Merz 28062012 - A Contribution to Design Foam

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A contribution to design foam fractionation processes Zur Erlangung des akademischen Grades eines Dr.-Ing. von der Fakultät Bio- und Chemieingenieurwesen der Technischen Universität Dortmund genehmigte Dissertation vorgelegt von Dipl.-Ing. Juliane Merz aus Linz am Rhein Tag der mündlichen Prüfung: 28.06.2012 1. Gutachter: Prof. Dr.-Ing. Gerhard Schembecker 2. Gutachter: Prof. Dr.-Ing. Andrzej G´ orak Dortmund 2012

Transcript of Merz 28062012 - A Contribution to Design Foam

Page 1: Merz 28062012 - A Contribution to Design Foam

A contribution to design

foam fractionation processes

Zur Erlangung des akademischen Grades eines

Dr.-Ing.

von der Fakultät Bio- und Chemieingenieurwesen

der Technischen Universität Dortmund

genehmigte Dissertation

vorgelegt von

Dipl.-Ing. Juliane Merz

aus

Linz am Rhein

Tag der mündlichen Prüfung: 28.06.2012

1. Gutachter: Prof. Dr.-Ing. Gerhard Schembecker

2. Gutachter: Prof. Dr.-Ing. Andrzej Gorak

Dortmund 2012

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Ein Gelehrter in seinem Laboratorium ist nicht nur ein Techniker;

er steht auch vor den Naturgesetzen wie ein Kind vor der Märchenwelt.

Marie Curie

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Abstract

In biochemical industry foams are generally undesired because foaming is mostly accompa-

nied by product loss and thus, effort is put into the prevention of foam formation especially

during downstream processing. However, the foaming tendency caused by amphiphilic or

surface-active substances in bioprocesses does not necessarily need to be disadvantageous.

A downstream separation technique, where foam is actually desired and used as the se-

paration medium, is foam fractionation. The principle of separation is the preferential

adsorption of surface-active substances at a gas-liquid interface. In this thesis, a fungal

hydrolytically active enzyme, biochemically characterized as a cutinase, is separated out

of the crude culture supernatant using foam fractionation. For batch and continuous

foam fractionation various process parameters, like pH value or temperature, and column

design parameters, such as length or diameter of the foaming column are systematically

investigated in regard to separation efficiency and foam stability. Impact parameters and

parameter interactions were identified using Design of Experiment and it was possible

to tailor the foam fractionation process to desired yields or enrichments. For continuous

foam fractionation enriching and stripping mode were examined, whereas stripping mode

is found to be more efficient for highly diluted or weak systems. Experiments with dyes

and surface tension measurements further increased the knowledge about the process.

It could be shown, that cutinase can be recovered and simultaneously concentrated and

purified. A recovery of 94 % and a 47-fold concentration and 19-fold purification of

cutinase in the foam phase could be achieved in a single process step. This, and the lack

of additives and the simple experimental procedure make foam fractionation a technique

for process intensification. However, before foam fractionation can be used in industry a

scale-up is necessary. Two traditional scale-up approaches were tested to enlarge the foam

fractionation system. However, the investigated and enlarged foam fractionation columns

were still lab-scale equipment. During the scale-up experiments, activity profiles of the

columns were analyzed. Relationships between gas superficial velocity and column length

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were found, which offer opportunities to configure a foam fractionation column to special

requirements.

Parallel to the cutinase system the industrial enzyme PLA2 was separated and purified

from complete culture broth of the recombinant Aspergillus niger via foam fractionation.

Batch foam fractionation and continuous foam fractionation in stripping and enriching

mode were investigated. Similar results as for the cutinase system were obtained.

As a consequence of the research presented a guideline was developed to adjust foam frac-

tionation fast and efficiently to general enzyme systems. Additionally, a pure component

system and non-foamable enzymes were investigated to validate the guideline.

