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Research Collection
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
Rights / License: In Copyright - Non-Commercial Use Permitted
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Diss. ETH Nr. 12082
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.
Characterization oforganisms by on-line analyses 17
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).
18 Characterization oforganisms by on-line analyses
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.
Characterization oforganisms by on-line analyses 19
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.
20 Characterization oforganisms by on-line analyses
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.
Characterization oforganisms by on-line analyses 21
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.
22 Characterization oforganisms by on-line analyses
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.
Characterization oforganisms by on-line analyses 23
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.
Development ofan optimal defined mediumfor HB101[pGEc47] 27
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.
28 Development ofan optimal defined mediumfor HB101[pGEc47]
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.
Development ofan optimal defined mediumfor HB101[pGEc47] 29
"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.
30 Development ofan optimal defined mediumfor HB101[pGEc47]
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).
Development ofan optimal defined mediumfor HB101[pGEc47] 31
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.
32 Development ofan optimal defined mediumfor HB101[pGEc47]
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.
Development ofan optimal defined mediumfor HB101[pGEc47] 33
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)-
34 Development ofan optimal defined mediumfor HB101[pGEc47]
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
Development ofan optimal defined mediumfor HB101[pGEc47] 35
^ 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.
36 Development ofan optimal defined mediumfor HB101[pGEc47]
,." 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.
Development ofan optimal defined mediumfor HB101[pGEc47] 37
-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
(3) continuous growth at a constant dilution rate of 0.35 h"1
(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
(5) continuous growth at a constant dilution rate of 0.05 h"1
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
38 Development ofan optimal defined mediumfor HB101[pGEc47]
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
Development ofan optimal defined mediumfor HB101[pGEc47] 39
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.
40 Development ofan optimal defined mediumfor HB101[pGEc47]
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.
42 Octanoate production and product inhibition
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.
44 Octanoate production and product inhibition
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
Octanoate production and product inhibition 45
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.
46 Octanoate production and product inhibition
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).
Octanoate production and product inhibition 47
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.
48 Octanoate production and product inhibition
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.
Octanoate production and product inhibition 51
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).
52 Octanoate production and product inhibition
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
Octanoate production and product inhibition 53
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).
54 Octanoate production and product inhibition
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).
Octanoate production and product inhibition 55
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.
56 Octanoate production and product inhibition
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-
Octanoate production and product inhibition 57
\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.
58 Octanoate production and product inhibition
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.
Octanoate production and product inhibition 59
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.
62 Process integrationfor the removal ofoctanoate
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.
Process integrationfor the removal ofoctanoate 65
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,
66 Process integration for the removal ofoctanoate
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.
Process integrationfor the removal ofoctanoate 67
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.
68 Process integrationfor the removal ofoctanoate
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.
Process integrationfor the removal ofoctanoate 69
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.
70 Process integration for the removal ofoctanoate
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 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 %.
76 Material and Methods
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
Material and Methods 77
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
78 Material and Methods
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.
Material and Methods 79
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
80 Material and Methods
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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Rothen S A, Saner M, S Meenakshisundaram, Sonnleitner B and Fiechter A (1996)Glucose uptake kinetics of Saccharomyces cerevisiae monitored with a newlydeveloped FIA. J Biotechnol 50,1-12
Sahm H (1991) Metabolic design in amino acid-producing bacteria. Biochem EngStuttgart, 54-62
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Sonnleitner B, Hahnemann U (1994) Dynamics of the respiratory bottleneck of
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22
Stanier R Y, Palleroni N J, Douderoff M (1966) The aerobic Pseudomonads: a
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Escherichia coli fermentations. Biotechnol Bioeng 27,818-824
van de Merbel N C, Lingeman H, Brinkman U A T (1996) Sampling and analytical
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van den Heuvel J C, Beeftink H H, Verschuren P G (1988) inhibition of the acidogenicpopulation during waste-water waste disposal. Appl Microbiol Biotechnol 29,
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(1995) On-line control of an immobilized hybridoma culture with multi-channel
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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
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some properties. J Biol Chem 218,97-106
von Zumbusch P, Brunner G, Meyer-Jens T, Maerkl H (1994) On-line monitoring of
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Weuster-Botz D, Pramatarova V, Spassov G, Wandrey C (1995) Use of a genetic
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133
Wubbolts M (1994) Xylene and alkane mono-oxygenases from Pseudomonas putida.Genetics, regulated expression and utilization in the synthesis of optically active
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90 References
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Yee L, Blanch HW (1993) Defined media optimization for growth of recombinant
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Zihao W, Kefeng Z (1995) Kinetics and mass transfer for lactic acid recovered with
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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
• Strain Designation
• Designation Source Person Choice
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
• Strain Designation
• Designation Source Person Choice
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
• Strain Designation
• Designation Source Person
HB16 Boyer H.
• CGSCtt: 2 601
• Sex: F-
• No. of Muts Carried: 8
• 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
• Strain Designation
• Designation Choice
HBlOl 1
• CGSC#: 652 4
• Sex: F-
• No. of Muts Carried: 13
• 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
• Strain Designation
• Designation Source Person Choice
W2 961 Lederberg J. 1
• CGSC#: 266
• Sex: F-
• No. of Muts Carried: 15
• 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!')
disp('C-bilanz (C) berechnet!')
break;
end % if
if sizegosizebm,
gcflag=l;
Appendix 103
disp('timebm und timegc sind unterschiedlich lang! GC-daten nicht in C-
bilanz integriert! '
)
disp('C-bilanz (C) berechnet!')
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.