

% bicarbonatconzentration aus C02-Daten berechnen, korrelation zwischen

% biomasse und CO2 im abgas mit modellrechnung bestimmt (bicarb.m)

Bc=0.12232*CO2ex; % [g/l]

% C-BILANZ

disp('C-bilanz wird gerechnet...')

% uberprufen, ob glucose-daten vorhanden. falls kem glc-vektor

% vorhanden 1st, wird glucose im ablauf als null angenommen

if -exist ('glc'),

disp('ACHTUNG: keine glucose-daten gefunden!')

disp('fur C-bilanz wird angenommen, dass in ablauf keine glucose mehr

vorhanden 1st')

S=zeros(size(timebm,1));

% uberprufen, ob size(bm)==size(glc), d.h. ob glc als off-line daten

% (Yellow Springs) oder als on-line Daten (FIA) vorliegt.% falls FIA-daten, muss glc-konzentration zur zeitpunkt der probenahme% bestimmt werden

elseif size(bm,1)~=size(glc,1), % glc als FIA-daten

% fur alle off-line daten

% probenahme-zeit ± 1 mm

% mittelwert

102 Appendix

disp('glc als FIA-daten')

for i = l:size(timebm, 1) ,

ii = find(timeglc<timebm(i)+5 & timeglotimebm(i) -5) ;

S(i,:) = mean(glc(ii)) ;

end % for

else, % glc als Yellow Springs-daten

disp('glc off-line')

S=glc;

end % if elseif else

% bestimmen von Fin und Pout zur zeitpunkt der probenahme:

for i = l:size(timebm,1) ,

ii=find(time<timebm(i)+1 & time>timebm(i)-1);

Fin(i,:) = mean(fin(ii));

Pout(i,:) = mean(pout(ii));

end % for

% berechnen der fliisse und C-anteile:

for i=l:size(timebm,1),

Fout(i,:) = Fin(i) - Pout(i);

if Fout(i,:)<0, Fout(i,:)=0;, end;

Cglc_in(i,:) = SO * Fin(i) * Aglc;

Cglc_out(i,:) = S(i) * Fout(i) * Aglc;

Cglc_per(i,:) = S(i) * Pout(i) * Aglc;

Cbm_out(i,:) = X(i) * Fout(i) * Abm;

Cbc_out(i,:) = C02ex(i)*0.12232*Fout(i)*Abc;

Cbc_per(i,:) = C02ex(i)*0.12232*Pout(i)*Abc,•

Cco2_out(i,:) = CPRex(i) * 12/1000;

end % for

% berechnen der C-bilanz:

Cin = Cglc_in;

Cout = Cglc_out + Cglc_per + Cbm_out + Cbc_out + Cbc_per + Cco2_out;

C=Cout./Cin*100;

% uberprufen, ob GC-daten vorhanden, sonst wird ausfuhrung abgebrochen:if ~exist('timegc'),

disp('C-bilanz (C) berechnet!')

break;

end % if

sizebm=size(timebm,1);

sizegc=size(timegc,1);

if sizebm==sizegc, idflag=l; end

if sizebm>sizegc,

bmflag=l;

disp('timebm und timegc sind unterschiedlich lang! GC-daten nicht in C-

bilanz integriert!')


break;

end % if

if sizegosizebm,

gcflag=l;

Appendix 103

disp('timebm und timegc sind unterschiedlich lang! GC-daten nicht in C-

bilanz integriert! '

)


break;

end % if

for i=l:size(timegc,1),

Cac_out(i,:) = Ac(i) * Fout(i) * Aac;

Cac_per(i,:) = Ac(i) * Pout(i) * Aac;

end % for

Cgc_out = Cac_out+Cac_per;

Couttot = Cout + Cgc_out;

Ctot = Couttot./Cin*100;

disp('C-bilanz (C und Ctot) berechnet!')

72.5 PID-controllers

The configuration of the process control system used for controlling the compact

loop bioreactor and the cell recycle contains 12 PID-controllers (TRPID, TCPID,

FLUXREG, WREG, PRESSPID, AIRPID, PHPID, PERMFPID, PTMPID, RFLUXPID,

PTMSTPID, FLUXSTPID). With one exception (PERMFPID) they are all configuredas Pi-controllers not using the differential part of the controller settings. They are

integrated in a configuration, that grew and was improved over time. All of these

controllers work perfectly under normal conditions, but there are some situations

where it is very useful to have some tricks in the backhand, if a controller gets stuck

in an undefined condition.

TCPID, TRPID. The configuration for controlling the temperature is divided into

two parts, the control of the temperature-circuit and the control of the reactor

temperature. The two PID controllers are therefore working in a multistage system(cascaded). It is not easy to find proper parameters for cascaded controllers, however

the parameters used in the present configuration worked always perfectly. It is

possible that the temperature of the temperature-circuit oscillates within a small

range, this does not affect the bioreactor temperature. If the bioreactor temperaturecan no longer be controlled within + 0.02 °C this could always be related to one of

two reasons. Either the valve for regulating the cooling water was not properlyworking or a T-connector in front of the heat exchanger got calcified.

FLUXREG. This PID-block controls the medium flow into the bioreactor. The

decrease of the weight of medium pumped out of an intermediate vessel over time is

used for calculation of the flow rate. The intermediate vessel has to be filled up

periodically (as soon as the weight of the medium in the intermediate vessel is lower

than 200 g), and during this time, the PID-controller is frozen to its last calculated

value before refill. After refilling to about 820 g, the controller begins to work againafter the weight has decreased below 800 g and a timer of 120 s has ran down. If a

cultivation is stopped during refill or before the weight decreased below 800 g after

refill, the controller has stopped in frozen state. This makes it impossible to start a

104 Appendix

new continuous cultivation just by setting the flowrate-setpoint, the feed-pump will

not start. In this situation, the pump has to be started manually with a pump settingcorresponding more or less the desired setpoint of the flowrate until the weightdecreases below 800 g and the timer has ran down. Now it is important to reset the

integral part of the controller (either via ALCOM/OPC: INT "YES" -> "NO" -> "YES"

of via VAX workstation: ALCONTROL - 'Send an Alert Block Parameter' -

FLUXREG.INT = 0. - FLUXREG.INT = 1.) in case that the error of the integral partlead to a negative output of the pump signal. A cultivation always has to be stoppedby first setting the flowrate-setpoint to zero. This prevents an accumulation of the

integral-error.WREG. This controller is responsible for closed-loop control of the bioreactor

weight and always worked well. A BHAN-block called WREGFREEZE has been

implemented to have the possibility to freeze this controller. This allows to performmanipulations on the bioreactor (change of exhaust-filter, pulses) during cultivations

without disturbing a steady-state.PERMFPID. This is the only PID-controller with an enabled differential part. This

was necessary mainly due to a insufficiently working pump that got blocked from

time to time, mainly at low pump speed. The differential part was needed chiefly for

a sudden increase of the pump-signal to deblock the pump and might not be

necessary otherwise. The calculation of the permeate flux is done gravimetricallysimilar to the calculation of the medium flow. The implementation of the blocks for

calculating the flow rate, however, is different. About 20 blocks are used for

calculation and control of the medium flux with the advantage, that no delay element

is involved. Calculation and control of the permeate flux is performed by one single(user written) block with the disadvantage, that a delay element is involved in the

calculations. This increases the tendency of this controller to oscillating behavior,

especially in startup situations. To prevent the controller from overshooting duringstartup, the setpoint has to be approached in several smaller steps instead of one bigone. Care has to be taken not to change the setpoint during the empty phase, where

the controller is blocked (again similar to FLUXREG). Otherwise the accumulatingerror makes the controller (mostly because of the differential part of the settings)behave like mad when it is reactivated and the bioreactor is emptied within minutes

via the permeate.

Appendix

12.6 Configuration FR3

SGROUP STERILIZE

TS = 2 ; RUN = YES ; PR = 1

SBLOCK STGRDBUT.BHAN

> S

SBLOCK STGRDBUT

S - "0-0-0-0",

Q "0 0-0-0",

NQ - "0-0-0-0",

QSET NO,

PULS - 5,

SBLOCK COOLSTAT.BHAN

-> S

SBLOCK COOLSTAT

S "0-0-0 0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

QSET - NO,

PULS 0,

SBLOCK TAKTGN.TIME

TAKTGN.NRDY -> RUN

-> RES

SBLOCK TAKTGN

RUN - "0-0-0-0",

RES "0-0 0-0",

RDY - "0-0-0-0",

NRDY - "0-0-0-0",

RUNV - NO,

RESV - NO,

TIME 30,

ZERO 0,

ACCU - YES,

PULS - 30,

SBLOCK COOLSET.OR

STERTIMER.RDY

INAKTIME.RDY

COOLSTAT.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK COOLSET

51 - "0-0-0-0".

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "1-2-2-8",

Q _ "0-0-0-0",

NQ "0-0-0-0",

SBLOCK COOLRES.OR

STERSTATE.Q

CULTSET.Q

INAKSTATE.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK COOLRES

si - 'O-o-o-O",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q _ "0-0-0-0",

NQ - '0-0-0-0",

SBLOCK SPWSNI.TIME

-> RUN

-> RES

SBLOCK SPWSNI

RUN - "1-2-1-2",

RES - "0-0-0-0",

RDY - "0-0-0-0",

NRDY - "0-0-0-0",

RUNV - NO,

RESV - NO,

TIME - 60,

ZERO - 0,

ACCU - YES,

PULS - 0,

SBLOCK COOLSTATE.SR

COOLSET.Q

COOLRES.Q

-> SET

-> RES

SBLOCK COOLSTATE

SET "0-0-0-0",

RES - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK STERGRDEX.OR

STGRDTI . RDY

CULTSTATE.NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK STERGRDEX

Si - "0-0-0-0",

52 - "0-0-0-0",

53 - "1-2-2-8",

54 - "1-2-2-8",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK NIVOK.AND

SPWSNI.RDY

SPWSNI.RDY

-> SI

-> S2

-> S3

-> S4

SBLOCK NIVOK

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "1-2-1-2",

54 "1-2-1-2",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK STERGRD SR

STGRDBUT.Q

STERGRDEX.Q

-> SET

-> RES

SBLOCK STERGRD

SET - "0-0-0-0",

RES - "0-0-0-0"

Q "0-0-0-0",

NQ "0-0-0-0",

SBLOCK TLTTBOIL.MIMA

TRCALIB.OUT

-> INI

-> IN2

SBLOCK TLTTBOIL

HI - "0-0-0-0",

NHI - "0-0-0-0",

IN1V - 98,

IN2V 0,

SBLOCK STERGRDNO.BSET

-> SI

-> S2

-> S3

STERGRD.NQ -> SEND

SBLOCK STERGRDNO

SEND "0-0-0-0",

BLK - "STGRDBUT",

PARI - "QSET" ,

S1V - NO,

PAR2 -""

,

S2V - NO,

PAR3 -""

,

S3V - NO,

SBLOCK TCULTGTT.MIMA

TRCALIB.OUT

-> INI

-> IN2

SBLOCK TCULTGTT

HI - "0-0-0-0",

NHI - "0-0-0-0",

IN1V - 45,

IN2V - 0,

SBLOCK TGTTSTER.MIMA

TRCALIB.OUT -> INI

-> IN2

SBLOCK TGTTSTER

HI - "0-0-0-0",

NHI - "0-0-0-0",

IN1V - 0,

IN2V - 123,

SBLOCK TAKTENA.AND

TAKTGN. RDY

TAKTGN.RDY

STERGRD.Q

STERGRD.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK TAKTENA

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 "0-0-0-0",

Q _ "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK STERTIMER.TIME

TGTTSTER.HI -> RUN

-> RES

SBLOCK STERTIMER

RUN - "0-0-0-0",

RES - "1-2-2-7",

RDY - "0-0-0-0",

NRDY - "0-0-0-0",

RUNV - NO,

RESV = NO,

TIME - 180 0,

ZERO - 0,

ACCU - YES,

PULS - 5,

SBLOCK SPWSGUIDE.OR

TAKTENA. Q

TAKTENA. Q

STERGRD.NQ

STERGRD.NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK SPWSGUIDE

51 "0-0-0-0",

52 - "0-0-0-0",

53 "0-0-0-0",

54 - "0-0-0-0",

Q - "1-2-4-5",

NQ - "0-0-0-0",

SBLOCK SPWOUT.SR

STERGRD.Q

STERGRD.NQ

-> SET

-> RES

SBLOCK SPWOUT

SET "0-0-0-0",

RES "0-0-0-0",

Q - "1-2-4-4",

NQ - "1-2-4-2",

SBLOCK STEREXIT.OR

STERTIMER. RDY -> SI

-> S2

-> S3

-> S4

SBLOCK STEREXIT

si - "O-o-o-O",

52 - "1-2-2-8",

53 = "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK SPWSPUMP SR

SPWOUT.NQ

SPWOUT Q

-> SET

-> RES

SBLOCK SPWSPUMP

SET - "0-0-0-0",

RES - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK STERSTART.AND

TCIRCPUMP.Q

TCIRCPUMP.Q

TCIRCPUMP.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK STERSTART

51 - "1-2-2-7",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK ENAMOT.AND

SPWSPUMP.Q

SPWSPUMP.Q

NIVOK.Q

NIVOK.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK ENAMOT

51 - "0-0-0-0",

52 "0-0-0-0",

53 "0-0-0-0",

54 - "0-0-0-0",

Q "0-0-0-0",

NQ - "0-0-0-0"

SBLOCK STERSTATE.SR

STERSTART.Q

STEREXIT.Q

-> SET

-> RES

SBLOCK STERSTATE

SET "0-0-0-0",

RES - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK REFLUXER.SR

CULTSTATE.Q

1 STERSTATE.Q

-> SET

-> RES

SBLOCK REFLUXER

SET - "0-0-0-0",

RES - "0-0-0-0",

Q "1-2-4-8",

NQ "0-0-0-0",

106 Appendix

SBLOCK VACUBREAK.AND

CULTSTATE.NQ

CULTSTATE.NQ

TLTTBOIL.HI

TLTTBOIL.HI

-> SI

-> S2

-> S3

-> S4

SBLOCK VACUBREAK

51 = "0-0-0-0",

52 - "0-0-0-0",

53 = "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK SPWSSTEAM.SR

STERGRD.Q

SPWSNI.RDY

-> SET

-> RES

SBLOCK SPWSSTEAM

SET = "0-0-0-0" ;

RES - "0-0-0-0" ;

Q = "1-2-4-1" ;

NQ - "0-0-0-0",

SBLOCK SPWSAIR.SR

SPWSSTEAM. NQ

STERGRD.Q

-> SET

-> RES

SBLOCK SPWSAIR

SET - "0-0-0-0" ;

RES - "0-0-0-0",

Q = "1-2-4-3" ;

NQ = "0-0-0-0",

SBLOCK EXHAUSTST. MPX

PRESSSUM.OUT

CULTSTATE.Q

TLTTBOIL.NHI

VACUBREAK.Q

PRESSSUM.OUT

INAKSTATE.Q

-> INI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

SBLOCK EXHAUSTST

51 - "0-0-0-0",

52 = "0-0-0-0",

53 "0-0-0-0",

54 = "0-0-0-0",

CHG = "0-0-0-0",

NCHG = "0-0-0-0",

INIV = 0 ;

IN2V - 95 ;

IN3V = 0 ;

IN4V - 0,

PULS - 20 ;

SBLOCK STERSTAT.BHAN

STERSTATE.Q -> S

SBLOCK STERSTAT

S - "0-0-0-0" ;

Q - "0-0-0-0",

NQ = "0-0-0-0" ;

QSET - NO ;

PULS - 0 ,-

SBLOCK TSWENARES.SR

-> SET

-> RES

SBLOCK TSWENARES

SET - "0-0-0-0",

RES = "0-0-0-0",

Q - "0-0-0-0" ;

NQ = "0-0-0-0",

SBLOCK EXHAUSTRV.AO

PRESMPX.OUT -> IN

SBLOCK EXHAUSTRV

IO = "1-1-5-1",

IMAX = 100 ;

IKIN = 0 ;

UNIT - "%" ;

ZERO - YES,

INV - NO ;

SBLOCK TSWENA.SR

-> SET

-> RES

SBLOCK TSWENA

SET - "0-0-0-0",

RES = "0-0-0-0",

Q = "0-0-0-0" ;

NQ = "0-0-0-0" ;

SBLOCK H20MIX.OR

STERGRD.Q

STERGRD.Q

STERSTATE. Q

STERSTATE. Q

-> SI

-> S2

-> S3

-> S4

SBLOCK H20MIX

51 = "0-0-0-0",

52 - "0-0-0-0",

53 = "0-0-0-0",

54 = "0-0-0-0",

Q - "1-2-5-5",

NQ - "0-0-0-0",

SGROUP TEMPERATURE

TS = .5 ; RUN = YES ; PR = 1

SBLOCK TRIN.AI SBLOCK TRIN SBLOCK TRCALIB.SFUN SBLOCK TRCALIB

IO

OMAX -

"1-1-1-1",

50,

TRIN.OUT -> IN N = 10.