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Zusammenfassung

In biotechnologischen Prozessen ist die Bildung von Schäumen grundsätzlich unerwünscht,

da diese meist mit Limitierungen und Produkteinbußen einhergeht. Allerdings muss die

Tendenz zur Schaumbildung, meist ausgelöst durch amphiphile beziehungsweise ober-

flächenaktive Substanzen nicht zwangsläufig negativ sein. Ein Aufreinigungsprozess der

gerade diesen physikalischen Unterschied der Substanzgruppen zur Trennung ausnutzt ist

die Zerschäumung. Das Trennprinzip beruht dabei darauf, dass sich die oberflächenak-

tiven Substanzen an eine Gas-Flüssigkeits-Grenzfläche anlagern beziehungsweise adsor-

bieren und mit dem Schaum ausgetragen werden. In dieser Arbeit wird ein hydrolytisch

aktives Pilzenzym, biochemisch identifiziert als eine Kutinase, mit Hilfe der Zerschäumung

aus unbehandeltem Kulturüberstand getrennt. Verschiedene Prozessgrößen, wie pH-Wert

oder Temperatur und Kolonnengrößen, wie der Durchmesser oder die Längen der Säule

wurden variiert und im Hinblick auf Trenneffizienz und Schaumstabilität systematisch

untersucht. Dabei wurde statistische Versuchsplanung als systematischer Ansatz gewählt

um absatzweise und kontinuierliche Zerschäumung zu untersuchen. Für die kontinuierliche

Zerschäumung wurden die Betriebsweisen „enriching mode” und „stripping mode” näher

untersucht. Für hochverdünnte oder schwach oberflächenaktive Substanzen führte die

„stripping” Fahrweise zu einem stabileren und effizienteren Prozess. Mit Hilfe der statis-

tischen Versuchsplanung konnten Einflussgrößen und deren Wechselwirkungen identifiziert

werden und die Zerschäumung so angepasst werden, dass die gewünschte Ausbeute oder

Anreicherung erlangt werden konnte. Zum weiteren Verständnis der Vorgänge während

der Zerschäumung wurden Farbstoffexperimente durchgeführt und die Oberflächenspan-

nung unter verschiedenen Bedingungen vermessen. Es konnte gezeigt werden, dass das

Enzym Kutinase aus dem Kulturüberstand getrennt und gleichzeitig konzentriert und

gereinigt werden konnte. In nur einem Schritt konnte eine Ausbeute von 94 % mit einer

47-fachen Aufkonzentrierung und 19-fachen Reinigung der Kutinase in der Schaumphase

erreicht werden. Des Weiteren kann die Zerschäumung aufgrund der einfachen Durch-

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führung und der Abwesenheit von Hilf- oder Zusatzstoffen als eine Technik zur Prozess-

intensivierung betrachtet werden. Um die Zerschäumung jedoch industriell einsetzen zu

können, ist eine Maßstabsvergrößerung notwendig. In dieser Arbeit wurden zwei tra-

ditionelle Ansätze zur Maßstabsvergrößerung der Zerschäumung betrachtet, wobei der

Labormaßstab nicht überschritten wurde. Zum einen wurde der Ansatz eines konstan-

ten Verhältnisses von Länge zu Breite und zum anderen der Ansatz konstanter Länger

aber variierender Breite untersucht. Das Scale-up Verfahren bei konstantem Längen-

und Breitenverhältnis führte zu einer verbesserten Trennleistung bei gleichbleibenden

Schaumeigenschaften. Die Maßstabsvergrößerung mit dem Ansatz konstante Länge und

variierender Durchmesser der Säule hingegen verschlechterte die Trenneffizienz und die

Schaumstruktur veränderte sich. Während dieser Versuche wurden zusätzlich Profile der

enzymatischen Aktivität über die Länge der Säulen vermessen. Es konnten Zusammen-

hänge zwischen der Gasleerrohrgeschwindigkeit und der Kolonnenlänge ermittelt werden,

die für die spätere Säulenkonfiguration von Bedeutung sind.

Parallel zu dem untersuchten Kutinase System wurde das im industriellen Maßstab herge-

stellte Enzym PLA2 aus der Kulturbrühe des rekombinanten Pilzes Aspergillus niger mit-

tels Zerschäumung getrennt und gereinigt. Auch hier wurde das System absatzweise und

kontinuierlich in „stripping” und „enriching” Fahrweise untersucht. Die Ergebnisse sind

denen des Kutinase Systems sehr ähnlich.

Als Folge der präsentierten Untersuchungen wurde ein Leitfaden zur schnellen und ef-

fizienten Übertragung herkömmlicher Enzymsysteme zur Zerschäumung entwickelt. Um

die Qualität des Leitfadens zu prüfen, wurden weiterhin ein Reinstoffsystem und nicht

zerschäumbare Enzyme untersucht.

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List of Publications

Journal Papers

Merz, J., Schembecker, G., Riemer, S., Nimtz, M., Zorn, H.: Purification and identification

of a novel cutinase from Coprinopsis cinerea by adsorptive bubble separation. Separation

and Purification Technology, 69 (1), 2009, 57-62.

Merz, J., Zorn, H., Burghoff, B., Schembecker, G.: Purification of a fungal cutinase by

adsorptive bubble separation: A statistical approach, Colloids and Surfaces A: Physico-

chemical and Engineering Aspects, 382, 2011, 81-87.

Merz, J., Burghoff, B., Schembecker, G.: Continuous foam fractionation: Performance

as a function of operating variables, Separation and Purification Technology, 82, 2011,

10-18.

Merz, J., Schembecker, G.: Foam fractionation: A technique for process intensification.

Submitted to Chemical Engineering and Processing: Process Intensification.

Oral Presentations

Merz, J., Van de Sandt, E., Meesters, G., Schaap, A., Schembecker, G.: Intensified

downstream processing of PLA2 using foam fractionation. 16th International Conference

on Biopartitioning and Purification (BPP), Puerto Vallarta, Mexico (2011)

Merz, J., Burghoff, B., Schembecker, G.: Continuous foam fractionation: Performance as

a function of operating variables. GVC/Vortrags- und Diskussionstagung: Bioverfahrens-

technik an Grenzflächen, Potsdam (2011)

Merz, J., Burghoff, B., Schembecker, G.: Purification of a fungal cutinase by adsorptive

bubble separation: A statistical approach. 8th Conference on Foams and foam-related sys-

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tems (fluid interfaces, thin films, surfactants, concentrated emulsions) Eufoam, Borovets,

Bulgaria (2010)

Merz, J., Schembecker, G., Zorn, H.: Isolierung und Anreicherung von Pilzenzymen mit-

tels fraktionierter Zerschäumung. GVC/Vortrags- und Diskussionstagung “Biokatalyse:

Neue Verfahren, neue Produkte”, Bad Schandau (2009)

Poster Presentations

Merz, J., Burghoff, B., Zorn, H., Schembecker, G.: Determination of impact factors for

continuous foam fractionation of the fungal enzyme cutinase. 8th European Symposium

on Biochemical Engineering Science, Bologna, Italy (2010)

Merz, J., Burghoff, B., Schembecker, G.: A Statistical approach for the purification of a

fungal cutinase by foam fractionation. GVC/Dechema Vortrags- und Diskussionstagung:

Bioprozessorientiertes Anlagendesign, Nürnberg (2010)

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Contents

Abstract i

Zusammenfassung iii

List of Publications v

Nomenclature xi

1 Introduction 1

2 Theoretical part 3

2.1 Principles of foam formation . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2 Surface adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.3 Foam stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.3.1 Liquid drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.3.2 Marangoni effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.3.3 Disproportionation or Ostwald ripening . . . . . . . . . . . . . . . . 9

2.3.4 Coalescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3.5 Foam structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.4 Influencing parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.5 Foaming process, devices and modes . . . . . . . . . . . . . . . . . . . . . 11

2.6 Model system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.7 State of the art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.8 Motivation and content of the work . . . . . . . . . . . . . . . . . . . . . . 16

3 Cutinase from C. cinerea: Screening experiments 19

3.1 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.1.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.1.2 Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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3.1.3 Cultivation and enzyme production . . . . . . . . . . . . . . . . . . 20

3.1.4 Protein quantification . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1.5 Activity assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1.6 Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1.6.1 Semi-native SDS-PAGE and activity measurement . . . . 21

3.1.6.2 Isoelectric focusing (IEF) and activity measurement . . . . 22

3.1.7 ESI-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

3.1.8 Configuration of equipment and experimental procedure for foam

fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.1.9 Separation performance . . . . . . . . . . . . . . . . . . . . . . . . 24