11 = 19.2,

OMIN = 0 ; Ol - 20,

UNIT = "GRD-C" ; 12 = 24.05,

ZERO - YES,

02 = 25 ;

INV - NO ; 13 = 28.85 ;

03 = 30 ;

14 = 33.8 ;

04 - 35 ;

15 = 38 4,

SBLOCK TRSELECT.MIMA

TRIN.OUT -> INI

-> IN2

SBLOCK TRSELECT

HI - "1-2-6-1",

NHI = "0-0-0-0",

INIV = 0 ;

IN2V = 47.5 ;05 - 40 ;

16 - 48.1 ;

06 - 50 ;

17 = 57.8 ;

07 - 60 ;

18 = 76.8,

08 - 80 ;

19 = 96.05 ;

09 = 100 ;

110 - 115.5 ;

010 - 120 ;

BIAS = 0 ;

SBLOCK TR150.SETP

TRSELECT.HI

-> INI

-> IN2

-> IN3

-> SEND

SBLOCK TR150

SEND =

BLK -

PARI =

INIV -

PAR2 =

IN2V =

PAR3 =

IN3V =

"0-0-0-0",

"TRIN" ;

"OMIN",

0 ;

"OMAX" ;

150 ;

0 ;

SBLOCK TR050.SETP

TRSELECT.NHI

-> INI

-> IN2

-> IN3

-> SEND

SBLOCK TR050

SEND =

BLK =

PARI -

INIV -

PAR2 -

IN2V =

PAR3 -

"0-0-0-0" ;

"TRIN" ;

"OMIN",

0,

"OMAX" ;

50 ;

SBLOCK TCIN.AI SBLOCK TCIN

IO - "1-1-1-2" ;

OMAX = 50 ;

OMIN = 0 ;

UNIT = "GRD-C" ;

ZERO = YES ;

IN3V = 0,

INV = NO ;

SBLOCK TCSELECT.MIMA SBLOCK TCSELECT

TCIN.OUT -> INI

-> IN2

HI = "1-2-6-2" ;

NHI - "0-0-0-0",

INIV - 0 ;

IN2V =47.5 ;

SBLOCK TC150.SETP SBLOCK TCI 50

TCSELECT.HI

-> INI

-> IN2

-> IN3

-> SEND

SEND - "0-0-0-0" ;

BLK = "TCIN" ;

PARI = "OMIN" ;

INIV - 0 ;

PAR2 = "OMAX" ;

IN2V - 150 ;

PAP3 =""

;

IN3V - 0 ;

Appendix 107

SBLOCK TC050.SETP SBLOCK TC050 SBLOCK INAKSTATE.SR SBLOCK INAKSTATE

-> INI SEND - "0-0-0-0", INAKSTAT.Q -> SET SET - "0-0-0-0"

,

-> IN2BLK - "TCIN"

, INAKRES.Q -> RESRES - "0-0-0-0"

.

-> IN3PARI - "OMIN"

,Q - "0-0-0-0"

,

TCSELECT.NHIINIV - 0

,NQ "0-0-0-0" ,

-> SENDPAR2 - "OMAX"

, SBLOCK CULTKILL.OR SBLOCK CULTKILL

IN2V 50, COOLSTAT.Q -> SI SI - "0-0-0-0"

,

PAR3 " "

,

STERSTATE.Q -> S2S2 - "0-0-0-0"

,

IN3V - 0,

INAKSTATE.Q -> S3S3 "0-0-0-0" ,

SBLOCK TCCALIB.SFUN SBLOCK TCCALIB-> S4

S4 - "0-0-0 0",

TCIN.OUT -> IN N 2,

11 - 1 04,

Ol - 0.

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK CULTSET.SR SBLOCK CULTSET

12 - 43 82, TCULTGTT.HI -> SET SET - "0-0-0-0"

,

02 _ 44 6,

CULTKILL.Q -> RESRES - "0-0-0-0"

,

13 - 0, Q - "0-0-0-0"

,

03 0,

14 0,

NQ - "0-0-0-0",

SBLOCK CULTRES.OR SBLOCK CULTRES

04 0,

STERSTATE.Q -> SI SI - '0-0-0-0",

15 - 0.

COOLSTATE.Q -> S2S2 - "0-0-0-0"

,

05 - 0,

INAKSTATE.Q -> S3S3 - "0-0-0-0"

,

16 - 0, S4 - "0-0-0-0" ,

06 - 0,

-> S4Q - "0-0-0-0"

,

17 - 0,

07 - 0,

NQ - "0-0-0-0",

SBLOCK CULTSTATE.SR SBLOCK CULTSTATE

18 0,

CULTSET.Q -> SET SET - "0-0-0-0",

08 - 0,

19 - 0,

CULTRES.Q -> RESRES "0-0-0-0"

,

Q "0-0-0-0",

09 - 0,

110 - 0,

NQ - "0-0-0-0",

SBLOCK TSETSLCT.MPX SBLOCK TSETSLCT

010 - 0,

-> INI SI - "0-0-0-0" ,

BIAS 0,

CULTSTATE.Q -> SI52 - "0-0-0-0"

,

53 - "0-0-0-0",

SBLOCK TLIN.AI SBLOCK TLIN

IO - "1-1-1-5",

-> IN2S4 "0-0-0-0"

,

OMAX - 150 ;STERSTATE.Q -> S2

CHG - "0-0-0-0",

OMIN - 0,

-> IN3NCHG = "0-0-0-0"

,

UNIT _ "GRD C", COOLSTATE.Q -> S3 INIV - 37

,

ZERO - YES, -> IN4 IN2V - 127

,

INV NO, INAKSTATE.Q -> S4 IN3V - 0

,

SBLOCK TLCALIB.SFUN SBLOCK TLCALIB IN4V - 80,

TLIN.OUT -> IN N - 10,

11 - 24 32,

PULS - 0,

SBLOCK TRPID.PID SBLOCK TRPID

Ol - 21,

TRCALIB.OUT -> MV STRA - "0-0-0-0",

12 - 36 34,

TSETSLCT.OUT -> SPMAN "0-0-0-0" ,

02 - 33 4, NMAN - "0-0-0-0"

,

13 - 43 52,

HOLDTR.Q -> STRASMAX 135

,

03 - 41,

TRTRA.OUT -> TRASMIN 0

,

14 49 93, MUNT - "GRD C"

,

04 - 47 8, OMAX _ 100

,

15 62 6 ; OMIN - -100,

05 - 60 8, OUNT -

" GRD C",

16 - 71 32, GAIN - 20

,

06 - 7 0.9, TI - 100

,

17 - 83 23, TD - 0

,

07 87 7, TFIL - 0

,

18 - 95 57, DZ - 0

,

08 - 100 4, BIAS - 0

,

19 - 113, INT - YES

,

09 - 119 5,

110 - 125 02,

MODE - 0,

SBLOCK TCSETPNT.SUM SBLOCK TCSETPNT

O10 - 132.2,

TRPID.OUT -> INI GAI1 - 1,

BIAS - 0,

TSETSLCT. OUT -> IN2GAI2 - 1

,

GAI3 - 1,

SBLOCK TSTERMIMA.MIMA SBLOCK TSTERMIMA

TRCALIB.OUT -> INI HI - "0-0-0-0",

-> IN3GAI4 - 1

,

TLCALIB.OUT -> IN2NHI _ "0-0-0-0"

,

INIV 0,

-> IN4BIAS - 0

,

SBLOCK TCPID.PID SBLOCK TCPID

IN2V - 0,

TCCALIB. OUT -> MV STRA - "0-0-0-0",

SBLOCK TRTLMEAN.SUM SBLOCK TRTLMEAN

TCSETPNT. OUT -> SPMAN - "0-0-0-0"

,

TRCALIB.OUT -> INI GAI1 - 5, NMAN - "0-0-0-0"

,

TLCALIB.OUT -> IN2GAI2 _ 5

,

-> STRASMAX - 150 ;

-> IN3GAI3 - 1

,

-> TRASMIN - 0

,

-> IN4GAI4 - 1

,

BIAS - 0,

MUNT - "GRD C",

OMAX 250,

OMIN - -250,SBLOCK INAKSTAT.BHAN SBLOCK INAKSTAT

-> S S - "0-0-0-0",

Q - "0-0-0-0",

NQ "0-0-0-0",

QSET - YES,

PULS _ 5.

OUNT -

" %" ;

GAIN - 20,

TI - 1000,

TD " 0,

TFIL - 0,

DZ - 0,SBLOCK INAKRES.OR SBLOCK INAKRES

INAKTIME.RDY -> SI SI - "0-0-0-0", BIAS - 0

,

COOLSTAT.Q -> S2

-> S3

52 - "0-0-0-0",

53 - "0-0-0-0",

INT - YES,

MODE - 0,

S4 - "0-0-0-0",

-> S4Q - "0-0-0-0"

,

NQ "0-0-0 0",

108 Appendix

SBLOCK STEAMRV.AO

TCPID.OUT -> IN

SBLOCK STEAMRV

IO = "1-1-4-2",

IMAX - 100,

IMIN = 0 ;

UNIT - "%",

ZERO - YES,

INV - NO,

SBLOCK ARRSTINTR.BSET

-> SI

-> S2

-> S3

STOPINTTR.Q -> SEND

SBLOCK ARRSTINTR

SEND - "0-0-0-0" ,

BLK - "TRPID" ,

PARI = "INT",

SIV - NO,

PAR2 =""

,

S2V - NO,

PAR3 =""

S3V = NO,

SBLOCK WATERRV.AO

TCPID OUT -> IN

SBLOCK WATERRV

IO - "1-1-4-1"

IMAX - 1,

IMIN -100,

UNIT = "%",

ZERO - YES,

INV - NO,

SBLOCK STARTINTR.BSET

-> SI

-> S2

-> S3

STOPINTTR.NQ -> SEND

SBLOCK STARTINTR

SEND = "0-0-0-0",

BLK = "TRPID",

PARI - "INT",

SIV = YES

PAR2 =""

,

S2V - NO,

PAR3 -

""

,

S3V - NO,

SBLOCK TEMPAUXV.REL

TCPID.OUT -> IN

SBLOCK TEMPAUXV

INC - "0-0-0-0"

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC = "0-0-0-0",

DZ - 150,

AMPL - 0,

SBLOCK STOPINTTC OR

WATERAUX.Q -> SI

STEAMAUX Q -> S2

-> S3

-> S4

SBLOCK STOPINTTC

51 - "0-0-0-0".

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0" ,

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK DELTATEMP.REL

TRPID E -> IN

SBLOCK DELTATEMP

INC - "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ = 65 ,

AMPL - 0,

SBLOCK ARRSTINTC BSET

-> SI

-> S2

-> S3

STOPINTTC.Q -> SEND

SBLOCK ARRSTINTC

SEND - "0-0-0-0",

BLK - "TCPID",

PARI - "INT",

SIV = NO,

PAR2 -

""

,

S2V - NO,

PAR3 -

""

,

S3V = NO,

SBLOCK STEAMAUX.AND

DELTATEMP. INC -> SI

DELTATEMP. INC -> S2

TEMPAUXV. INC -> S3

HOLDTR.NQ -> S4

SBLOCK STEAMAUX

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0" ,

54 - "0-0-0-0",

Q = "1-2-4-6",

NQ - "0-0-0-0", SBLOCK STARTINTC.BSET

-> SI

-> S2

-> S3

STOPINTTC.NQ -> SEND

SBLOCK STARTINTC

SEND - "0-0-0-0",

BLK = "TCPID",

PARI - "INT",

SIV - YES,

PAR2 =""

,

S2V = NO,

PAR3 -""

,

S3V - NO,

SBLOCK WATERAUX.AND

HOLDTR.NQ -> SI

DELTATEMP. DEC -> S2

TEMPAUXV.DEC -> S3

STERSTATE.NQ -> S4

SBLOCK WATERAUX

51 - "0-0-0-0",

52 - "0-0-0-0",

53 = "0-0-0-0" ,

54 - "0-0-0-0"

Q - "1-2-4-7",

NQ = "0-0-0-0"

SBLOCK STOPINTTR.OR

DELTATEMP. INC -> SI

DELTATEMP. DEC -> S2

-> S3

-> S4

SBLOCK STOPINTTR

51 = "0-0-0-0"

52 = "0-0-0-0"

53 = "0-0-0-0"

54 - "0-0-0-0"

Q = "0-0-0-0"

NQ - "0-0-0-0",

SBLOCK DELTAT SUM

TRCALIB.OUT -> INI

TCCALIB.OUT -> IN2

-> IN3

-> IN4

SBLOCK DELTAT

GAI1 = 1,

GAI2 - -1,

GAI3 = 1,

GAI4 - 1,

BIAS - 0,

SGROUP MOTORS

TS = 2 ; RUN = YES ; PR = 1

SBLOCK NSTSET.AI SBLOCK NSTSET

IO - "0-0-0-0"

OMAX - 6000,

OMIN = 0,

UNIT = "RPM",

ZERO = YES,

INV = NO,

SBLOCK NSTENABLE.REL

STIRRSET.OUT -> IN

SBLOCK NSTENABLE

INC - "0-0-0-0" ,

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ - 100,

AMPL - 0.

SBLOCK STIRRSET.SFUN

NSTSET.OUT -> IN

SBLOCK STIRRSET

N - 8,

11 - 216,

01 - 200,

12 = 417,

02 - 400,

13 - 823,

03 - 800,

14 - 1234,

04 = 1200,

15 = 1650,

05 - 1600,

16 = 2075 ,

06 - 2000,

17 - 2505,

07 = 2400,

18 - 2720,

08 = 2600,

19 = 0,

09 = 0,

110 = 0,

oio = o,

BIAS - 0,

SBLOCK NSTREAD.Al SBLOCK NSTREAD

IO - "1-1-1-3",

OMAX - 6000.

OMIN - 0,

UNIT - "RPM",

ZERO = YES,

INV = NO,

SBLOCK TCIRCPUMP.OR

STERSTATE.Q -> SI

CULTSTATE.Q -> S2

COOLSTATE.Q -> S3

NSTENABLE.INC -> S4

SBLOCK TCIRCPUMP

51 = "0-0-0-0" ,

52 - "0-0-0-0",

53 - "0-0-0-0",

54 = "0-0-0-0" ,

Q = "1-2-5-3",

NQ - "0-0-0-0",

SBLOCK ENASTI.AND

NSTENABLE.INC -> SI

ENAMOT.Q -> S2

ENAMOT.Q -> S3

TCIRCPUMP.Q -> S4

SBLOCK ENASTI

51 - "0-0-0-0" ,

52 - "0-0-0-0",

53 - "0-0-0-0",

54 = "0-0-0-0",

Q - "1-2-5-7",

NQ = "0-0-0-0",

Appendix 109

SBLOCK PROTST.MPX SBLOCK PROTST SBLOCK NFSENABLE.REL SBLOCK NFSENABLE

STIRRSET.OUT

ENASTI.Q

ENASTI.NQ

-> INI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

51 "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

CHG - "0-0-0-0",

NCHG "0-0 0-0",

INIV - 0,

IN2V - 0,

IN3V 0,

IN4V 0,

PULS - 0,

FOAMSET. OUT -> IN INC - "0-0-0-0",

NINC - "0-0-0-0",

DEC = "0-0-0-0",

NDEC - "0-0-0-0",

DZ - 25,

AMPL - 0,

SBLOCK ENAFOS.AND

ENAMOT.Q

STERGRD.NQ

NFSENABLE.INC

STERSTATE. NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK ENAFOS

51 - "0-0-0-0",

52 "0-0-0-0",

53 "0-0-0-0",

54 "0-0-0-0",

Q - "1-2-5-8",SBLOCK NSTDLIMIT.DLIM SBLOCK NSTDLIMIT

PROTST. OUT -> IN DMAX _ 100,

DMIN - -100,

NQ "0-0-0-0",

SBLOCK PROTFS.MPX

FOAMSET.OUT -> INI

SBLOCK PROTFS

SI "0-0-0-0",SBLOCK NSTOUT.AO SBLOCK NSTOUT

NSTDLIMIT.OUT -> IN IO - "1-1-6-1",

IMAX 6000,

IMIN - 0,

UNIT - 'RPM',

ZERO - YES,

INV - NO,

ENAFOS.Q

ENAFOS.NQ

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

CHG "0-0-0-0",

NCHG "0-0-0-0",

INIV 0,

IN2V 0,

IN3V 0,

IN4V 0,

PULS - 0,

SBLOCK STIRRLR.SR

-> SET

-> RES

SBLOCK STIRRLR

SET - "1-2-1-4",

RES _ "1-2-1-5" ;

Q - "0-0-0-0",

NQ - "0-0-0-0" SBLOCK NFSDLIMIT.DLIM

PROTFS OUT -> IN

SBLOCK NFSDLIMIT

DMAX - 100,

DMIN - -100,

SBLOCK NSTIN.SFUN

NSTREAD.OUT -> IN

SBLOCK NSTIN

N - 8,

11 - 122, SBLOCK NFSOUT.AO SBLOCK NFSOUT

Ol - 216,

12 _ 356,

NFSDLIMIT.OUT -> IN IO - "1-1-6-2",

IMAX 6000,

02 - 417,

IMIN - 0,

13 _ 846,

UNIT - "RPM",

03 - 823,

ZERO - YES,

14 _ 1360,

04 1234,

15 - 1880,

INV - NO,

SBLOCK NFSREAD.AI SBLOCK NFSREAD

IO - "1-1-1-4",

05 - 1650, OMAX 6000

,

16 - 2404, OMIN 0

,

06 _ 2075, UNIT - "RPM"

,

17 - 2926, ZERO - YES

,

07 _ 2505,

18 - 3193,

08 _ 2720,

INV - NO,

SBLOCK FOAMSEPLR.AI SBLOCK FOAMSEPLR

io - "O-o-o-O",

19 - 0, OMAX - 100

,

09 0.