3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.2.1 Influence of medium composition on the production of esterase type

enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.2.2 Isolation and concentration of extracellular enzymes from culture

supernatant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.2.3 Characterization of enzymes in the foam phase . . . . . . . . . . . . 26

3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.3.1 Separation of active enzyme . . . . . . . . . . . . . . . . . . . . . . 28

3.3.2 Influence of pH value . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.3.3 Influence of gas flow rate . . . . . . . . . . . . . . . . . . . . . . . . 29

3.3.4 Influence of additives . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.3.5 Comparison to alternative separation processes . . . . . . . . . . . 30

3.3.6 Biochemical characterization . . . . . . . . . . . . . . . . . . . . . . 30

3.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

4 Statistical investigation of batch foam fractionation 31

4.1 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.4.1 pH dependence (A) . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.4.2 Temperature dependence (B) . . . . . . . . . . . . . . . . . . . . . 40

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4.4.3 Gas flow rate (C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4.4.4 Foam volume (D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.4.5 Starting volume (E) . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.4.6 Pore diameter (F) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

5 Statistical investigation of continuous foam fractionation 43

5.1 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.1.1 Surface tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.1.2 Configuration of equipment and experimental procedure for foam

fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

5.1.3 Determination of liquid holdup (ϕ) . . . . . . . . . . . . . . . . . . 46

5.1.4 Design of Experiments (DoE) . . . . . . . . . . . . . . . . . . . . . 46

5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.2.1 Configuration of equipment . . . . . . . . . . . . . . . . . . . . . . 48

5.2.2 Design of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.2.3 Surface tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

5.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

5.3.1 Statistical analysis of single and quadratic effects . . . . . . . . . . 55

5.3.1.1 Recovery of activity (R) . . . . . . . . . . . . . . . . . . . 55

5.3.1.2 Enrichment factor (EC) . . . . . . . . . . . . . . . . . . . 56

5.3.1.3 Liquid holdup (ϕ) . . . . . . . . . . . . . . . . . . . . . . 56

5.3.2 Statistical analysis of factor interactions . . . . . . . . . . . . . . . 57

5.3.2.1 Recovery of activity (R) . . . . . . . . . . . . . . . . . . . 57

5.3.2.2 Enrichment factor (EC) . . . . . . . . . . . . . . . . . . . 57

5.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6 Statistical investigation of column dimensions 61

6.1 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

6.2 Design of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

6.3.1 Surface tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

6.3.2 Configuration of equipment . . . . . . . . . . . . . . . . . . . . . . 66

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6.3.3 Design of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . 68

6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

6.4.1 Configuration of equipment . . . . . . . . . . . . . . . . . . . . . . 72

6.4.2 Discussion of single effects . . . . . . . . . . . . . . . . . . . . . . . 73

6.4.3 Discussion of factor interactions . . . . . . . . . . . . . . . . . . . . 75

6.5 Foam fractionation as process intensification . . . . . . . . . . . . . . . . . 76

6.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

7 A contribution to the scale-up of foam fractionation 79

7.1 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

7.1.1 Feed preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

7.1.2 Configuration of equipment and experimental procedure . . . . . . 80

7.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

7.2.1 Dependency between surface tension of feed and performance of

foam fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

7.2.2 Operating range of column (D30) . . . . . . . . . . . . . . . . . . . 83

7.2.3 Activity profiles of column (D30) . . . . . . . . . . . . . . . . . . . . 87

7.2.4 Scale-up approaches . . . . . . . . . . . . . . . . . . . . . . . . . . 89

7.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

8 Design guideline for foam fractionation processes 93

8.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

8.2 Design guideline for foam fractionation . . . . . . . . . . . . . . . . . . . . 94

9 Conclusion and Outlook 109

List of Figures 112

List of Tables 117

References 119

Curriculum Vitae 132

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Nomenclature

AbbreviationsAPS Ammonium peroxodisulfate

ATR-FTIR Attenuated total reflection Fourier transform infrared spectroscopy

bp base pairs

CBS Centraalbureau voor Schimmelcultures

CD Circular dichroism spectroscopy

CMC Critical micelle concentration

CRYO-SEM Cryo scanning electron microscopy

Da Dalton

DoE Design of Experiments

EDTA 2,2’,2”,2” ’-(Ethane-1,2-diyldinitrilo)tetraacetic acid

ESI MS/MS Electrospray ionization linked mass spectrometry

eV electron Volt

HPLC High pressure liquid chromatography

IEF Isoelectric focusing

IRRAS Infrared reflection absorption spectroscopy

MM Mineral salt medium

PCH Phanerochaete chrysosporium

pI Isoelectric point

SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis

SNL Standard nutrient medium

TEMED N,N,N’,N’-tetramethyl-ethane-1,2-diamine

Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol

Latin symbolsVFeed Feed flow rate

VGas Gas flow rate

AF Enzymatic activity of foamate

AI Enzymatic activity of the initial liquid

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xii Nomenclature

ai Activity of component i

CF Protein concentration in the foamate

CI Protein concentration in the initial liquid

csurfactants Concentration of surface-active molecules

DColumn Diameter of whole foam fractionation column

Dfoam Diameter foam column

Dliquid Diameter liquid column

DPore Diameter of frit pore

EC Enrichment factor

G Gibbs energy

H Enthalpy

kia Associating rate of component i

kid Disasociating rate of component i

Lfoam Length foam column

Lliquid Length liquid column

P Purification factor

R Gas constant

R Recovery of activity

S Entropy

T Temperature

tfoaming Foaming time

U Unit

VFixtures Volume of fixtures (glass bowl, column enlargement)

Vfoamcolumn Volume of foam column

Vfoamliquid Liquid volume in the foam

VF Foamate volume

VI Volume of initial solution

Vliquidcolumn Volume of liquid column

Vtotalliquid Volume of emptied foam fractionation column

Greek symbolsα Star value for Design of Experiments

Δ Difference symbol

ε410nm Molar extinction coefficient at a wavelength of 410 nm

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Nomenclature xiii

Γ Concentration of component at the interface

Γi Concentration of component i at the interface

π Spreading pressure

σ Surface tension of solution with surface active components

σ0 Surface tension of pure solution

ϕ Liquid hold up in the foam phase

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1 Introduction

History of modern enzyme technology goes back to the 19th century where scientists likeBuchner and Kühne discovered the existence of biocatalytic proteins, so called enzymes[1]. But enzymes have been used by man throughout the ages without knowledge oftheir nature and abilities. In parts of microorganisms they were used in baking, in theproduction of alcoholic beverages, or in leather tanning. These days we know enzymesas biocatalysts, which catalyze various chemical reactions. According to Buchholz etal. [2], enzymes can cover the production of all desired human material needs, such asfood, pharmaceuticals, fine and bulk chemicals. In comparison to inorganic catalystssuch as acids, bases, metals, or metal oxides, enzymes typically act highly specific. Thisspecificity allows high yields of product accompanied by a minimum of undesirable by-products. Unlike most of the chemical catalysts, enzymes work under mild conditionswith respect to temperature, pressure, and acidity (pH). Furthermore, enzymes are partof the sustainable environment [3, 4]. They work on renewable raw materials like fruits,milk, fats, and wood. Products and waste of most enzymatic reactions are non-toxic andin some cases recyclable as fertilizer or usable as animal feed. Due to their specificity, highbiodegradability, ecology, and mild reaction terms enzymes are used in a wide range ofindustrial applications, like washing industry or food industry [2]. According to the report“Enzymes in Industrial Applications: Global markets” [5] by BCC Research, formerlyknown as Business Communications Company, Inc., the market of enzymes is estimatedto increase to 4.4 billion US$ in the year 2015 (see figure 1.1).

Figure 1.1: Global industrial enzymes market: Recent development and forecast (reprintedfrom [5])