110 - 0,

010 - 0,

OMIN 0,

UNIT -

""

,

ZERO - NO,

BIAS - -30, INV - NO

SBLOCK CCIRCPUMP.OR SBLOCK CCIRCPUMP SBLOCK FOAM.SR SBLOCK FOAM

CULTSTATE.Q -> SI SI - "0-0-0-0",

-> SET SET - "1-2-1-1",

STERSTATE.Q

COOLSTATE.Q

INAKSTATE.Q

-> S2

-> S3

-> S4

52 _ "0-0-0-0",

53 - "0-0-0-0",

54 _ "0-0-0-0",

Q - "1-2-5-4",

NQ _ -0-0-0-0",

-> RESRES - "0-0-0-0"

,

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK NFSIN.SFUN

NFSREAD.OUT -> IN

SBLOCK NFSIN

N - 8,

11 - 220 ;SBLOCK NFSSET.AI SBLOCK NFSSET

io _ "o-o-o-O", Ol - 460

,

OMAX - 6000, 12 - 740

,

OMIN - 0, 02 898

,

UNIT "RPM",

ZERO - YES,

13 - 1330,

03 - 1343 ;

INV - NO, 14 1950

,

04 - 1792,SBLOCK FOAMSET.SFUN SBLOCK FOAMSET

NFSSET.OUT -> IN N - 8,

11 - 460,

01 - 400,

12 898,

02 = 800,

13 1343,

03 1200,

14 - 1792,

04 _ 1600,

15 - 2250,

05 - 2000,

16 - 2710,

06 _ 2400,

17 = 2942,

15 2575,

05 2250,

16 - 3180,

06 - 2710,

17 - 3475 ;

07 - 2942,

18 - 4025,

08 3416,

19 - 0,

09 - 0,

110 - 0,

010 - 0,

BIAS - 0,

07 _ 2600 ;

18 - 3416,

08 3000,

19 - 0,

09 - 0,

110 - 0,

O10 - 0,

BIAS - 0,

110 Append]

SGROUP WEIGHTFEED

TS - 2 ; RUN = YES ; PR = 1

SBLOCK BALANCE.Al SBLOCK BALANCE

IO = "1-1-1-6",

OMAX - 8,

OMIN - 0,

UNIT = "KG",

ZERO - YES,

INV NO

SBLOCK MEDFLUX.Al SBLOCK MEDFLUX

IO = "0-0-0-0",

OMAX = 5000,

OMIN = 0,

UNIT - "ML/H" ,

ZERO = NO ,

INV = NO,

SBLOCK WEIGHTIN.SFUN SBLOCK WEIGHTIN SBLOCK FINCALIB SFUN SBLOCK FINCALIB

BALANCE.OUT -> IN N - 6,

11 -12,

01 - 0,

12 = 2 75

02 - 2,

13 3 15,

03 -25,

14 - 3 35,

04 - 2 75.

15 = 3 55,

05 - 3,

16 - 3 95,

06 -35,

17 - 0,

07 = 0,

18 - 0,

08 - 0,

19 - 0,

09 = 0,

110 - 0,

010 - 0,

BIAS - 0,

MEDFLUX. OUT -> IN N - 10,

11 - 0,

01 - -1000,

12 - 35,

02 = 1,

13 - 190

03 - 4 73,

14 - 380,

04 - 9 45,

15 - 570,

05 - 14 18,

16 - 760,

06 - 18 24,

17 - 950,

07 - 22 7,

18 = 1140,

08 = 26 64,

19 = 1520,

09 - 35 2,

110 = 1900,

010 - 43 7,

BIAS - 0 ,

SBLOCK WEICALC.SFUN SBLOCK WEICALC SBLOCK TOHEAVY.MIMA SBLOCK TOHEAVY

WEIGHTIN OUT -> IN N = 2, WEIGHTIN.OUT -> INI HI - "0-0-0-0"

,

11 - 3,

-> IN2NHI - "0-0-0-0"

,

01 - 200,

INIV = 0,

12 - 5,

02 - 800,

IN2V =65,

SBLOCK FEEDEMER.OR SBLOCK FEEDEMER

13 = 0, TOHEAVY.HI -> SI SI - "1-2-1-3"

03 = 0,

TOHEAVY.HI -> S2S2 = "0-0-0-0"

,

14 - 0,

TOHEAVY.HI -> S3S3 - "0-0-0-0"

,

04 - 0, S4 - "0-0-0-0"

,

15 = 0,

TOHEAVY.HI -> S4Q - "0-0-0-0"

,

05 = 0,

16 = 0,

NQ - "0-0-0-0",

SBLOCK BATCH.BHAN SBLOCK BATCH

06 = 0,

-> S S = "0-0-0-0",

17 - 0, Q - "0-0-0-0"

,

07 - 0, NQ = "0-0-0-0"

,

18 - 0, QSET = NO

,

08 = 0,

19 = 0,

PULS - 0,

SBLOCK BATCHSTAT.SR SBLOCK BATCHSTAT

09 = 0,

BATCH. Q -> SET SET = "0-0-0-0",

110 = 0,

010 - o,

BATCH.NQ -> RESRES = "0-0-0-0"

,

Q - "0-0-0-0",

BIAS - 0, NQ = "0-0-0-0"

,

SBLOCK WSIGNAL.AI SBLOCK WSIGNALSBLOCK PRUNSTP.SETP SBLOCK PRUNSTP

IO = "1-1-1-8",

-> INI SEND - "0-0-0-0",

OMAX - 1000,

-> IN2BLK - "PINSUM"

,

OMIN = 0, PARI - "BIAS"

,

UNIT = "G",

-> IN3INIV = 0

,

ZERO = NO,

BATCHSTAT.NQ -> SENDPAR2 =

""

,

INV = NO, IN2V = 0

,

PAR3 -

""

,SBLOCK WCALIB.SFUN SBLOCK WCALIB

WSIGNAL.OUT -> IN N = 10,

11 =1 52625,

IN3V - 0,

SBLOCK PSTOPSTP.SETP SBLOCK PSTOPSTP

01 = 0,

-> INI SEND = "0-0-0-0",

12 - 101 343,

-> IN2BLK = "PINSUM"

,

02 - 100 1,

-> IN3 [PARI - "BIAS",

13 = 200 855, IIN1V

- -1000,

03 = 200 1,

BATCHSTAT.Q -> SEND1mm

PAR2 =""

,

14 = 300 366, IN2V - 0

,

04 - 300 1, PAR3 -

""

,

15 - 399 878,

05 = 400 1,

IN3V - 0,

SBLOCK BATCONT.MPX SBLOCK BATCONT

16 - 499 389,

06 = 500 1,

17 = 599 206,

WEICALC.OUT

BATCHSTAT.Q

-> INI

-> SI

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

07 = 600 1,

WCALIB.OUT -> IN2S4 - "0-0-0-0"

,

18 - 698 718,

BATCHSTAT.NQ -> S2CHC = "0-0-0-0"

,

08 - 700 1, WCALIB.OUT -> IN3

NCHG - "0-0-0-0",

19 - 798 23, BATCHSTAT.NQ -> S3 INIV - 0

,

09 - 800 1, WEICALC. OUT -> IN4 IN2V = 0

,

110 897 741,

BATCHSTAT.Q -> S4 IN3V = 0,

OlO = 900 1, IN4V = 0

,

BIAS - 0, PULS - 0

,

Appendix 111

SBLOCK SETMEDFL. SETP

BATCHSTAT. Q

-> INI

-> IN2

-> IN3

-> SEND

SBLOCK SETMEDFL

SEND "0-0-0-0",

BLK -""

,

PARI -

""

,

INIV - 0,

PAR2 -""

,

IN2V - 0,

PAR3 -""

,

IN3V - 0,

SBLOCK STOPFLOW.REL

MEDFLUX. OUT -> IN

SBLOCK STOPFLOW

INC _ "0-0-0-0" ,

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC "0-0-0-0",

DZ 1,

AMPL 10,

SBLOCK COUDOC02.AND

STOPFLOW.INC

BATCHSTAT. NQ

FISDIFF.NQ

STERSTATE.NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK COUDOC02

51 "0-0-0-0",

52 _ "0-0-0-0" ,

53 "0-0-0-0",

54 _ "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0" ,

SBLOCK WEIGHTSET.AI SBLOCK WEIGHTSET

io "O-o-o-O",

OMAX 7,

OMIN - 0,

UNIT - "KG",

ZERO - NO ,

INV - NO.

SBLOCK COUDOCO.AND

UPLIM.NHI

LOLIM.NHI

BUFFALLOW.NQ

COUDOC02.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK COUDOCO

51 - "0-0-0-0" ,

52 "0-0-0-0",

53 _ "0 0-0-0",

54 - "0-0-0-0",

Q "0-0-0-0",

NQ "0-0-0-0",

SBLOCK UPLIM.MIMA

BATCONT. OUT -> INI

-> IN2

SBLOCK UPLIM

HI - "0 0-0-0",

NHI - "0-0-0-0",

INIV - 0,

IN2V 800,

SBLOCK LOLIM.MIMA

BATCONT. OUT

-> INI

-> IN2

SBLOCK LOLIM

HI "0-0-0-0",

NHI "0-0-0-0",

INIV - 200,

IN2V - 0,

SBLOCK RESCO.OR

STERSTATE.Q

BATCHSTAT.Q

UPLIM.HI

FISDIFF.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK RESCO

51 - "0-0-0-0" ,

52 - "0-0-0-0",

53 - "0-0-0-0" ,

54 "0-0-0-0",

Q "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK FILLLOG.AND

STERSTATE.NQ

STERSTATE.NQ

LOLIM.HI

LOLIM.HI

-> SI

-> S2

-> S3

-> S4

SBLOCK FILLLOG

si - "o-o-o-O",

52 - "0-0-0-0',

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK COUNTDOWN.TIME

COUDOCO.Q -> RUN

RESCO.Q -> RES

SBLOCK COUNTDOWN

RUN "0-0-0-0",

RES - "0-0-0-0",

RDY - "0-0-0-0" ,

NRDY - "0-0-0-0",

RUNV - NO,

RESV NO,

TIME 0,

ZERO - 0,

ACCU - YES,

PULS - 0,

SBLOCK REFILL.SR

FILLLOG.Q

UPLIM HI

-> SET

-> RES

SBLOCK REFILL

SET - "0 0-0-0",

RES - "0-0 0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK BUFFALLOW.AND

REFILL.Q

REFILL.Q

FEEDEMER.NQ

FEEDEMER.NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK BUFFALLOW

51 - "0-0-0-0",

52 - "0-0-0-0",

53 "0-0-0-0" ,

54 - "0-0-0-0",

Q - "1-2-6-7",

NQ - "0-0-0-0",

SBLOCK FEEDTIME.AI SBLOCK FEEDTIME

IO "0-0-0-0",

OMAX 1 e+06,

OMIN - 0,

UNIT "SEC",

ZERO - NO

INV - NO,SBLOCK FCHANGE.GEDY

MEDFLUX.OUT -> INI

-> IN2

-> TRA

-> STRA

SBLOCK FCHANGE

STRA - "0-0-0-0",

MAN - "0-0-0-0",

NMAN - "0-0-0-0",

I1MA - 10000,

I1MI - 0,

I1UN - "ML/H" ,

I2MA - 100,

I2MI - 0,

I2UN -""

,

OMAX - 500,

OMIN - -500,

OUNT - "ML/H" ,

Al 0,

A2 - 0,

A3 - 0,

B0 - 1,

Bl - 0,

B2 - 0,

B3 - -1,

CO - 0,

CI - 0,

C2 - 0,

C3 - 0,

MODE - 0,

SBLOCK DECRTIME.SUM

COUNTDOWN. RETI

FEEDTIME.OUT

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK DECRTIME

GAI1 - -1,

GAI2 - 1,

GAI3 - 0,

GAI4 - 0,

BIAS - 0,

SBLOCK SENDWEI.SETP

WCALIB. OUT

COUDOCO.Q

-> INI

-> IN2

-> IN3

-> SEND

SBLOCK SENDWEI

SEND - "0-0-0-0" ,

BLK - "CALCDW",

PARI _ "J3IAJ3" ,

INIV - 0,

PAR2 - ""

,

IN2V 0,

PAR3 -

""

,

IN3V - 0,

SBLOCK DELFSP.GEDY

MEDFLUX. OUT -> INI

-> IN2

-> TRA

-> STRA

SBLOCK DELFSP

STRA - "0-0-0-0" ;

MAN - "0-0-0-0",

NMAN - "0-0-0-0",

I1MA _ 10000 ,-

I1MI - 0,

HUN "ML/H" ,

I2MA _ 100,

I2MI - 0,

I2UN -

""

,

OMAX - 10000,

OMIN - 0,

OUNT "ML/H" ,

Al - 0,

A2 - -.2,

A3 - 0,

B0 0,

Bl - 0,

B2 8,

B3 - 0,

CO - 0,

CI - 0,

C2 - 0,

C3 - 0,

MODE - 0

SBLOCK CHANGEF.REL

FCHANGE. OUT -> IN

SBLOCK CHANGEF

INC - "0-0-0-0",

NINC - "0-0-0-0" ,

DEC - "0-0-0-0",

NDEC = "0-0-0-0" ,

DZ - .1,

AMPL - 50,

SBLOCK FISDIFF.OR

CHANGEF.INC

CHANGEF.DEC

-> SI

-> S2

-> S3

-> S4

SBLOCK FISDIFF

51 - "0-0-0-0",

52 - "0-0-0-0".

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK CALCW.MULT

DELFSP.OUT

DECRTIME. OUT

-> INI

-> IN2

SBLOCK

\

CALCW

GAIN - 000277778,

BIAS - 0,

112 Appendix

SBLOCK CALCDW.SUM

CALCW.OUT -> INI

-> IN2

-> IN3

-> IN4

SBLOCK CALCDW

GAI1 - -1,

GAI2 - 0,

GAI3 - 0,

GAI4 - 0,

BIAS = 799 179,

SBLOCK TOFPUMP.GEDY

FLUXREG.OUT -> INI

-> IN2

-> TRA

-> STRA

SBLOCK TOFPUMP

STRA = "0-0-0-0",

MAN = "0-0-0-0",

NMAN = "0-0-0-0" ;

I1MA - 100,

I1MI = -100,

HUN ""

,

I2MA = 100

I2MI = 0,

I2UN -

""

,

OMAX = 100,

OMIN = -100,

OUNT =""

,

Al -- 5

,

A2 - 0.

A3 - 0,

B0 - 5,

Bl - 0,

B2 - 0,

B3 = 0,

CO = 0,

CI - 0,

C2 - 0,

C3 - 0,

MODE - 0,

SBLOCK SENDFTIME.SETP

FEEDTIME.OUT -> INI

-> IN2

-> IN3

LOLIM.NHI -> SEND

SBLOCK SENDFTIME

SEND "0-0-0-0"

BLK - "COUNTDOWN",

PARI - "TIME",

INIV - 0,

PAR2 -""

,

IN2V - 0,

PAR3 -""

,

IN3V - 0,

SBLOCK FREGINTDEL.TIME

COUDOCO.Q -> RUN

RESCO.Q -> RES

SBLOCK FREGINTDEL

RUN - "0-0-0-0",

RES - "0-0-0-0",

RDY - "0-0-0-0",

NRDY - "0-0-0-0",

RUNV - NO,

RESV - NO,

TIME = 60,

ZERO - 0,

ACCU - YES

PULS - 0, SBLOCK PINSUM.SUM

FINCALIB.OUT -> INI

TOFPUMP.OUT -> IN2

-> IN3

-> IN4

SBLOCK PINSUM

GAI1 1,

GAI2 = 1,

GAI3 - 0,

GAI4 - 0,

BIAS = 0,

SBLOCK FREGINT.BSET

-> SI

-> S2

-> S3

COUDOCO.Q -> SEND

SBLOCK FREGINT

SEND - "0-0-0-0",

BLK - "FLUXREG",

PARI - "INT",

SIV - YES,

PAR2 -""

S2V - NO,

PAR3 -""

,

S3V - NO,

SBLOCK MEDPUMP.AO

PINSUM.OUT -> IN

SBLOCK MEDPUMP

IO = "1-1-7-1",

IMAX - 100,

IMIN - 0,

UNIT - "%",

ZERO = YES,

INV - NO,

SBLOCK FREGFREEZE.AND

FREGINTDEL.RDY -> SI

FREGINTDEL.RDY -> S2

COUDOCO.Q -> S3

STOPFLOW.INC -> S4

SBLOCK FREGFREEZE

51 "0-0-0-0",

52 - "0-0-0-0*,

53 - "0-0-0-0",

54 - "0-0-0-0",

Q = "0-0-0-0",

NQ = "0-0-0-0",

SBLOCK SENDWEI2.SETP

WCALIB.OUT -> INI

-> IN2

-> IN3

COUDOCO.Q -> SEND

SBLOCK SENDWEI2

SEND = "0-0-0-0",

BLK - "MFLUXSUM" ,

PARI - "BIAS",

INIV - 0,

PAR2 -""

,

IN2V = 0,

PAR3 =""

,

IN3V - 0,

SBLOCK FLUXREG.PID

WCALIB.OUT -> MV

CALCDW.OUT -> SP

FREGFREEZE.NQ -> STRA

-> TRA

SBLOCK FLUXREG

STRA - "0-0-0-0",

MAN - "0-0-0-0",

NMAN - "0-0-0-0",

SMAX - 1000,

SMIN - 0,

MUNT = "GRAMM",

OMAX 100,

OMIN - -100,

OUNT = "%PUMP",

GAIN = - 01,

TI = 10000,

TD - 0,

TFIL = 0,

DZ - 0,

BIAS - 0,

INT - NO,

MODE = 3,

SBLOCK MFLUXSUM.SUM

WCALIB.OUT -> INI

-> IN2

-> IN3

-> IN4

SBLOCK MFLUXSUM

GAI1 - -1,

GAI2 - 1,

GAI3 - 1,

GAI4 - 1,

BIAS = 799 179,

SBLOCK MFLUXCALC.DIV

MFLUXSUM.OUT -> NUM

DECRTIME.OUT -> DEN

SBLOCK MFLUXCALC

GAIN = 3600,

BIAS - 0,

SBLOCK DILUCALC.DIV

MFLUXCALC.OUT -> NUM

WEIGHTIN.OUT -> DEN

SBLOCK DILUCALC

GAIN = 001,

BIAS = 0,

SBLOCK NOINTCO.OR

RESCO.Q -> SI

RESCO.Q -> S2

STOPFLOW.NINC -> S3

STOPFLOW.NINC -> S4

SBLOCK NOINTCO

51 - "0-0-0-0",

52 = "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK MFLUXSEND.SETP

MFLUXCALC.OUT -> INI

-> IN2

-> IN3

LOLIM.HI -> SEND

SBLOCK MFLUXSEND

SEND = "0-0-0-0",

BLK - "MFLUXHOLD",

PARI - "BIAS",

INIV = 0,

PAR2 -""

,

IN2V - 0,

PAR3 -

""

,

IN3V - 0,

SBLOCK FREGNOINT.BSET

-> SI

-> S2

-> S3

NOINTCO.Q -> SEND

SBLOCK FREGNOINT

SEND = "0-0-0-0" ,

BLK - "FLUXREG",

PARI - "INT",

SIV = NO,

PAR2 =""

,

S2V NO,

PAR3 -

""