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2 1 Introduction

The market growth is enabled by gene technologies which improve productivity and en-zyme stability on the one hand and techniques, like site-directed mutagenesis, directedevolution, or gene shuffling which offer possibilities to modify selectivity and specificityon the other hand [2]. Compared to the fast growing techniques for the specification andproduction of enzymes, the downstream processing strategies to purify the target moleculeout of complex and highly diluted culture broths lag behind. To meet the steadily in-creasing demand for microbial enzymes, cost-effective methods are required. Therefore,downstream processing may determine overall process economy [6] and thus, influencewhether the enzyme production becomes economically viable or not [7]. Hence, the im-provement of existing or the development of new purification techniques are crucial for thesuccess of biochemical production concepts. Traditionally, the recovery of enzymes in food,pharmaceutical, and cosmetics industry is carried out in so-called multistage processes.They include standard steps like precipitation, ultrafiltration, and chromatography. Thedisadvantages of these processes are, that they are time-consuming, expensive, and oftentainted with high losses of enzymatic activity. Because different solvents and additives areused in the multistage processes several intermediate and pretreatment steps are necessaryto operate the next process step. Therefore, the development of ecologically compatible,efficient, and selective processes or process intensification strategies, like the reduction ofprocess steps, the decrease of equipment volume, or waste streams is of high interest. Anexample for a separation technique for the isolation of extracellular enzymes from micro-bial culture media, which does not need additives and is operated under gentle conditions,is foam fractionation. This technology known in principle but new in application is basedon the adsorption of surface-active components to a gas-liquid interface. It is regarded aseconomically and ecologically compatible method for the separation and/or concentrationof proteins, including active enzymes [8–15]. Foam fractionation is particularly appropri-ate for early stages of downstream processing, where the separation and concentration ofa dilute protein from a large volume of crude starting material is required.

The thesis presented was financially supported by the Federal Ministry of Education andResearch (“Technology platform: Innovative Downstream Processes”, FKZ 0315520).

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2 Theoretical part

According to Lemlich [16], foam fractionation belongs to the group of adsorptive bubbleseparation techniques. These methods comprise various mechanisms of separating dis-solved or suspended materials by means of adsorption or attachment at the surfaces ofbubbles rising through a feed solution [16]. Adsorptive bubble separation methods are sub-divided into foam separation and non-foaming adsorptive bubble separation techniques.For the first group foaming is required for separation, while the second group does notneed foam generation to carry off the target materials. Both foam separation and non-foaming adsorptive bubble separation techniques are further subdivided as depicted inFigure 2.1. Foam separation methods, for instance, are divided into foam fractionationand flotation, where flotation is defined as the removal of coarsely dispersed material andfoam fractionation as the separation of dissolved and surface-active components by foam.

Foam separation Non-foaming adsorptive bubble separation

Foam fractionation

Flotation Solvent sublation

Bubble fractionation

mineral precipitate adsorbing colloid

ion molecular colloid micro- macro- ore

flotation

Adsorptive bubble separation

Flotation Flotation Flotation

Figure 2.1: Schematic presentation of adsorptive bubble separation methods (reprintedfrom [16])

2.1 Principles of foam formation

Foams consist of thousands of bubbles and are generated by dispersing gas (air, nitrogen,oxygen, CO2, or noble gas) in liquid. If the bubbles rise to the surface of the liquid

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and accumulate faster as they decay, a foam is formed [17]. Foams are also defined asa gas-liquid dispersion with a dominating gas content [17]. With increasing interfacialarea of a foam the overall free energy of the system increases compared to the free energyof the separated gas and liquid phases. Hence, foams are thermodynamically unstableand classified as lyophobic colloidal dispersions [18]. That means that foams are notformed spontaneously and the gas and liquid phases tend to separate fast to decreasethe free energy of the system. To stabilize foams the interfacial tension between thecontinuous liquid phase and the dispersed gas phase needs to be minimized. This can bedone by adding amphiphilic molecules which adsorb to the gas-liquid interface. Moleculeswhich spontaneously adsorb to a gas-liquid interface, like flavors, antioxidants, pigments,proteins, or microorganism [17, 19] are called surface-active. Surface-active, amphiphilicmolecules will probably adsorb to the gas bubble with their hydrophobic parts orientedto the inner of the bubble and the hydrophilic parts oriented to the continuous aqueousphase (see Fig. 2.2). The film of surface-active molecules at the interface stabilizes thefoam kinetically, but a true thermodynamic equilibrium cannot be realized due to thefoams metastable character [20].

Figure 2.2: Schematic formation of surface-active molecules at a gas bubble. • hydrophilicpart, - hydrophobic part

For efficient separation using foam fractionation the surface adsorption as well as the foamstability are of main importance and will be discussed in the following chapters.