,

S3V - NO,

SBLOCK MFLUXHOLD.SUM

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK MFLUXHOLD

GAI1 - 1,

GAI2 = 1,

GAI3 - 1,

GAI4 - 1,

BIAS = -22273 6,

SBLOCK DILUSEND.SETP

DILUCALC.OUT -> INI

-> IN2

-> IN3

LOLIM.HI -> SEND

SBLOCK DILUSEND

SEND - "0-0-0-0" ,

BLK - "DILUHOLD",

PARI - "BIAS",

INIV - 0,

PAR2 -

""

,

IN2V - 0,

PAR3 -

""

,

IN3V - 0,

Appendix 113

SBLOCK DILUHOLD.SUM SBLOCK DILUHOLD SBLOCK BOTTALLOW.AND SBLOCK BOTTALLOW

-> INI GAI1 - 1, REFILL.Q -> SI SI - "0-0-0-0"

,

->

->

->

IN2

IN3

IN4

GAI2 - 1,

GAI3 1,

GAI4 - 1,

BIAS 14 5031,

REFILL.Q -> S2

FEEDEMER.NQ -> S3

FEEDEMER.NQ -> S4

52 - "0-0-0-0",

53 - "0-0-0-0",

54 "0-0-0-0",

Q "1-2-6-6",

NQ - "0 0-0-0",SBLOCK CALCDELAY.TIME

COUDOCO.Q -> RUN

SBLOCK CALCDELAY

RUN "0-0-0-0', SBLOCK WREGFREEZE.BHAN SBLOCK WREGFREEZE

LOLIM.HI -> RESRES "0-0-0-0

RDY - "0-0-0-0,

NRDY "0-0-0-0',

RUNV - NO,

RESV NO,

TIME - 60,

-> S S "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

QSET NO,

PULS 120,

SBLOCK WREG.PID SBLOCK WREG

ZERO 0, WEIGHTIN.OUT -> MV STRA "0-0-0-0"

,

ACCU - NO,

PULS - 0,

WEIGHTSET.OUT -> SP

WREGFREEZE Q -> STRA

-> TRA

MAN - "0-0 0-0",

NMAN "0-0-0-0",

SMAX - 7,

SMIN - 0,

SBLOCK DILUCLC.MPX

DILUCALC.OUT -> INI

SBLOCK DILUCLC

SI - "0-0-0-0",

CALCDELAY.RDY

DILUHOLD.OUT

LOLIM.HI

->

->

->

SI

IN2

S2

52 "0 0 0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

CHG "0-0-0-0",

MUNT""

,

OMAX 100,

OMIN - 0,

OUNT -

""

,

-> IN3NCHG "0-0-0-0"

,GAIN - -500

,

-> S3 INIV - 0,

TI - 100,

-> IN4 IN2V - 0 TD - 0,

-> S4 IN3V 0,

IN4V - 0,

PULS - 0,

TFIL 0,

DZ 0,

BIAS 0,

INT YES,SBLOCK MFLUXCLC.MPX SBLOCK MFLUXCLC

MFLUXCALC.OUT INI SI "0-0-0-0",

MODE 0,

CALCDELAY.RDY

MFLUXHOLD.OUT

LOLIM.HI

->

->

->

->

SI

IN2

S2

IN3

52 - "0-0-0-0",

53 "0-0-0-0",

54 "0-0-0-0",

CHG "0-0-0-0',

NCHG "0-0-0-0",

-> S3 INIV - 0,

-> IN4 IN2V - 0,

-> S4 IN3V - 0

IN4V - 0,

PULS - 0,

SGROUP RENEWBATCH

TS = 1 ; RUN = YES PR - 1

SBLOCK START.BHAN

-> S

SBLOCK START

S - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

QSET - NO,

PULS - 0,

SBLOCK TRTRA.AI SBLOCK TRTRA

io - "O-o-o-O",

OMAX - 20,

OMIN - -20,

UNIT - "GRAD C",

ZERO - NO,

INV - NO,SBLOCK STARTENAB.AND

START.Q

START.Q

CULTSTATE.Q

BATCHSTAT.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK STARTENAB

51 - "0-0-0-0",

52 - "0-0-0-0"

53 - "0-0-0-0",

54 - -0-0-0-0",

Q "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK PHTRA.AI SBLOCK PHTRA

IO - "0-0-0-0",

OMAX - 100,

OMIN - -100.

UNIT - "%",

ZERO - NO,

INV - NO,SBLOCK RESEMPT.AND

LOLIM.HI

LOLIM.HI

BATCH.Q

BATCH. Q

-> SI

-> S2

-> S3

-> S4

SBLOCK RESEMPT

51 "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK HOLDENAB.OR

BUFFALLOW.Q

EMPTYSTAT. Q

-> SI

-> S2

-> S3

-> S4

SBLOCK HOLDENAB

si - "O-o-o-O",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0" ;SBLOCK EXITOR.OR

RESEMPT.Q -> SI

-> S2

-> S3

-> S4

SBLOCK EXITOR

si = "O-o-o-O",

52 - "1-2-2-8",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK DELAYTR.TIME

BUFFALLOW.Q

HOLDTR.NQ

-> RUN

-> RES

SBLOCK DELAYTR

RUN - "0-0-0-0",

RES "0-0-0-0",

RDY = "0-0-0-0",

NRDY - "0-0-0-0",

RUNV - YES,

RESV - NO,

TIME - 600,

ZERO - 0,

ACCU - NO,

PULS - 0,

SBLOCK EMPTYSTAT.SR

STARTENAB.Q

EXITOR.Q

-> SET

-> RES

SBLOCK EMPTYSTAT

SET - "0-0-0-0",

RES - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK RESET.BSET

EMPTYSTAT.NQ

-> SI

-> S2

-> S3

-> SEND

SBLOCK RESET

SEND - "0-0-0-0",

BLK - "START",

PARI - "QSET" ,

SIV - NO,

PAR2 -""

,

S2V - NO,

PAR3 -" "

,

S3V NO,

SBLOCK DELAYPH.TIME

BUFFALLOW.Q

HOLDPH.NQ

-> RUN

-> RES

SBLOCK DELAYPH

RUN "0-0-0-0",

RES - "0-0-0-0",

RDY - "0-0-0-0",

NRDY "0-0-0-0",

RUNV - NO

RESV NO,

TIME - 180,

ZERO - 0,

ACCU - NO,

PULS 0,

114 Appendix

SBLOCK DELAYTROR.OR

HOLDENAB.NQ

DELAYTR.RDY

-> SI

-> S2

-> S3

-> S4

SBLOCK DELAYTROR

51 - "0-0-0-0",

52 - "0-0-0-0"

53 = "1-2-2-8",

54 = "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK MAXWEI.Al SBLOCK MAXWEI

IO - "0-0-0-0",

OMAX - 800

OMIN = 100

UNIT = "G"

ZERO - NO

INV - NO

SBLOCK DELAYPHOR.OR

HOLDENAB NQ

DELAYPH.RDY

-> SI

-> S2

-> S3

-> S4

SBLOCK DELAYPHOR

51 - "0-0-0-0"

52 - "0-0-0-0",

53 = "1-2-2-8",

54 = "0-0-0-0"

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK HARVCALC SUM

MINWEI.OUT

WEIGHTIN. OUT

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK HARVCALC

GAIL - -1,

GAI2 - 1000,

GAU - 1,

GAI4 = 1,

BIAS - 0,

SBLOCK HARVSTOP.REL

HARVCALC.OUT -> IN

SBLOCK HARVSTOP

INC = "0-0-0-0",

NINC = "0-0-0-0" ,

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ = 100

AMP-j - 50,

SBLOCK HARVSEL.MPX

EMPTYSTAT Q

WREG.OUT

EMPTYSTAT NQ

WREG.OUT

BATCHSTAT.NQ

WREG.OUT

BATCHSTAT. Q

-> INI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

SBLOCK HARVSEL

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

CHG - "0-0-0-0",

NCHG - "0-0-0-0",

INIV - 100,

IN2V - 0,

IN3V - 0,

IN4V - 0,

PULS - 0,

SBLOCK FASTHARV AND

EMPTYSTAT.Q

EMPTYSTAT.Q

HARVSTOP INC

HARVSTOP. INC

-> SI

-> S2

-> S3

-> S4

SBLOCK FASTHARV

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0" ,

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",SBLOCK HARVEST AO

HARVSEL OUT -> IN

SBLOCK HARVEST

IO = "1-1-7-2"

IMAX - 100,

IMIN - 0,

UNIT - %",

ZERO = YES,

INV = NO,

SBLOCK FASTPUMP OR

FASTHARV.Q

FASTHARV.Q

TOHEAVY. HI

TOHEAVY. HI

-> SI

-> S2

-> S3

-> S4

SBLOCK FASTPUMP

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",SBLOCK MINWEI.AI SBLOCK MINWEI

IO - "0-0-0-0",

OMAX - 800,

OMIN = 100,

UNIT - "G",

ZERO = NO,

INV - NO

SGROUP PRESSAIR

TS = 1 ; RUN = YES ; PR = 1

SBLOCK PRESSIN.AI SBLOCK PRESSIN SBLOCK PRESSPID.PID SBLOCK PRESSPID

IO - "1-1-3-1",

OMAX - 2,

OMIN - 0,

UNIT -

" BAR",

ZERO = NO,

PRESSCAL. OUT

PRESSSET. OUT

-> MV

-> SP

-> STRA

-> TRA

STRA - "0-0-0-0",

MAN - "0-0-0-0" ,

NMAN - "0-0-0-0",

SMAX - 2,

SMIN = 0,

INV = NO,

MUNT = "BAR",

OMAX = 100

OMIN = -100,

OUNT = "*"

SBLOCK PRESSCAL SFUN

PRESSIN.OUT -> IN

SBLOCK PRESSCAL

N = 2,

11 = 01125,

Ol = 0,

GAIN = 150

12 =2 00875,

TI - 8,

02 = 2,

TD = 0,

13 - 0,

TFI^ - 0,

03 - 0,

DZ - 0,

14 = 0,

BIAS - 0,

04 - 0,

INT - YES

15 - 0,

05 - 0,

MODE - 0,

SBLOCK PRESSREL.REL SBLOCK PRESSREL

16 = 0,

06 = 0,

PRESSPID.E -> IN INC - "0-0-0-0",

NINC - "0-0-0-0",

17 - 0,

DEC - "0-0-0-0",

07 - 0,

NDEC - "0-0-0-0",

18 - 0,

DZ - 1,

08 - 0,

19 - 0,

AMPL - 50,

SBLOCK PRESSOR.OR SBLOCK PRESSOR

09 - 0,

110 - 0,

010 - o,

BIAS - 0,

PRESSREL.INC

PRESSREL.INC

PRESSREL.DEC

PRESSREL.DEC

-> SI

-> S2

-> S3

-> S4

51 - "0-0-0-0",

52 = "0-0-0-0",

53 - "0-0-0-0" ,

s4 = "O-o-o-O",

Q - "0-0-0-0",SBLOCK PRESSSET.AI SBLOCK PRESSSET

IO - "0-0-0-0",

OMAX - 2,

NQ - "0-0-0-0",

SBLOCK PINTNO.BSET SBLOCK PINTNO

OMIN - 0,

UNIT - "BAR",

ZERO - YES,

INV - NO,

PRESSOR.Q -> SI

-> S2

-> S3

-> SEND

SEND - "0-0-0-0",

BLK - "PRESSPID",

PARI - "INT",

SIV - NO,

PAR2 -""

,

S2V = NO,

PAR3 =""

,

S3V - NO,

Appendix 115

SBLOCK PINTYES.BSET SBLOCK PINTYES SBLOCK AIRCALIB SFUN SBLOCK AIRCALIB

PRESSOR.NQ

-> SI

-> S2

-> S3

-> SEND

SEND - "0-0-0-0",

BLK - "PRESSPID",

PARI - "INT",

SIV - YES,

PAR2 -

""

,

S2V - NO,

PAR3 -

" "

,

S3V - NO,

BROOKSIN.OUT -> IN N - 6,

11 - 051,

01 0,

12 1,

02 1 009,

13 -25,

03 2 54,

14 5,

04 - 5 039,SBLOCK PRESFAK.SUM SBLOCK PRESFAK

PRESSSET.OUT -> INI

-> IN2

-> IN3

-> IN4

GAI1 -11,

GAI2 1,

GAI3 - 1,

GAI4 1,

BIAS - 0,

15 -75,

05 - 7 486,

16 10,

06 - 10 024,

17 - 0,

07 0,SBLOCK PRESMM.MIMA SBLOCK PRESMM

PRESSCAL.OUT

PRESFAK. OUT

-> INI

-> IN2

HI - "0-0-0-0",

NHI - "0-0-0-0",

INIV - 0,

IN2V - 0,

18 - 0,

08 - 0,

19 - 0,

09 - 0,

110 - 0,

SBLOCK BLOTAKT TIME SBLOCK BLOTAKT

BLOTAKT.NRDY -> RUN

-> RES

RUN "0-0-0-0",

RES "0 0-0-0",

RDY "0-0-0-0",

BIAS - 0,

SBLOCK AIRSET.AI SBLOCK AIRSET

NRDY "0-0-0-0",

IO - "0-0-0-0",

RUNV - NO,

RESV - NO,

OMAX - 10,

OMIN 0,

TIME - 15,

ZERO - 0,

UNIT "0",

ZERO NO,

ACCU - NO,

PULS - 4,

INV - NO,

SBLOCK AIRMPX.MPX

AIRSET.OUT -> INI

SBLOCK AIRMPX

SI - "0-0-0-0",SBLOCK PEMFAK.SUM SBLOCK PEMFAK

PRESSSET OUT -> INI

-> IN2

-> IN3

-> IN4

GAI1 - 1 35,

GAI2 - 1,

GAI3 - 1,

GAI4 - 1,

BIAS 0,

PEMMM.NHI

AIRSET.OUT

CULTSTATE.Q

AIRSET.OUT

PEMMM.NHI

PEMMM.HI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

52 _ "0-0-0-0",

53 "0-0-0-0",

54 _ "0-0-0-0",

CHG - "0-0-0-0",

NCHG - "0-0-0-0",

INIV - 0,

IN2V - 0,

IN3V - 0,

IN4V - 5,

PULS - 0,

SBLOCK PEMMM.MIMA

PRESSCAL.OUT

PEMFAK.OUT

-> INI

-> IN2

SBLOCK PEMMM

HI - "0-0-0-0",

INIV - 0,

IN2V - 0,

SBLOCK CUEM.OR

CULTSTATE.Q

CULTSTATE.Q

EMPTYSTAT.Q

EMPTYSTAT.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK CUEM

SI - "0-0-0-0",

53 - "0-0-0-0",

54 "0-0-0-0",

Q - "0-0-0-0",

NQ "0-0-0-0",

SBLOCK PRESSBIAS.SFUN

AIRMPX.OUT -> IN

SBLOCK PRESSBIAS

N 3,

11 = 2,

01 - 45,

12 4 5,

02 - 10,

13 6,

03 - 52 5,

14 - 0,

04 - 0,

15 - 0,

05 - 0,

16 - 0,

06 - 0,

17 - 0,

07 _ 0,

18 - 0,

08 - 0,

19 - 0,

09 - 0,

110 _ 0,

010 - o,

BIAS - 0,

SBLOCK BLOWENA OR

EMPTYSTAT.Q

EMPTYSTAT. Q

PRESMM.HI

PRESMM.HI

-> SI

-> S2

-> S3

-> S4

SBLOCK BLOWENA

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK PASTAKT.AND

PEMMM.NHI

BLOTAKT. RDY

CUEM.Q

BLOWENA.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK PASTAKT

51 "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 "0-0-0-0",

q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK PRESMPX.MPX

PASTAKT.Q

EXHAUSTST.OUT

CULTSTATE. Q

EXHAUSTST. OUT

EMPTYSTAT. NQ

EXHAUSTST. OUT

PASTAKT.NQ

-> INI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

SBLOCK PRESMPX

si - "O-o-o-O",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 "0-0-0-0",

CHG - "0-0-0-0",

NCHG - "0-0-0-0",

INIV = 100 ;

IN2V 0,

IN3V - 0,

IN4V - 0,

PULS - 0,

SBLOCK PRESSSUM.SUM

PRESSBIAS.OUT

PRESSPID.OUT

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK PRESSSUM

GAI1 1,

GAI2 - 1,

GAI3 - 3,

GAI4 _ 1,

BIAS - 0,

SBLOCK AIRPID.PID

AIRCALIB.OUT

AIRMPX.OUT

-> MV

-> SP

-> STRA

-> TRA

SBLOCK AIRPID

STRA - "0-0-0-0",

MAN - "0-0-0-0",

NMAN - "0-0-0-0",

SMAX - 10,

SMIN 0,

MUNT - "NL/MIN" ,

OMAX - 50,

OMIN - -50,

OUNT -

" %",

GAIN - 4,

TI - 50,

SBLOCK BROOKSIN.AI SBLOCK BROOKSIN

IO - "1-1-1-7",

OMAX 10 ;

OMIN - 0,

UNIT - "NL/MIN" ,

ZERO YES,

INV = NO,

|

TD - 0,

TFIL - 5,

DZ - 0,

BIAS - 0,

INT - YES

MODE - 0,

116 Appendix

SBLOCK AIRBIAS.SFUN

AIRMPX.OUT -> IN

SBLOCK AIRBIAS

N - 9,

11 = 0,

01 = 0,

12 - 0,

02 5,

13 - 35,

SBLOCK AIROR.OR

AIRREL.INC -> SI

AIRREL.INC -> S2

AIRREL.DEC -> S3

AIRREL.DEC -> S4

SBLOCK AIROR

51 - "0-0-0-0" ,

52 = "0-0-0-0" ,

53 = "0-0-0-0" ;

54 = "0-0-0-0",

Q = "0-0-0-0",

NQ - "0-0-0-0",

03 - 10

14 - 97,

04 - 15,

15 - 1 87,

05 = 20,

16 - 3 47,

06 = 25,

17 = 5 66,

07 - 30,

18 - 7 99,

08 - 35,

19 - 9 71,

09 = 40.