2.2 Surface adsorption

As mentioned before, the adsorption of surface-active molecules happens due to the factthat systems strive for a minimum in enthalpy and a maximum in entropy. In aqueoussolutions protein molecules are surrounded by water clusters. When the molecules ad-sorb to the interface they partly lose their water cluster and the entropy of the systemincreases compared to the system without adsorption [21]. Additionally, the surface pres-sure or spreading pressure π, which is defined as π = σ0 - σ (σ0 is the surface tension ofthe pure solution and σ the surface tension of the solution with surfactants) reduces thesurface tension and the free energy of the system respectively. Aveyard and Haydon [22]

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2 Theoretical part 5

describe the spreading pressure π as a two dimensional pressure exerted by the adsorbedmolecules in the plane of the surface. To sum up, protein adsorption increases the entropyand decreases the free enthalpy of the system. Thus, according to the Gibbs-Helmholtz-Equation for constant T and p: ΔG = ΔH - T · ΔS the process of surface adsorption isexergonic and with it spontaneous (ΔS>0 and ΔH<0 → ΔG<0) [20]. In figure 2.3 thechange of surface tension in presence of surface-active molecules is depicted schematically.If the concentration of surfactants in the bulk phase increased, at constant surface areaand temperature, the spreading pressure π and the surface tension σ decreased. If theconcentration exceeds a critical value, the so called critical micelle concentration (CMC),surface tension reaches a plateau. This plateau corresponds to the formation of completesurfactant monolayers [14]. Above the CMC molecules will aggregate to micelles, whichare in dynamical equilibrium with the monomers. Further increase in surfactant con-centration increases the possibility of forming lamellar, cubic, or hexagonal mesophases[23].

Surf

ace

tens

ion

[mN

m-1

]

log csurface-active molecules

CMC

Figure 2.3: Dependency of surface tension at varying surfactant concentration (reprintedfrom [23]) (• hydrophilic part, - hydrophobic part)

The relationship between surface tension and adsorption of solute molecules at the inter-face can be expressed by the Gibbs adsorption equation 2.1. Under equilibrium conditionsand constant gas constant R and temperature T the equation is written as:

Γi = − 1

RT

dσdlnai

(2.1)

Γi is the so called surface excess or the concentration of the adsorbed component i atthe surface. R is the gas constant, T the absolute temperature, and ai the activity ofcomponent i. If the concentration of surface-active molecules is below the critical micelleconcentration, the relationship between concentration of surface active molecules and

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6 2 Theoretical part

surface excess Γ can be displayed graphically with a Langmuir isotherm (Fig. 2.4) [24].A constant value of Γ is equivalent to a monolayer of molecules adsorbed at the surface.

C surface-active molecules

co st

CMC

Figure 2.4: Dependency of surface excess Γ and the concentration of surface-active com-ponents in the bulk solution

To enable the surface-active molecules or proteins to form a protective film around thebubbles the hydrophobicity and with it adsorption strength is the determining factor. Dueto the uneven distribution of hydrophobic groups in a protein the adsorption strength isinfluenced as can be seen in figure 2.5. The easiest way to manipulate the hydrophobicityof proteins is the variation of pH in the liquid phase. Proteins have their maximal hy-drophobicity and minimum solubility in the aqueous phase at their isoelectric point (pI)[12, 13, 25].

No adsorption

Weak adsorption

High adsorption

Gas phase

Liquid phase

Hydrophobic part Hydrophilic part

Figure 2.5: Schematic adsorption behavior of surface-active molecules in dependence oftheir hydrophobic properties. Adsorption strength increases with increasing hydropho-bicity oriented to the outer surface of the protein (reprinted from [8])

The adsorption process itself is diffusion-controlled. The mobility of the surface-activemolecules is expected to decrease strongly with increasing film size. According to Minton[26] two distinct kinetic pathways can explain the growth behavior of protein films orprotein clusters (see Fig. 2.6).