110 = 0,

010 = o,

BIAS - 0,

SBLOCK AIRINTNO. BSET

AIROR.Q -> SI

-> S2

-> S3

-> SEND

SBLOCK AIRINTNO

SEND - "0-0-0-0",

BLK = "AIRPID",

PAFl - "INT",

SIV - NO,

PAP2 -""

,

S2V - NO,

PAF3 =""

,

S3V = NO,

SBLOCK AIRINTYES.BSET

-> SI

-> S2

-> S3

AIROR.NQ -> SEND

SBLOCK AIBINTYES

SEND - "0-0-0-0",

BLK = "AIRPID",

PAFl = "INT",

SIV = YES,

PAR2 =""

,

S2V - NO,

PAP3 -""

,

S3V - NO,

SBLOCK AIRSUM.SUM

AIRPID OUT -> INI

AIRBIAS.OUT -> IN2

-> IN3

-> IN4

SBLOCK AIRSUM

GAI1 - 1,

GAI2 - 1,

GAI3 - 0

GAI4 - 0,

BIAS - 0,

SBLOCK AIRRV.AO

AIRSUM.OUT -> IN

SBLOCK AIRRV

IO - "1-1-5-2",

IMAX 100,

IMIN - 0,

UNIT - "%",

ZERO - YES,

INV - NO,

SBLOCK AIRREL.REL

AIRPID.E -> IN

SBLOCK AIRREL

INC = "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ = 1,

AMPL - 50,

SGROUP PH

TS = 1 ; RUN = YES ; PR = 1

SBLOCK PHMEASURE.AI SBLOCK PHMEASURE

IO = "1-1-2-1",

OMAX - 12,

OMIN = 2,

UNIT - "PH",

ZERO = YES,

INV - NO,

SBLOCK PHPID.PID

PHIN.OUT -> MV

PHSET.OUT -> SP

HOLDPH.Q -> STRA

PHTRA.OUT -> TRA

SBLOCK PHPID

STRA = "0-0-0-0" ,

MAN = "0-0-0-0",

NMAN - "0-0-0-0" ,

SMAX - 12,

SMIN = 2,

MUNT = "PH",

OMAX - 100,

OMIN - -100,

OUNT =" %" ,

GAIN - 200,

TI = 1000,

TD = 0,

TFIL = 0,

DZ - 0,

BIAS = 0,

IN" - NO,

MODE - 0,

SBLOCK PHSET.AI SBLOCK PHSET

IO = "0-0-0-0",

OMAX = 12,

OMIN - 2,

UNIT -

" PH",

ZERO - YES,

INV - NO,

SBLOCK PHIN SFUN

PHMEASURE. OUT -> IN

SBLOCK PHIN

N - 3,

11 = 3 34,

01 - 4 01,

12 - 5 97,

02 - 7,

13 - 7 85,

03 - 9 21,

14 - 0,

04 = 0,

15 = 0,

05 - 0,

16 - 0,

06 = 0,

17 = 0,

07 - 0,

18 - 0,

08 = 0,

19 0,

09 = 0,

110 - 0,

010 - o,

BIAS - 0,

SBLOCK PHBIAS.SFUN

MEDFLUX.OUT -> IN

SBLOCK PHBIAS

N - 2,

11 = 0,

01 - o,

12 = 1600,

02 = 14,

13 = 0,

03 - 0,

14 - 0,

04 - 0,

15 - 0,

05 = 0,

16 - 0,

06 - 0,

17 - 0,

07 - 0,

18 - 0,

08 - 0,

19 - 0,

09 = 0,

110 - 0,

010 = 0,

BIAS - 0,

Appendix 117

SBLOCK PHSUM.SUM

PHPID.OUT -> INI

PHBIAS.OUT -> IN2

-> IN3

-> IN4

SBLOCK PHSUM

GAIl = 1,

GAI2 1,

GAI3 - 0,

GAI4 - 0,

BIAS - 0,

SBLOCK PHMPX.MPX

PHSUM.OUT -> INI

BATCH.NQ -> SI

PHPID.OUT -> IN2

BATCH.Q -> S2

-> IN3

-> S3

-> IN4

-> S4

SBLOCK PHMPX

51 - "0-0-0-0",

52 - "0-0-0-0" ,

53 "0-0-0-0",

54 "0-0-0-0" ,

CHG = "0-0-0-0",

NCHG - "0-0-0-0" ,

INIV - 0,

IN2V - 0,

IN3V - 0,

IN4V 0,

PULS - 0.

SBLOCK PHREL.REL

PHPID E -> IN

SBLOCK PHREL

INC "0-0-0-0" ,

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ - 25,

AMPL - 50 , SBLOCK ALKALI.AO

PHMPX.OUT -> IN

SBLOCK ALKALI

IO - "1-1-8-1",

IMAX 100,

IMIN 0,

UNIT "%,

ZERO - YES,

INV - NO,

SBLOCK PHOR.OR

PHREL.INC -> SI

PHREL.INC -> S2

PHREL.DEC -> S3

PHREL.DEC -> S4

SBLOCK PHOR

51 "0-0-0-0" ,

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0" ,

Q - "0-0-0-0",

NQ - "0-0-0-0" , SBLOCK SAEURE.AO

PHMPX.OUT -> IN

SBLOCK SAEURE

IO "1-1-8-2",

IMAX 1,

IMIN -100,

UNIT - "%",

ZERO - YES,

INV YES.

SBLOCK PHINTNO.BSET

-> SI

-> S2

-> S3

PHOR.Q -> SEND

SBLOCK PHINTNO

SEND - "0-0-0-0" ,

BLK - "PHPID",

PARI - "INT",

SIV - NO,

PAR2 -

""

,

S2V - NO.

PAR3 -

"",

S3V - NO,

SBLOCK PHINTYES.BSET

-> SI

-> S2

-> S3

PHOR.NQ -> SEND

SBLOCK PHINTYES

SEND = "0-0-0-0" ,

BLK "PHPID",

PARI = "INT",

SIV YES

PAR2 " "

,

S2V - NO,

PAR3 -

""

,

S3V = NO,

SGROUP GAS

TS = 1 ; RUN = YES ; PR = 1

SBLOCK 02IN.AI SBLOCK 02 IN

IO - "1-1-2-6",

OMAX - 21,

OMIN - 11,

UNIT - "%"

ZERO - YES,

INV - NO

SBLOCK C02CALIB.SFUN

C02IN.OUT -> IN

SBLOCK C02CALIB

N - 2,

11 = 031746 ,

01 - 033,

12 - 7 926 ;

02 - 8 06,

13 - 0,

03 - 0,

14 - 0,

04 - 0,

15 - 0,

05 - 0,

16 = 0,

06 = 0,

17 - 0,

07 - 0,

18 - 0,

08 - 0 ;

19 - 0,

09 - 0,

110 - 0,

010 _ o,

BIAS - 0,

SBLOCK 02CALIB.SFUN

02IN.OUT -> IN

SBLOCK 02CALIB

N - 2,

11 - 13 42 ,

01 - 13 1,

12 = 20 4359,

02 - 20 95 ,

13 - 0,

03 - 0,

14 - 0,

04 - 0,

15 - 0,

05 - 0,

16 - 0,

06 - 0,

17 _ 0,

07 - 0,

18 = 0,

08 - 0,

19 0,

09 - 0,

110 - 0,

010 - 0 ;

BIAS - 0,

SBLOCK GACHAl.OR

-> SI

-> S2

-> S3

-> S4

SBLOCK GACHAl

51 - "1-2-2-3",

52 - "0-0-0-0",

53 = "0-0-0-0",

54 _ "0-0-0-0",

Q = "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK C02IN.AI SBLOCK C02IN

10 - "1-1-2-7",

OMAX - 10,

OMIN = 0,

UNIT - "%",

ZERO - YES,

INV - NO,

SBLOCK GACHA2.0R

-> SI

-> S2

-> S3

-> S4

SBLOCK GACHA2

51 = "1-2-2-4",

52 = "0-0-0-0" ,

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - -0-0-0-0",

NQ - "0-0-0-0" ,

118 Appendix

SBLOCK C020UT.MPX

C02CALIB.OUT

MEASTIMER.RDY

CO20FF.0UT

CHGCHA.Q AAAAAAAAINI

SI

IN2

S2

IN3

S3

IN4

S4

SBLOCK C020UT

51 = "0-0-0-0",

52 = "0-0-0-0",

53 = "0-0-0-0" ;

54 - "0-0-0-0",

CHG = "0-0-0-0" ;

NCHG = "0-0-0-0",

INIV = 0,

IN2V - 0 ;

IN3V - 0 ;

IN4V = 0,

PULS = 0 ;

SBLOCK OUR.MULT

02CALIB.OUT

INERTGAS2.0UT

->

->

INI

IN2

SBLOCK OUR

GAIN =

BIAS -

-1 ;

20.95,

SBLOCK CPR.MULT

C02CALIB.OUT

INERTGAS2.0UT

->

->

INI

IN2

SBLOCK CPR

GAIN =

BIAS -

1 ;

-.033 ,

SBLOCK CPRSUM.SUM

C02CALIB.OUT

02CALIB.OUT

CREATE1. OUT

->

->

->

->

INI

IN2

IN3

IN4

SBLOCK CPRSUM

GAIl =

GAI2 -

GAI3 =

GAI4 =

BIAS

.79054,

.00033 ;

-.033,

1 ;

0,

SBLOCK 02OUT.MPX

02CALIB.0UT

MEASTIMER.RDY

020FF.0UT

CHGCHA.Q

->

->

->

->

->

->

->

->

INI

SI

IN2

S2

IN3

S3

IN4

S4

SBLOCK 02OUT

51 - "0-0-0-0" ;

52 - "0-0-0-0",

53 = -0-0-0-0",

54 = "0-0-0-0",

CHG - "0-0-0-0" ;

NCHG - "0-0-0-0",

INIV - 0 ;

IN2V - 0,

IN3V - 0 ;

IN4V - 0 ;

PULS = 0,

SBLOCK OURSUM.SUM

C02CALIB.OUT

02CALIB.OUT

CREATE1 . OUT

->

->

->

->

INI

IN2

IN3

IN4

SBLOCK OURSUM

GAIl =

GAI2 -

GAI3 -

GAI4 =

BIAS =

-.20946 ;

- 99967 ;

20 946,

1,

0,

SBLOCK RQ.DIV

CPRSUM.OUT

OURSUM.OUT

->

->

NUM

DEN

SBLOCK RQ

GAIN -

BIAS =

1,

0 ;

SBLOCK INERTGASl.SUM

02CALIB.0UT

C02CALIB.0UT

->

->

->

->

INI

IN2

IN3

IN4

SBLOCK INERTGASl

GAIl = -1,

GAI2 = -1 ;

GAI3 = 1 ;

GAI4 = 1 ;

BIAS = 1,

SBLOCK INERTGAS2.DIV

CREATE1. OUT

INERTGASl. OUT

->

->

NUM

DEN

SBLOCK INERTGAS2

GAIN = -19.983 ;

BIAS - 0 ;

SGROUP GASAUTOCAL

TS = 1 ; RUN = YES ; PR = 1

SBLOCK GACALSTOP.BHAN

-> S

SBLOCK GACALSTOP

S - "0-0-0-0",

Q - "0-0-0-0" ,

NQ - "0-0-0-0",

QSET = NO ;

PULS = 0 ;

SBLOCK C02CORR.SETP

C02IN.OUT

CALTIMER. RDY

-> INI

-> IN2

-> IN3

-> SEND

SBLOCK C02CORR

SEND - "0-0-0-0" ;

BLK = "C02CALIB" ;

PARI - "11" ;

INIV - 0 ;

PAR2 -"

,

IN2V = 0 ;

PAR3 =""

;

IN3V = 0,

SBLOCK GACALENA.AND

GACALSTOP.NQ

GACHA2.NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK GACALENA

51 = "0-0-0-0",

52 = "0-0-0-0",

53 = "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ = "0-0-0-0",

SBLOCK CREATE1.AI SBLOCK CREATE1

IO - "0-0-0-0" ;

OMAX = 1 ;

OMIN = 0 ;

UNIT =""

;

ZERO = NO ;

INV = NO ;

SBLOCK CHGCHA.SR

GACALENA.Q

GACALENA.NQ

-> SET

-> RES

SBLOCK CHGCHA

SET - "0-0-0-0",

RES = "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0" ,

SBLOCK C02OFF.MULT

C02CALIB.OUT

CREATE1.OUT

-> INI

-> IN2

SBLOCK C020FF

GAIN = -1 ;

BIAS - 0 ;SBLOCK CALTIMER.TIME

CHGCHA.Q

CHGCHA. NQ

-> RUN

-> RES

SBLOCK CALTIMER

RUN - "0-0-0-0",

RES = "0-0-0-0",

RDY = "0-0-0-0",

NRDY = "0-0-0-0",

RUNV = NO ;

RESV - NO ;

TIME - 300,

ZERO = 0 ;

ACCU - YES ;

PULS = 0 ;

SBLOCK 020FF.MULT

02CALIB.OUT

CREATE1. OUT

-> INI

-> IN2

SBLOCK 02CFF

GAIN - -1,

BIAS = 0 ;

SBLOCK MEASTIMER.TIME

CHGCHA.NQ

CHGCHA.Q

-> RUN

-> RES

SBLOCK MEASTIMER

RUN - "0-0-0-0"

RES - "O-0-0-0"

RD-, - "0-0-0-0"

NRDY - "0-0-0-0"

RUNV - NO ;

SBLOCK 02CORR.SETP

02IN.OUT

CALTIMER. RDY

-> INI

-> IN2

-> IN3

-> SEND

SBLOCK 02CORR

SEND = "0-0-0-0",

BLK = "02CALIB" ;

PARI = "12" ;

INIV = 0 ;

PAR2 -""

;

IN2V = 0 ;

PAR3 -""

;

IN3V - 0,

RESV = NO ;

TIME = 120,

ZERO - 0 ;

ACCU - NO,

PULS = 0 ;

SGROUP SENSORS

TS = .5 ; RUN = YES ,- PR == 1

SBLOCK FLUOROIN.AI SBLOCK FLUOROIN 1 SBLOCK FILTER.FIL SBLOCK FILTER 1

IO = "0-0-0-0" ;

OMAX - 5,

| FLUOROIN. OUT -> IN TIME = 5,

1

OMIN - 0 ;

UNIT - "V",

ZERO - YES ;

INV = NO

Appendix 119

SBLOCK AQUASANT.AI SBLOCK AQUASANT SBLOCK REDOXCAL. SFUN SBLOCK REDOXCAL

IO - "1-1-2-5", REDOXIN.OUT -> IN N 2

,

OMAX 100 11 - 0,

OMIN 0,

Ol - 0,

UNIT -

"-",

12 - 232 6,

ZERO - NO,

02 220 ;

INV NO,

13 - 0,

03 - 0,

SBLOCK REDOXIN.AI SBLOCK REDOXIN

IO "1-1-2-4",

14 - 0,

OMAX - 500,

04 0,

OMIN - -500,

15 - 0,

UNIT "MV",

05 - 0,

ZERO - YES,

16 0,

INV NO,

06 - 0 ;

17 - 0,

07 0,

18 0,

08 - 0,

19 - 0,

09 0,

110 - 0,

OlO - 0,

BIAS - 0,

SGROUP PC02P02

TS = 10 ; RUN = YES ; PR = 1

SBLOCK PC02IN.AI SBLOCK PC02IN SBLOCK PMIMA1.MIMA SBLOCK PMIMA1

IO "1-1-2-3", P02 IN. OUT -> INI HI "0-0-0-0"

,

OMAX 2, -> IN2

NHI = "1-2-6-4",

OMIN - 0,

INIV - 0,

UNIT - "<MV>",

ZERO YES ,

IN2V = 24,

SBLOCK PSUMl.SUM SBLOCK PSUM1

INV - NO P02IN.OUT -> INI GAIl - 1,

SBLOCK PC02CALIB.SFUN SBLOCK PC02CALIB-> IN2

GAI2 - 1,

PC02IN.0UT -> IN N - 2,

-> IN3GAI3 - 1

,

11 84,

-> IN4GAI4 - 1

,

01 - 57978,

12 - 1 88,

BIAS - -30,

SBLOCK PREL1.REL SBLOCK PREL1

02 =1 8195, PSUM1. OUT -> IN INC - "0-0-0-0"

,

13 0,

NINC - "0-0-0-0",

03 - 0,

DEC - "0-0-0-0",

14 - 0,

NDEC - "0-0-0-0",

04 - 0,

DZ 18 5,

15 - 0,

05 0,

AMPL - 0,

SBLOCK PANDl.AND SBLOCK PAND1

16 - 0, PRELl.NINC -> SI SI "0-0-0-0"

,

06 - 0,

PRELl.NDEC -> S2S2 "0-0-0-0"

,

17 = 0,

PMIMAl.NHI -> S3S3 0-0-0-0"

,

07 - 0, S4 "0-0-0-0" ;

18 = 0,

PMIMAl.NHI -> S4Q "0-0-0-0"

,

08 - 0,

19 - 0,

NQ "0-0-0-0",

SBLOCK PSETP3.SETP SBLOCK PSETP3

09 - 0,

-> INI SEND - "0-0-0-0",

110 - 0,

-> IN2BLK - "P02IN"

,

010 - o. PARI - "OMAX"

,

BIAS - 0,

PAND1.Q

-> IN3

-> SENDINIV -

PAR2 -

100,

SBLOCK P02IN.AI SBLOCK P02IN

IO - "1-1-2-2", IN2V - 0

,

OMAX - 100, PAR3 -

..

_

OMIN - 0,

UNIT - "% SAT",

IN3V - 0,

SBLOCK PAND2.AND SBLOCK PAND2

ZERO - YES,

PRELl.NINC -> SI SI "0-0-0-0",

INV - NO ;

PRELl.NDEC -> S2S2 0-0-0-0"

,

SBLOCK P02CAL.SFUN SBLOCK P02CALPMIMAl.HI -> S3

S3 0-0-0-0",

P02IN.OUT -> IN N - 2, S4 "0-0-0-0"

,

11 - 0,

PMIMAl.HI -> S4Q "0-0-0-0"

,

01 = 0,

12 - 100,

NQ "0-0-0-0",

SBLOCK PSETP4.SETP SBLOCK PSETP4

02 - 100,

-> INI SEND - "0-0-0-0",

13 = 10,

-> IN2BLK - "P02IN"

,

03 = 50, PARI - "OMAX"

,

14 - 15,

-> IN3INIV - 100

,

04 - 75,

PAND2.Q -> SENDPAR2 -

15 - 21, IN2V - 0

,

05 = 100, PAR3 -

""

16 - 0,

06 - 0,

IN3V - 0,

SBLOCK PORI.OR SBLOCK POR1

17 - 0,

07 - 0,

18 - 0,

08 - 0,

PRELl.DEC

PRELl.DEC

-> SI

-> S2

r-t

CN

Ui

w

0-0-0-0",

"0-0-0-0",

PAND2.Q -> S3S3 0-0-0-0"

,

S4 "0-0-0-0",

19 - 0,

PAND2.Q -> S4Q "1-2-6-3"

,

09 - 0,

110 0,

NQ "0-0-0-0",

OlO - 0,

BIAS 0,

120 Appendix

SBLOCK PSETP2.SETP SBLOCK PSETP2 SBLOCK PSETPl.SETP SBLOCK PSETP1

-> INI SEND = "0-0-0-0", -> INI SEND = "0-0-0-0" ;

-> IN2BLK = "P02IN"

, -> IN2BLK - "P02IN" ;

PREL1.DEC

-> IN3

-> SEND

PARI =

INIV -

PAR2 =

"OMAX" ;

100 ;

PREL1.INC

-> IN3

-> SEND

PARI =

INI J -

PAR2 =

"OMAX" ;

100,

IN2V = 0 ; IN2/ = 0 ;

PAR3 =""

,PAR3 =

""

,

IN3V - 0 ; IN3V - 0 ;

SGROUP FILTERSLOW

TS = 2 ; RUN = YES ; PR = 1

SBLOCK STARTFILT.BHAN

-> S

SBLOCK STARTFILT

S - "0-0-0-0",

Q - "0-0-0-0" ;

NQ = "0-0-0-0",

QSET - YES ;

PULS - 10,

SBLOCK PERMFPID.PID

PERMIN.F

PERMFLUX. OUT

-> MV

-> SP

-> STRA

-> TRA

SBLOCK PERMFPID

STRA - "0-0-0-0",

MAN = "0-0-0-0",

NMAN - "0-0-0-0" ,

SMAX = 10000,

SMIN - 0,

MUNT - "ML/H" ;

OMAX = 120 ;

OMIN = 0,

OUNT - "MAIN" ;

GAIN = 006 ;

TI = 300 •

TD - 20 ;

TFIL = 0,

DZ - 0 ;

BIAS - 49.75 ;

INT - NO ;

MODE - 0 ;

SBLOCK STARTRUN. BHAN

-> s

SBLOCK STARTRUN

S = "0-0-0-0" ;

Q = "0-0-0-0" ;

NQ = "0-0-0-0" ;

QSET = YES ;

PULS = 15 ;

SBLOCK RUNFILT.SR

STARTRUN.Q

RUNOR.Q

-> SET

-> RES

SBLOCK RUNFILT

SET - "0-0-0-0",

RES - "0-0-0-0" ;

Q - "0-0-0-0" ;

NQ = "0-0-0-0" ;

SBLOCK STOPFILT.BHAN

-> S

SBLOCK STOPFILT

S - "0-0-0-0",

Q = "0-0-0-0",

NQ = "0-0-0-0" ;

QSET = YES,

PULS = 15,

SBLOCK PERMPUMP.AO

PPUMPMPX. OUT -> IN

SBLOCK PERMPUMP

IO = "0-0-0-0" ;

IMAX - 100,

IMIN - 0 ;

UNIT - "%" ;

ZERO - YES ;

INV = NO ;SBLOCK STARTUP.SR

STARTFILT.Q

STARTUPOR.Q

-> SET

-> RES

SBLOCK STARTUP

SET = "0-0-0-0" ;

RES = "0-0-0-0" ;

Q = "0-0-0-0" ;

NQ - "0-0-0-0",

SBLOCK PFLUXCONT.REL

PERMFPID.E -> IN

SBLOCK PFLJXCONT

INC = "0-0-0-0" ;

NINC - "0-0-0-0" ,

DEC - "0-0-0-0" ;

NDEC = "0-0-0-0" ,

DZ - 1000 ;

AMPL - 0 ;

SBLOCK PERMFLUX.Al SBLOCK PERMFLUX

IO - "0-0-0-0" ;

OMAX = 10000 ;

OMIN = 0 ;

UNIT - "ML/H" ;

ZERO - YES ;

INV - NO,

SBLOCK PFLUXNORM.OR

PFLUXCONT. INC

PFLUXCONT. DEC

-> SI

-> S2

-> S3

-> S4

SBLOCK PFLUXNORM

51 = "0-0-0-0" ;

52 = "0-0-0-0" ;

53 - "0-0-0-0" ;

54 - "0-0-0-0".

Q = "0-0-0-0" ;

NQ = "0-0-0-0" ;

SBLOCK METPERMIN.AI SBLOCK METPERMIN

IO = "1-1-3-6" ;

OMAX = 1000 ;

OMIN = 0 ;

UNIT = "G" ;

ZERO - NO ;

INV - NO ;

SBLOCK RFLUXSET.AI SBLOCK RFLUXSET

IO - "0-0-0-0" ;

OMAX = 5 ;

OMIN - 0 ;

UNIT = "M"3/H" ;

ZERO = NO,

INV - NO ;

SBLOCK PERMIN. FINR

METPERMIN.OUT

WEIGHTIN. OUT

CULTSTATE. Q

-> W

-> WR

-> CC

SBLOCK PERMIN

CC - "0-0-0-0" ;

STEP = "STOPPED" ,-

FILL - "1-2-6-6" ;

WORK = "0-0-0-0" ;

TDIF - 12 ;

MINW = 100 ;

MAXW - 900 ;

UHRT = 180000 ;

UHRA = NO ;

UHRG = YES ;

UHRD = NO,

UHRR = 177052 ;

DIFT = 12 ;

DIFA = NO ;

DIFG = YES,

DIFD = NO ;

DIFR = 4 ;

SBLOCK PFLUXOR.OR

PFLUXCONT.DEC

PPROBLEM.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK PFLUXOR

51 = "0-0-0-0" ;

52 - "0-0-0-0",

53 = "0-0-0-0" ;

54 = "0-0-0-0" ;

Q = "0-0-0-0" ;

NQ = "0-0-0-0" ;

SBLOCK PTMFIX.AI SBLOCK PTMFIX

IO = "0-0-0-0",

OMAX = 2 ;

OMIN = 0 ;

UNIT =" BAR" ;

ZERO = NO ;

INV - NO ;

SBLOCK PTMSET.AI SBLOCK PTMSET

IO = "0-0-0-0" ;

OMAX = 3 ;

OMIN = 0 ,-

UNIT = "BAR" ;

ZERO - NO ;

INV = NO ;

SBLOCK PFLUXAND.AND

PFLUXCONT. INC

PPROBLEM.NQ

PFLUXCONT.INC

PPROBLEM.NQ

-> SI

-> S2

-> S3

-> S4

SBLOCK PFLUXAND

51 - "0-0-0-0" ;

52 - "0-0-0-0" ;

53 = "0-0-0-0",

54 = "0-0-0-0" ;

Q = -0-0-0-0" ;

NQ = "0-0-0-0" ;

Appendix 121

SBLOCK FLUXFIX.AI SBLOCK FLUXFIX SBLOCK PTMPID.PID SBLOCK PTMPID

10 - "0-0-0-0", PTM.OUT -> MV STRA - "0-0-0-0"

,

OMAX 5, PTMMPX.OUT -> SP

MAN "0-0-0-0",

OMIN - 0,

-> STRANMAN - "0-0-0-0"

,

UNIT - "M~3",

-> TRASMAX - 3

,

ZERO - NO,

SMIN 0,

INV NO,

MUNT - "BAR",

OMAX - 7 0,SBLOCK PTMMINUS.INT SBLOCK PTMMINUS

PTMSET.OUT -> IN RUN - "0-0-0-0" ,OMIN -200

,

PFLUXOR.Q -> RUNRES - "0-0-0-0"

,

RUNV - NO,

OUNT - •%,

GAIN - 1,

ANDFLUX.Q -> RESRESV - NO

,

GAIN - 01,

BIAS - 0,

TI 10,

TD 0,

TFIL 0,

DZ 0,SBLOCK MIMAPTM.MIMA SBLOCK MIMAPTM

PTMFIX.OUT -> INI HI - "0-0-0-0".

BIAS - 0,

PTMMINUS. OUT -> IN2NHI "0-0-0-0"

,

INIV 0,

IN2V 0,

INT - NO,

MODE - 0,

SBLOCK RFLUX.AI SBLOCK RFLUX

IO - "1-1-3-5",SBLOCK FLUXMINUS. INT SBLOCK FLUXMINUS

RFLUXSET.OUT -> IN RUN - "0-0-0-0",

OMAX 5,

ANDPTM.Q -> RUNRES - "0-0-0-0" ,

RUNV - NO

OMIN - 0,

UNIT - "M~3/H" ,

PFLUXAND.Q -> RESRESV NO

,

GAIN - - 01,

BIAS - 0,

ZERO - NO,

INV - NO,

SBLOCK FLUXCALIB.SFUN

RFLUX.OUT -> IN

SBLOCK FLUXCALIB

N - 7,SBLOCK FLUXPLUS INT SBLOCK FLUXPLUS

RFLUXSET.OUT -> IN RUN - "0-0-0-0",

11 226,

PFLUXAND. Q -> RUNRES - "0-0-0-0"

,

RUNV - NO,

Ol - 438,

12 - 941,

ANDPTM.Q -> RESRESV - NO

,

GAIN - 01,

BIAS - 0,

02 - 1 873,

13 - 1 052,

03 =2 135,

14 - 1 233,

SBLOCK MIMAFLUX.MIMA SBLOCK MIMAFLUX

FLUXFIX.OUT -> INI HI "0-0-0-0",

04 - 2 456,

FLUXPLUS.OUT -> IN2

NHI - "0-0-0-0",

INIV 0,

IN2V - 0,

15 - 1 397,

05 - 2 742,

16 - 1 702,

06 - 3.436,

SBLOCK FLUXTRIG.OR SBLOCK FLUXTRIG

PFLUXNORM.NQ

PFLUXAND.Q

-> SI

-> S2

51 - "0-0-0-0",

52 "0-0-0-0",

17 - 1 846,

07 - 3 631,

S3 - "0-0-0-0" ,

-> S3S4 - "0-0-0-0"

,

08 - 0

-> S4Q - "0-0-0-0"

,

NQ - "0-0-0-0",

19 - 0

09 - 0

110 0

010 = 0SBLOCK RFLUXMPX.MPX SBLOCK RFLUXMPX

FLUXMINUS. OUT -> INI 51 - "0-0-0-0",

52 "0-0-0-0",

53 - "0-0-0-0",

BIAS - 0

ANDPTM.Q -> SISBLOCK RFLUXPID.PID SBLOCK RFLUXPID

RFLUXSET.OUT -> IN2S4 = "0-0-0-0"

,

FLUXCALIB. OUT -> MV STRA "0-0-0-0",

PFLUXNORM NQ -> S2CHG - "0-0-0-0"

,RFLUXMPX. OUT -> SP

MAN - "0-0-0-0",

FLUXPLUS. OUT -> IN3NCHG - "0-0-0-0"

, -> STRANMAN - "0-0-0-0" ;

PFLUXAND.Q -> S3 INIV - 0, -> TRA

SMAX -38,

FLUXMINUS. OUT -> IN4 IN2V 0,

SMIN - 0,

FLUXTRIG.NQ -> S4 IN3V - 0,

IN4V - 0,

PULS - 0,

MUNT - "M^/H" ,

OMAX - 7 0,

OMIN - 55,

OUNT - "ft",

SBLOCK PTMPLUS.INT SBLOCK PTMPLUSGAIN - 1

,

PTMSET.OUT -> IN RUN - "0-0-0-0",

TI - 20 ;

ANDFLUX. Q -> RUNRES - "0-0-0-0"

,

TD - 0,

PFLUXOR.Q -> RESRUNV - NO

,

RESV - NO,

GAIN 01 ;

BIAS - 0,

TFIL - 0,

DZ ~ 0,

BIAS - 55,

INT - YES,

SBLOCK PTMTRIG.OR

PFLUXOR.Q -> SI

SBLOCK PTMTRIG

51 "0-0-0-0",

52 "0-0-0-0",

MODE - 0,

SBLOCK RPSETSTER.AI SBLOCK RPSETSTER

ANDFLUX.Q -> S2 IO - "0-0-0-0" ;S3 - "0-0-0-0"

,

-> S3S4 - "0-0-0-0"

,

OMAX - 100,

-> S4Q - "0-0-0-0"

,

NQ "0-0-0-0",

OMIN - 0,

UNIT - »%",

ZERO - NO,

SBLOCK PTMMPX.MPX

PTMSET.OUT -> INI

SBLOCK PTMMPX

51 - "0-0-0-0",

52 - "0-0-0-0",

INV - NO,

SBLOCK RPSTANDBY.AI SBLOCK RPSTANDBY

PFLUXNORM.NQ -> SI IO - "0-0-0-0",

S3 - "0-0-0-0",

PTMMINUS. OUT -> IN2S4 - "0-0-0-0"

,

OMAX - 100,

PFLUXOR.Q -> S2CHG - "0-0-0-0"

,

OMIN - 0,

PTMPLUS. OUT -> IN3NCHG - "0-0-0-0"

,

UNIT - "%",

ANDFLUX.Q -> S3 INIV - 0,

ZERO - NO,

PTMSET. OUT -> IN4 IN2V - 0,

IN3V - 0,

INV - NO,

PTMTRIG.NQ -> S4 SBLOCK STANDBY.SR SBLOCK STANDBY

IN4V - 0,

STOPFILT.Q -> SET SET - "0-0-0-0",

PULS 0, STANDBYOR.Q -> RES

RES - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

122 Appendix

SBLOCK PSTBYOR.OR

PSECURITY Q -> SI

STANDBY.Q -> S2

-> S3

-> S4

SBLOCK PSTBYOR

51 - "0-0-0-0"

52 = "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0"

SBLOCK RVSTAT.MPX

RVSETSTER.OUT -> INI

CULTSTATE.NQ -> SI

RVSTANDBY.OUT -> IN2

PSTBYOR.Q -> S2

PTMSUM OUT -> IN3

STARTUP.Q -> S3

PTMPID.OUT -> IN4

RUNFILT.Q -> S4

SBLOCK RVSTAT

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0-,

54 = "0-0-0-0"

CHG - "0-0-0-0",

NCHG = "0-0-0-0",

INI/ - 0,

IN2/ - 0,

IN3/ - 0,

IN4/ - 0,

PULS - 0,

SBLOCK FLUXSTAT.MPX

RPSETSTER.OUT -> INI

CULTSTATE.NQ -> SI

RPSTANDBY.OUT -> IN2

PSTBYOR.Q -> S2

FLUXSUM.OUT -> IN3

STARTUP.Q -> S3

RFLUXPID.OUT -> IN4

RUNFILT Q -> S4

SBLOCK FLUXSTAT

51 - "0-0-0-0",

52 - -0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0"

CHG - "0-0-0-0",

NCHG - "0-0-0-0",

INIV = 0,

IN2V - 0,

IN3V - 0,

IN4V - 0,

PULS - 0,

SBLOCK RVLOOP.AO

RVSTAT.OUT -> IN

SBLOCK RVLOOP

IO - "1-2-8-1",

IMAX = 100

IMIN - 0,

UNIT _ "%"

ZERO - YES

INV - NO,

SBLOCK RPMDLIM.DLIM

FLUXSTAT.OUT -> IN

SBLOCK RPMDLIM

DMAX 3,

DMIN = -5,

SBLOCK VSTER.OR

STARTUP.Q -> SI

RUNFILT.Q -> S2

STANDBY.Q -> S3

-> S4

SBLOCK VSTZR

51 - "0-0-0-0",

52 - "0-0-0-0-,

53 "0-0-0-0",

54 = "0-0-0-0",

Q - -0-0-0-0-,

NQ - -0-0-0-0-,

SBLOCK RPUMP.AO

RPMDLIM.OUT -> IN

SBLOCK RPUMP

IO - "1-2-8-2",

IMAX - 100,

IMIN = 0,

UNIT - "%" ,

ZERO = YES,

INV - NO,

SBLOCK VIN.SR

CULTSTATE.NQ -> SET

VSTER.Q -> RES

SBLOCK VIN

SET = -0-0-0-0-,

RES - "0-0-0-0",

Q - "1-2-7-1",

NQ - "0-0-0-0",

SBLOCK RVSETSTER.Al SBLOCK RVSETSTER

IO = "0-0-0-0",

OMAX = 100,

OMIN - 0,

UNIT - "%",

ZERO - NO,

INV - NO,

SBLOCK VOUT.SR

CULTSTATE.NQ -> SET

VSTER.Q -> RES

SBLOCK VOUT

SET - "0-0-0-0",

RES - "0-0-0-0",

Q = "1-2-7-2",

NQ = "0-0-0-0",

SBLOCK RVSTANDBY.AI SBLOCK RVSTANDBY

IO = "0-0-0-0",

OMAX - 100,

OMIN - 0,

UNIT - "%",

ZERO - NO,

INV - NO,

SGROUP FILTERFAST

TS = 1,RUN = YES ; PR = 1

SBLOCK PIN.AI SBLOCK PIN SBLOCK PPCALIB.SFUN SBLOCK PPCALIB

IO - "1-1-3-4", PP.OUT -> IN N - 2

,

OMAX - 10,

11 - 0,

OMIN = 0,

oi = o,

UNIT - "BAR",

12 - 5 021,

ZERO = NO,

02 * 5.

INV - NO,

13 - 0

03 = 0,

SBLOCK PINCALIB.SFUN SBLOCK PINCALIB

PIN.OUT -> IN N = 2,

14 = 0,

11 = 0,

04 = 0,

Ol - 0,

15 = 0,

12 - 10 021,

05 - 0,

02 = 10,

16 - 0,

13 = 0,

06 = 0,

03 - 0,

17 = 0,

14 - 0,

07 = 0,

04 - 0,

18 - 0,

15 = 0,

08 - 0,

05 - 0,

19 - 0,

16 = 0,

09 - 0,

06 - 0,

110 - 0,

17 = 0,

010 - o,

07 = 0,

18 = 0,

BIAS = 0,

SBLOCK POUT.AI SBLOCK POUT

08 - 0,

IO - "1-1-3-3",

19 - 0,

OMAX - 5,

09 - 0,

OMIN - 0,

110 = 0,

UNIT - "BAR",

OlO - 0,

ZERO - NO,

BIAS - 04,

INV - NO,

SBLOCK PP.AI SBLOCK PP

IO - "1-1-3-2"

OMAX - 5,

OMIN - 0,

UNIT - "BAR" ,

ZERO = NO,

INV - NO ,

Appendix 123

SBLOCK POUTCALIB.SFUN

POUT.OUT -> IN

SBLOCK POUTCALIB

N = 2,

11 = 0,

01 - 0,

12 - 5 021,

02 - 5,

13 - 0,

03 - 0,

14 - 0,

04 - 0,

15 - 0,

05 - 0,

16 - 0,

06 - 0,

17 - 0,

07 - 0,

18 - 0,

08 - 0,

19 - 0,

09 - 0,

110 - 0,

010 - 0,

BIAS - 0,

SBLOCK PPREL.REL

PPCALIB.OUT -> IN

SBLOCK PPREL

INC - "0-0-0-0" ,

NINC "0-0-0-0",

DEC = "0-0-0-0",

NDEC - "0-0-0-0" ,

DZ -22,

AMPL - 1,

SBLOCK PSECURITY.OR

PINREL.INC

PPREL.INC

STOPFILT.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK PSECURITY

51 - "0-0-0-0" ,

52 - "0-0-0-0",

53 "0-0-0-0" ,

54 - "0-0-0-0",

Q "0-0-0-0",

NQ - "0-0-0-0" ,

SBLOCK RPMINRPM.REL

FLUXSTAT.OUT -> IN

SBLOCK RPMINRPM

INC "0-0-0-0- .

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0" ,

DZ 5,

AMPL - 1,

SBLOCK FILTSTAT.OR

CULTSTATE.NQ

STARTUP.Q

RUNFILT.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK FILTSTAT

51 - "0-0-0-0- ,

52 - -0-0-0-0",

53 - "0-0-0-0",

54 -0-0-0-0",

Q - -0-0-0-0" ,

NQ = "0-0-0-0",

SBLOCK ADDP.SUM

PINCALIB.OUT

POUTCALIB.OUT

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK ADDP

GAIl - 5,

GAI2 - 5,

GAI3 1,

GAI4 - 1,

BIAS - 0, SBLOCK RPAND.AND

RPMINRPM.INC

FILTSTAT.Q

RPMINRPM.INC

FILTSTAT.Q

-> SI

-> S2

-> S3

-> S4

SBLOCK RPAND

51 "0-0-0-0",

52 "0-0-0-0-,

53 = "0-0-0-0-,

54 = "0-0-0-0",

Q - "0-0-0-0" ,

NQ - "0-0-0-0",

SBLOCK PTM.SUM

ADDP.OUT

PPCALIB. OUT

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK PTM

GAIl - 1,

GAI2 - -1,

GAI3 - 1,

GAI4 - 1,

BIAS - 0,

SBLOCK SUBP.SUM

PINCALIB.OUT

POUTCALIB.OUT

-> INI

-> IN2

-> IN3

-> IN4

SBLOCK SUBP

GAIl - 1,

GAI2 - -1,

GAI3 - 1,

GAI4 - 1,

BIAS 0,

SBLOCK RPENABLE.SR

RPAND.Q

PSECURITY.Q

-> SET

-> RES

SBLOCK RPENABLE

SET - "0-0-0-0" ,

RES - "0-0-0-0" ,

Q - "1-2-7-3",

NQ = "0-0-0-0-,

SBLOCK MEDINREL.REL

-> IN

SBLOCK MEDINREL

INC = "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ - 500,

AMPL - 50,

SBLOCK CONTRSUBP.REL

SUBP.OUT -> IN

SBLOCK CONTRSUBP

INC - "0-0-0-0" ;

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ - 1,

AMPL - 1, SBLOCK SPSETPERM.AI SBLOCK SPSETPERM

IO - "0-0-0-0",

OMAX - 100,

OMIN - 0,

UNIT -

" %",

ZERO - NO ;

INV = NO,

SBLOCK CONTRPTM.REL

PTM.OUT -> IN

SBLOCK CONTRPTM

INC - "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC = "0-0-0-0",

DZ - 2,

AMPL - 1, SBLOCK MEDNORM.OR

MEDINREL.INC

MEDINREL.DEC

-> SI

-> S2

-> S3

-> S4

SBLOCK MEDNORM

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0" ;

54 - "0-0-0-0",

q - -o-O-O-O",

NQ = "0-0-0-0",

SBLOCK CONTRPIN.REL

PINCALIB.OUT -> IN

SBLOCK CONTRPIN

INC - "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ - 5,

AMPL - 1, SBLOCK PPUMPMPX.MPX

SPSETPERM.OUT

MEDINREL. INC

PERMFPID. OUT

MEDNORM.NQ

PERMFPID. OUT

MEDINREL.DEC

-> INI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

SBLOCK PPUMPMPX

51 - "0-0-0-0",

52 = "0-0-0-0" ,

53 - "0-0-0-0",

54 - "0-0-0-0" ,

CHG - "0-0-0-0",

NCHG - "0-0-0-0",

INIV - 0 ;

IN2V = 0,

IN3V - 0,

IN4V - 0,

PULS - 0,

SBLOCK CONTRPP.REL

PPCALIB. OUT -> IN

SBLOCK CONTRPP

INC - "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ -15,

AMPL - 1.

SBLOCK PPROBLEM.OR

CONTRSUBP.INC

CONTRPTM.INC

CONTRPIN.INC

CONTRPP.INC

-> SI

-> S2

-> S3

-> S4

SBLOCK PPROBLEM

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0",

54 - "0-0-0-0",

Q - "0-0-0-0",

NQ - "0-0-0-0",

SBLOCK WEIGHTREL.REL

WEIGHTIN.OUT -> IN

SBLOCK WEIGHTREL

INC - "0-0-0-0-,

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ = 4,

AMPL - 50,

SBLOCK PINREL.REL

PINCALIB.OUT -> IN

SBLOCK PINREL

INC - "0-0-0-0",

NINC - "0-0-0-0",

DEC - "0-0-0-0",

NDEC - "0-0-0-0",

DZ -35,

AMPL - 1,

SBLOCK WEIGHTOR.OR

WEIGHTREL.INC

WEIGHTREL.DEC

-> SI

-> S2

-> S3

-> S4

SBLOCK WEIGHTOR

51 - "0-0-0-0",

52 - "0-0-0-0",

53 - "0-0-0-0-,

54 - -0-0-0-0" ,

Q = -0-0-0-0",

NQ - "0-0-0-0" ,

124 Appendix

SBLOCK FEEDAI Al SBLOCK FEEDAI

IO - "0-0-0-0",

OMAX - 100,

OMIN - 0,

UNIT - %",

ZERO - NO,

INV - NO,

SBLOCK FEEDMPX MPX

FEEDAI. OUT

WEIGHTREL.INC

WEIGHTOR.NQ

-> INI

-> SI

-> IN2

-> S2

-> IN3

-> S3

-> IN4

-> S4

SBLOCK FEEDMPX

si = "O-o-o-O",

52 - "0-0-0-0",

53 = "0-0-0-0",

54 - "0-0-0-0",

CHG - "0-0-0-0",

NCHG - "0-0-0-0",

INIV - 0,

IN2V = 0,

IN3V = 0,

IN4V - 0,

PULS = 0,

SGROUP SPCREATION

TS = 1 ; RUN = YES ; PR = 1

SBLOCK PTMSTART.AI SBLOCK PTMSTART

IO "0-0-0-0",

OMAX - 5,

OMIN - 0,

UNIT = "BAR",

ZERO - NO,

INV - NO,

SBLOCK FLUXSTART.AI SBLOCK FLUXSTART

IO - -0-0-0-0",

OMAX - 5,

OMIN - 0,

UNIT = "M~3/H" ,

ZERO - NO

INV - NO

SBLOCK PTMSTDLIM.DLIM SBLOCK PTMSTDLIM SBLOCK FSTDLIM.DLIM SBLOCK FSTDLIM

PTMSTART. OUT -> IN DMAX = 01,

DMIN -

- 01,

FLUXSTART.OUT -> IN DMAX - 2,

DMIN -

- 2,

SBLOCK PTMSFUN.SFUN SBLOCK PTMSFUN SBLOCK FLUXSFUN.SFUN SBLOCK FLUXSFUN

PTMSTDLIM.OUT -> IN N = 3,

11 = 23,

01 - 50,

12 - 26,

02 = 80,

13 - 45,

03 - 100 ,

14 - 0,

04 - 0,

15 = 0,

05 - 0,

16 = 0,

06 - 0,

17 = 0,

07 = 0,

18 = 0,

08 = 0,

19 = 0,

09 - 0,

110 - 0,

010 - 0,

BIAS = 0,

FSTDLIM.OUT -> IN N - 7,

11 - 44,

01 - 50,

12 - 1 87,

02 - 55,

13 =2 135,

03 - 60,

14 - 2 456,

04 - 65,

15 =2 742,

05 = 70,

16 - 3 436,

06 - 80,

17 =3 631,

07 = 90,

18 = 0,

08 - 0,

19 = 0,

09 = 0,

110 = 0,

010 - o,

BIAS = 0,

SBLOCK PTMSTPID.PID SBLOCK PTMSTPID SBLOCK FLUXSTPID.PID SBLOCK FLUXSTPID

PTM.OUT -> MV STRA = "0-0-0-0", FLUXCALIB. OUT -> MV STRA = "0-0-0-0"

,

PTMSTDLIM.OUT -> SP

-> STRA

-> TRA

MAN - "0-0-0-0",

NMAN - "0-0-0-0",

SMAX - 5,

SMIN = 0,

MUNT - "BAR",

OMAX = 500,

OMIN - -500,

OUNT = "%",

GAIN = 1,

TI - 100,

TD - 0,

TFIL - 10,

DZ - 0,

BIAS = 0,

INT = YES,

MODE - 0,

FSTDLIM.OUT -> SP

-> STRA

-> TRA

MAN - "0-0-0-0",

NMAN = -0-0-0-0",

SMAX - 5,

SMIN = 0,

MUNT - "M~3/H" ,

OMAX - 100,

OMIN - -100 ,

OUNT - "%",

GAIN - 20,

TI = 100,

TD = 0,

TFIL - 3,

DZ = 0,

BIAS - 0,

INT - NO,

MODE = 0,

SBLOCK PTMSUM.SUM SBLOCK PTMSUM SBLOCK FLUXSUM.SUM SBLOCK FLUXSUM

PTMSTPID.OUT -> INI GAIl = 1, FLUXSTPID.OUT -> INI GAIl = 1

,

PTMSFUN. OUT -> IN2

-> IN3

-> IN4

GAI2 - 1,

GAI3 - 1,

GAI4 = 1,

BIAS - 0,

FLUXSFUN.OUT -> IN2

-> IN3

-> IN4

GAI2 = 1,

GAI3 = 1,

GAI4 = 1,

BIAS = 0,

SBLOCK PTMSTREL.REL SBLOCK PTMSTREL SBLOCK FLUXSTREL.REL SBLOCK FLUXSTREL

PTMSTPID.E -> IN INC = "0-0-0-0",

NINC - "0-0-0-0"

DEC = "0-0-0-0"

NDEC = "0-0-0-0",

DZ = 2,

AMPL - 1,

FLUXSTPID.E -> IN INC = "0-0-0-0" ,

NINC = "0-0-0-0",

DEC = "0-0-0-0",

NDEC = "0-0-0-0" ,

DZ - 5,

AMPL = 1,

SBLOCK PTMSTOR OR SBLOCK PTMSTOR SBLOCK FLUXSTOR.OR SBLOCK FLUXSTOR

PTMSTREL. INC -> SI SI - "0-0-0-0" FLUXSTREL.INC -> SI SI - "0-0-0-0",

PTMSTREL DEC -> S2

-> S3

-> S4

52 = "0-0-0-0"

53 - "0-0-0-0"

54 = "0-0-0-0"

Q - "0-0-0-0"

NQ - "0-0-0-0"

FLUXSTREL.DEC -> S2

-> S3

-> S4

52 - "0-0-0-0",

53 - "0-0-0-0" ,

54 = "0-0-0-0",

Q - "0-0-0-0" ,

NQ - -0-0-0-0" ,

Appendix 125

SBLOCK PTMNOINT.BSET SBLOCK PTMNOINT SBLOCK FLUXINT.BSET SBLOCK FLUXINT

-> SI SEND - "0-0-0-0" , -> SI SEND - "0-0-0-0",

-> S2BLK - "PTMSTPID"

, -> S2BLK - "FLUXSTPID" ,

-> S3

PTMSTOR.Q -> SEND

PARI "INT",

SIV NO,

PAR2 ""

,

S2V - NO

PAR3 ""

,

S3V NO,

FLUXSTOR.NQ

-> S3

-> SEND

PARI - "INT",

SIV - YES ,

PAR2 -""

,

S2V - NO,

PAR3 -""

,

S3V - NO,

SBLOCK FLUXNOINT BSET SBLOCK FLUXNOINT SBLOCK PTMINT.BSET SBLOCK PTMINT

-> SI SEND "0-0-0-0", -> SI SEND "0-0-0-0" ,

-> S2BLK - "FLUXSTPID" , -> S2

BLK - "PTMSTPID",

-> S3

FLUXSTOR Q -> SEND

PARI "INT",

SIV NO

PAR2 -""

,

S2V NO,

PAR3 -""

,

S3V NO

PTMSTOR.NQ

-> S3

-> SEND

PARI = "INT" ,

SIV - YES,

PAR2 -""

,

S2V - NO ;

PAR3 -""

,

S3V NO,

SGROUP DUMMY

TS = .5 ; RUN = YES ; PR = 1

SBLOCK RUNOR.OR SBLOCK RUNOR

CULTSTATE.NQ -> SI SI "0-0-0-0" ,

STARTFILT.Q

STANDBY.Q

STOPFILT.Q

-> S2

-> S3

-> S4

52 = "0-0-0-0" ,

53 - "0-0-0-0" ,

54 "0-0-0-0",

Q "0-0-0-0" ,

NQ - "0-0-0-0" ,

SBLOCK STARTUPOR.OR SBLOCK STARTUPOR

CULTSTATE. NQ -> SI SI "0-0-0-0" ,

STARTRUN. Q

STANDBY.Q

STOPFILT.Q

-> S2

-> S3

-> S4

52 "0-0-0-0",

53 - "0-0-0-0" ,

54 - "0-0-0-0",

Q "0-0-0-0" ,

NQ - "0-0-0-0" ,

SBLOCK STANDBYOR.OR SBLOCK STANDBYOR

CULTSTATE.NQ -> SI SI - "0-0-0-0" ,

RUNFILT.Q

STARTFILT.Q

STARTRUN. Q

-> S2

-> S3

-> S4

52 - "0-0-0-0" .

53 - "0-0-0-0" ,

54 - "0-0-0-0",

Q - "0-0-0-0" ,

NQ - "0-0-0-0",

SBLOCK ANDFLUX.AND SBLOCK ANDFLUX

PFLUXAND.Q -> SI SI - "0-0-0-0" ,

MIMAFLUX.NHI

PFLUXAND. Q

MIMAFLUX.NHI

-> S2

-> S3

-> S4

52 - "0-0-0-0",

53 - "0-0-0-0" ,

54 = "0-0-0-0" ,

Q - "0-0-0-0" ,

NQ - "0-0-0-0",

SBLOCK ANDPTM.AND SBLOCK ANDPTM

MIMAPTM.HI -> SI SI - "0-0-0-0",

PFLUXOR.Q

MIMAPTM.HI

PFLUXOR.Q

-> S2

-> S3

-> S4

52 - "0-0-0-0" ,

53 - "0-0-0-0",

54 "0-0-0-0" ,

Q - "0-0-0-0" ,

NQ - "0-0-0-0- ,

SBLOCK HOLDTR.SR SBLOCK HOLDTR

EMPTYSTAT.Q -> SET SET - "0-0-0-0" ,

DELAYTROR.Q -> RESRES - "0-0-0-0"

,

Q = "0-0-0-0" ,

NQ - "0-0-0-0" ,

SBLOCK HOLDPH.SR SBLOCK HOLDPH

EMPTYSTAT.Q -> SET SET - "0-0-0-0" ,

DELAYPHOR.Q -> RESRES - "0-0-0-0"

,

Q - "0-0-0-0" ,

NQ - "0-0-0-0",

SBLOCK STGRDTI.TIME SBLOCK STGRDTI

STERGRD.Q -> RUN RUN - "0-0-0-0" ,

STERGRD. NQ -> RESRES - "0-0-0-0" ,

RDY - "0-0-0-0-,

NRDY - "0-0-0-0",

RUNV - NO,

RESV - NO,

TIME - 1800,

ZERO - 0,

ACCU - NO,

PULS - 0,

SBLOCK INAKTIME.TIME SBLOCK INAKTIME

INAKSTATE.Q

COOLSTAT.Q

-> RUN

-> RES

RUN - "0-0-0-0" ,

RES "0-0-0-0",

RDY - "0-0-0-0" ,

NRDY - "0-0-0-0" ,

RUNV - NO,

RESV - NO,

TIME - 1600,

ZERO = 0,

ACCU - NO,

PULS - 10,

126 Appendix

Hardwareadressen FR3

D Digitale AnzeigeG Gerat

K Kabelanschluss

R ReglerS Schalter

1-1-1-1 K Signal der Reaktor-Temperatur Sonde [°C]

1-1-1-2 K Signal der Kreislauf-Temperatur Sonde [°C]

1-1-1-3 K Motorendrehzahl des Riihrers [U/min]

1-1-1-4 K Motorendrehzahl des Schaumabscheiders [U/min]

1-1-1-5 K LOOP: Signal der Loop-Temperatur Sonde [°C]

1-1-1-6 K Signal der Reaktor-Waage (Busch) [kg]1-1-1-7 K Signal des Airflow-Meters (Brooks) [nl/min]

1-1-1-8 K Signal der Puffergefass-Waage (Mettler) [g]

1-1-2-1 K Signal der pH-Sonde1-1-2-2 K Signal der p02-Sonde1-1-2-3 K Signal der pC02-Sonde1-1-2-4 K Signal der Redox-Sonde

1-1-2-5 K Signal der Fluoreszenz-Sonde

1-1-2-6 K 02-Signal der Abgasanalyse1-1-2-7 K C02-Signal der Abgasanalyse1-1-2-8 K Signal der Aquasant-Sonde

1-1-3-1 K Signal der Reaktordmck-Sonde

1-1-3-2 K LOOP: Druck Permeat-seitig (PP.AI / FILTERFAST)

1-1-3-3 K LOOP: Druck Filterausgag (POUT.AI / FILTERFAST)

1-1-3-4 K LOOP: Druck Filtereingang (PIN.AI / FILTERFAST)

1-1-3-5 K LOOP: Flussmenge Rezirkulations-Loop (RFLUX.AI / FILTERSLOW)

1-1-3-6 K LOOP: Gewicht Mettler Permeat-Waage (METPERMIN.AI / F.SLOW)

1-1-3-7 K LOOP: Drehzahl Rezirkulationspumpe1-1-3-8 K

1-1-4-1 R V2: Regelventil Wasser des Temperatur-Kreislaufs1-1-4-2 R VI: Regelventil Dampfdes Temperatur-Kreislaufs

1-1-5-1 R V4: Regelventil Reaktorabluft (Reaktordruck)1-1-5-2 R V3: Brooks-Regelventil Reaktorzuluft

1-1-6-1 R Regelung der Ruhrer-Drehzahl

1-1-6-2 R Regelung der Schaumabscheider-Drehzahl

1-1-7-1 R Regelung der Mediumszufluss-Pumpe1-1-7-2 R Regelung der Mediumsabfluss-Pumpe

1-1-8-1 R Regelung der Basen-Pumpe

Appendix 127

1-1-8-2 R Regelung der Sauren-Pumpe (LOOP: Regelung Permeat-Pumpe)

1-2-1-1 D Lichtschranke Schaum-Detektor (nicht mehr vorhanden)

1-2-1-2 D SPW-Niveau Fuhler

1-2-1-3 D

1-2-1-4 D Drehrichtung Riihrer links

1-2-1-5 D Drehrichtung Riihrer rechts

1-2-1-6 D Drehrichtung Schaumabscheider links

1-2-1-7 D Drehrichtung Schaumabscheider rechts

1-2-1-8 D

1-2-2-1 D

1-2-2-2 D Motorstorung potentialfrei1-2-2-3 D Gasanalyse Kanal 1

1-2-2-4 D Gasanalyse Kanal 2

1-2-2-5 D

1-2-2-6 D

1-2-2-7 D Start Sterilisation

1-2-2-8 D Stop Sterilisation

1-2-4-1 S V5: Digitalventil Dampfuberlagerung Sperrwasserkreislauf1-2-4-2 S V6: Digitalventil Kiihlwasser Kondensatbildung Sperrwasserkreislauf1-2-4-3 S V7: Digitalventil Druck(Pressluft)iiberlagerung Sperrwasserkreislauf1-2-4-4 S V8: Digitalventil Ablass Sperrwasser1-2-4-5 S V9: Digitalventil Dampfumlenkung wahrend GRD-Sterilisation

1-2-4-6 S V10: Digitalventil Dampfzufuhrung Warmetauscher

1 -2-4-7 S V11: Digitalventil Wasserzufuhrung Warmetauscher

1-2-4-8 S V12: Digitalventil Wasserzufuhrung Riickflusskuhler

1-2-5-1 S Riihrer Drehrichtung (on = links)1-2-5-2 S Schaumabscheider Drehrichtung (on = links)1-2-5-3 S Temperatur-Kreislauf-Pumpe1-2-5-4 S Sperrwasser-Kreislauf-Pumpe1-2-5-5 S VO: Digitalventil Kiihlwasser Abfluss

1-2-5-6 S

1-2-5-7 S Riihrer einschalten ermoglichen1-2-5-8 S Schaumabscheider einschalten ermoglichen

1-2-6-1 S Verstarkung Umschaltung Reaktortemperatur-Sonde1-2-6-2 S Verstarkung Umschaltung Kreislauftemperatur-Sonde1-2-6-3 S Verstarkung Umschaltung tief p02-Sonde1-2-6-4 S Verstarkung Umschaltung hoch p02-Sonde1-2-6-5 S Verstarkung Umschaltung Redox-Sonde

1-2-6-6 S Mediumsflaschen Umfull-Pumpe1-2-6-7 S Puffergerfass Auffull-Pumpe1-2-6-8 S

128 Appendix

1-2-7-1 S

1-2-7-2 s

1-2-7-3 s

1-2-7-4 s

1-2-7-5 s

1-2-7-6 s

1-2-7-7 s

1-2-7-8 s

1-2-8-1 R

1-2-8-2 R

1-3-1 G

1-3-2 G

1-3-3 G

1-3-4 G

1-3-5 G

1-3-6 G

1-3-7 G

1-3-8 G

1-4-1 G

1-4-2 G

1-4-3 G

1-4-4 G

1-4-5 G

1-4-6 G

1-4-7 G

1-4-8 G

1-4-9 G

1-4-10 G

1-5-1 G

1-5-2 G

Ventile FR3

V0 1-2-5-5

VI 1- 1-4-2

V2 1- 1-4-1

V3 1- 1-5-2

V4 1-1-5-1

V5 1-2-4-1

V6 1-2-4-2

V7 1-2-4-3

V8 1-2-4-4

LOOP: Digitalventil Eingang Rezirkulations-LoopLOOP: Digitalventil Ausgang Rezirkulations-LoopLOOP: Rezirkulations-Pumpe einschalten ermoglichen (LPright)LOOP: (LPleft)

LOOP: Regelung RegelventilLOOP: Regelung Rezirkulations-Pumpe

Verstarker Reaktortemperatur-SondeVerstarker Kreislauftemperatur-SondeVerstarker pH-SondeVerstarker 02-Sonde

Verstarker C02-Sonde

Verstarker Redox-Sonde

Stromversorgung + 15V DC

Trennverstarker Riihrer und Schaumabscheider

(Weight VG und P.Tw.)

LOOP: Temperatur-Verstarker (Temp. F.)

samson i/p-Wandler Regelventile Kaltwasser/Dampfsamson i/p-Wandler Regelventile Zuluft/Abluft

Speisung Busch-Waage

Speisung Brooks-Airflow-Meter

Digitalventil Kiihlwasser Abfluss

Regelventil Dampf des Temperatur-Kreislaufs

Regelventil Wasser des Temperatur-Kreislaufs

Regelventil Reaktorzuluft (Brooks)

Regelventil Reaktorabluft (Reaktordruck)

Digitalventil Dampfiiberlagerung Sperrwasserkreislauf

Digitalventil Kiihlwasser Kondensatbildung Sperrwasserkreislauf

Digitalventil Druck(Pressluft)uberlagerung Sperrwasserkreislauf

Digitalventil Ablass Sperrwasser

Appendix

V9 1-2-4-5

V10 1-2-4-6

Vll 1-2-4-7

V12 1-2-4-8

Ventile LOOP

V13 1-2-7-1

V14 1-2-7-2

V15 1-2-8-1

Digitalventil Dampfumlenkung wahrend GRD-Sterilisation

Digitalventil Dampfzufiihrung Warmetauscher

Digitalventil Wasserzufuhrung Warmetauscher

Digitalventil Wasserzufuhrung Ruckflusskiihler

Digitalventil Filtereingang

Digitalventil Filterausgang

Regelventil Rezirkulationsfiuss

130 Curriculum vitae

Simon Andreas Rothen

Date of Birth: 23. September 1962

Native Village: Wahlern BE

Marital Status: married

Children: Dorninik Alexander, born 23. November 1996

Education

Memberships

Publications

1993 -1997 Ph.D. thesis, Institute of Biotechnology, ETH Zurich

1992 -1993 Scientific research-fellow, Institute of Biotechnology, ETH Zurich

1992 Diploma as natural scientist (Dipl. Natw. ETH)

1988 -1992 Study of biotechnology, department of biology, ETH Zurich

1982 -1987 Study of medicine, medical faculty, University of Berne

1981 Maturity type C

Swiss Society of Microbiology (SGM)

Rothen S A, Sauer M, Sonnleitner B, Witholt B (1997) Biotransformation of octane

by E. coli HB101[pGEc47] on defined medium: octanoate production and productinhibition. Biotech Bioeng, submitted

Rothen S A, Sauer M, Sonnleitner B, Witholt B (1997) Growth characteristics of E.

coli HB101[pGEc47] on defined medium. Biotech Bioeng, submitted

Sonnleitner B, Rothen S A, Kuriyama H (1997) Dynamics of glucose consumption in

yeast. Biotechnol Prog, 13, 8-13

Rothen S A, Saner M, Meenakshisundaram S, Sonnleitner B, Fiechter A (1996)Glucose uptake kinetics of Saccharomyces cerevisiae monitored with a newlydeveloped FIA. J Biotechnol 50, 1, 1-12

Busch M, Schmidt J, Rothen S A, Leist C, Sonnleitner B, Verpoorte E (1996) uTASmeets biotechnology: Micromachined flow systems combined with biosensor arrays

for bioprocess monitoring. 2nd uTAS Symposium '96, SACh-NSCS, Basel, CH

Rothen S A, Sonnleitner B, Witholt B (1996) Biotransformation of octane to

octanoate by recombinant E. coli HB101[pGEc47]: Product inhibition and

productivity increase. Abstract book, 1st ESBES, Dublin, Irleand

Rothen S A, Sonnleitner B, Witholt B (1996) Growth inhibition of E. coli

HB101[pGEc47] by octanoic and acetic acid. SGM annual meeting, Bern

Rothen S A, Sonnleitner B, Witholt B (1995) Dynamics of recombinant E. coli

HB101[pGEc47] in transient experiments. ECB7, Nice, F

Sonnleitner B, Rothen S A, Hahnemann U (1994) Dynamics of yeast cultures.

Proceedings of 1st Asian Control Conference, Tokyo, J

Rothen S A, Miinch T, Sonnleitner B, Fiechter A (1993/4) On-line interfacinginstrumental analysis to monoseptic bioprocesses. NSCS-SACh, Basel and SGM

annual meeting, Luzern

Miinch T, Rothen S A, Sonnleitner B, Fiechter A (1993) DNA-flow-analysis system:a method for on-line monitoring cell cycle activities in bioprocesses. ECB6, Firenze,I

Acknowledgement 131

Wahrend meiner Studienzeit am Institut fiir Biotechnologie hatte ich das Gliick, in

der Gruppe von PD Dr. Bernhard Sonnleitner unter Prof. Dr. Arrnin Fiechter meine

Semester- und Diplomarbeit durcrifuhren zu konnen. Wahrend dieser Zeit habe ich

einen ersten Einblick in die faszinierende Welt der Bio-Verfahrenstechnik erhalten,

was nicht unwesentlich dazu beigetragen hat, dass ich mich entschloss, eine

Doktorarbeit auf diesem Gebiet in Angriff zu nehmen. Nach einer kurzen

Verzogerung, bedingt durch den Riicktritt von Prof. Fiechter, konnte ich unter Prof.

Bernard Witholt, weiterhin in der Gruppe von Bernhard Sonnleitner, mit meiner

Doktorarbeit beginnen.Was dann folgte, war eine turbulente Zeit gepragt von diversen Umziigen (ging

der Schrank beim letzten Mai nicht noch durch diese Tiire?), fieberhaftem

Experimentieren (welche Limitation ist es denn diesmal?), Kampf mit der Technik

(was halt wohl langer, der Gleitring oder die Einstellung des D/A-Wandlers?),

Verirrungen in die Tiefen von VMS (Boooooooongo!), Besprechung von Resultaten

am Kaffeetisch (wieso geht nur die Pfeife immer aus?), Linien entwirren beim

Simulieren (warum nur produzieren diese Viecher plotzlich Zucker?), kreieren von

Homepages mit kryptischen Tags (<META name="GENERATOR" content="sar"

http-equiv="REFRESH,, content="0,URL=http://www.rothen.ch/">), betreuen einer

Semester- und Diplomarbeit (wie war das doch gleich mit den Schoggistangeli?),Sticheleien rund um PC's und Apple (Imagine that!), endlosem Vorbereiten von

Seminarien und Prasentationen ('aber ich war doch erst letzte Woche dran!', 'I know,

but...') sowie von genussreichem Pflegen der Gruppendynamik.Ich mochte Bernard Witholt dafur danken, dass er es mir ermoglicht hat, in seiner

Gruppe eine Dissertation durchzufuhren. Ich habe wahrend unserer Gesprache viel

gelernt und seine Anregungen waren immer sehr konstruktiv und hilfreich.

Herrn Heinzle mochte ich ganz herzlich fiir die Ubernahme des Korreferates

sowie fur das sehr gewissenhafte Redigieren des Manuskriptes danken.

Bernhard Sonnleitner war der ruhende Pol wahrend all der Turbulenzen in den

letzten Jahren. Seine unerschutterliche Ruhe hat er auch in fiir ihn schwierigen Zeiten

nie abgelegt. Es war immer wieder erstaunlich, woher er sein unerschopflichesWissen hervorzauberte. Ganz speziell mochte ich ihm dafur danken, dass er mir alle

nur erdenklichen Freiheiten liess, jedoch zu jeder Zeit ansprechbar und bereit war,

bei alien Problemen nach Losungen mitzusuchen. Ich habe diesen Fuhrungsstil sehr

geschatzt.Mit Michael Sauer habe ich, wahrend dem er bei mir seine Semester- und

Diplomarbeit durchfuhrte, eine schone Zeit in der Halle verlebt. Seine Arbeit findet

sich in wichtigen Teilen meiner Dissertation wieder und ich mochte ihm fur seinen

Einsatz, und naturlich auch fur die Schoggistangeli, danken.

Ebenfalls erwahnen mochte ich all diejenigen, die durch ihre Mitarbeit am Institut

mir das Arbeiten ganz wesentlich erleichterten: Erika, Hanny, Marjud und ganzbesonders naturlich Helena im Sekretariat, Gertrud und Helene in der Waschkiiche,

Myrtha, Christoph und Rahel bei der Materialverwaltung, sowie Werni, Hans und

Peter in der Werkstatt.

Speziellen Dank verdienen Christian Weikert, der es mir ermoglicht hat, auf einem

anstandigen Gaschromatographen meine Oktanoat-Messungen durchzufuhren,sowie Manfred Zinn, der mir seine HPLC-Saule ausgeliehen hat.

132 Acknowledgement

Interessante Diskussionsabende verdanke ich dem 'Company-Club', zu dessen

Kernmitgliedern Peter David, Michael Kotik, Peter Rothlisberger, Andrew Schmid,Ivo Staijen, Christian Weikert und Manfred Zinn gehorten, wobei ein spezieller DankAndrew Schmid, dem Mitinitiator dieser Runde, gilt.

Interessant war es auch immer, einen Abstecher zur ISCB und der LUNAMED zu

machen. Peter Rothlisberger war immer fur einen Meinungsaustausch zu haben und

einem Abstecher zu Katrin, Jenny, Lydia und Tobias haftete immer ein wenig der

Hauch des Exotischen an. Dass Katrin wahrend ihrer Besuche in Indien gelernt hat,

sich durchzusetzen, konnte ich in Dublin erleben, wo sie in einem Laden fur irische

Wollsachen kurzentschlossen einem Dieb nachrannte, der meine Kongressunterlagenstehlen wollte. Dank ihrem Einsatz brachte ich nicht nur Wollpullover, sondern auch

meine Kongressnotizen sicher in die Schweiz zurtick.

Allen Mitgliedern der Witholt-Gruppe, stellvertretend seien hier nur Marcel,

Biggi, Jan, Ivo, Maarten, Martin, Ruth, Michele, Silke, Wil und Sven erwahnt, mochte

ich fur die Kameradschaft danken, die sie mir entgegengebracht haben.

Mein innigster Dank geht jedoch an meine Frau Barbara. Die ganze Zeit meiner

Ausbildung hier in Zurich habe ich zusammen mit ihr verbracht. Sie hat mir

geholfen, immer den notwendigen Abstand zu meiner Arbeit zu wahren. Wenn ich

am Abend oder sogar oft spat in der Nacht nach Hause kam, es war immer jemandda, mit dem ich meine kleinen Siege, aber auch meine Frustrationen teilen konnte.

Sie hat mir die Ausgeglichenheit verliehen, die notwendig war, damit ich mich im

komplexen Umfeld des Institutes bewegen konnte. Am Ende meiner

Dissertationszeit schenkte sie mir Dominik, unseren Sohn. Dominik hat die Zeit,

wahrend der diese Arbeit niedergeschrieben wurde, wesentlich mitgepragt. Sei es,

dass er im Tragtuch schlafend mit am Computer zugegen war, oder auch, dass ich

dank ihm zu vielen freiwilligen und unfreiwilligen Schreibpausen kam. Der

Umstand, dass Dominik schon mit vier Wochen die Nacht durchschlief hat es

schlussendlich wohl ermoglicht, dass ich zu geniigend Schlaf kam, wodurch diese

Arbeit in sinnvoller Zeit fertiggestellt werden konnte.

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