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2 Theoretical part 7

Figure 2.6: Schematic depiction of the kinetic pathways for protein adsorption. P1: directdeposition of soluble protein onto available surface; P2: “piggyback” deposition of solu-ble protein onto cluster species i; P3: accretion/incorporation of adsorbed protein ontocluster species i to form cluster species i+1. kia: associating rate, kid: disassociatingrate (reprinted from [26])

The first pathway is the adsorption of soluble protein to vacant surface positions (P1),followed by diffusion of adsorbed protein to the edge of the cluster species i and subsequentaccretion to form the cluster species i+1 (P2). The second pathway consists of directdeposition of a protein in solution onto the solution-facing surface of a preexisting clusterspecies i ("piggyback" deposition) (P3) and subsequent incorporation into the cluster ito form the new cluster species i+1. The parameter kia is the association rate and kid

the disassociation rate of the cluster species. For a multicomponent system additionalinteractions and competitive forces need to be taken into account during the adsorptionprocess. Amount and composition of the resulting molecule film are influencing the foamstability.

2.3 Foam stability

If a liquid phase containing surface-active molecules is aerated with a gas the adsorptiontakes place in the liquid phase and molecule films are formed around the rising gas bubbles.Due to the stabilization of the gas bubbles foam can emerge above the liquid phase. Severalfactors affect the stability of the emerging foam, such as gravitational and capillary forces(liquid drainage), pressure differences (Ostwald ripening), or collisions of bubbles witheach other or the column wall (coalescence).

2.3.1 Liquid drainage

Drainage results from pressure differences between lamella and Plateau Border. ThePlateau Border or Gibbs-canal is a system of slit-shaped delta-canals. In equilibrium

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8 2 Theoretical part

condition three lamellae abut in an angle of 120° (cf. Fig. 2.7) [27]. The curvature of the

Figure 2.7: Cryo-SEM picture of a Plateau border (reprinted from [28])

interface at the Plateau Border causes a pressure difference in direction of the Gibbscanal and liquid will flow out of the lamellae into the canals. Beside capillary forces alsogravitational forces can cause drainage. Due to drainage, surface-active components canadsorb to the rising bubble surfaces, and surface-inactive components drain back to theinitial solution [8, 9]. Thus, a chromatographic effect occurs inside the column [29, 30].Additionally, the liquid fraction in the foam decreases. The decreasing liquid contentcauses thinning of the lamellae until they break. Hydrophobic interactions between ad-joining molecule films support drainage effects and film thinning. If a high number ofhydrophilic parts is oriented in the adjoining molecule films electric double layers can beformed. Due to the electrostatic effects drainage is slowed and the lamellae are stabilized.

2.3.2 Marangoni effect

An important effect, which acts against drainage, is the Marangoni effect. During drainagelamellae are stretched whereby the adsorbed molecules are dispersed. Thus, some parts ofthe interface are depleted in surface-active components resulting in regions with higher sur-face tension and an interfacial tension gradient. If surface-active components are presentin the lamella liquid and adsorb quickly to the region of high surface tension, drainage andthus film thinning can continue. However, if no surface-active components are present,the adsorbed surfactant film moves from regions of low surface tension to the region ofhigh surface tension. The movement of the surfactant layer drags the lamella liquid along[20] as can be seen in figure 2.8. Thus, the Marangoni effect is the ability of a surfactantlayer to rapidly respond to a local interfacial tension gradient and retards drainage effectsor film thinning [16, 20, 31]. The Marangoni effect is driven by thermodynamic processesto decrease the energy of the system and can be realized due to the dilatational elasticityof the surfactant film. The dilatational elasticity is related to the ability of the molecule

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2 Theoretical part 9

film to stretch to regions of high surface tension. In case of protein films the dilatationalelasticity is high compared to low molecular weight surfactants and the stability of thelamellae higher [20].

Figure 2.8: A lamella under influence of drainage. The lamella is stretched and regionswith high and low surface tension are formed. If no Marangoni effect retards the flow,the lamella is thinning till it ruptures. If Marangoni effect retards drainage, part of thedrained lamellar liquid is dragged with the stretching surfactant film and the lamellais temporally stable

2.3.3 Disproportionation or Ostwald ripening

Disproportionation or Ostwald ripening is the diffusion of gas from small bubbles to largebubbles. The driving force is the pressure difference in the bubbles. The Laplace pressurein small bubbles is higher than in big bubbles. Thus, gas diffuses from small to big bubblesand the foam will become coarse. Disproportionation can be retarded by generating ahomogeneous bubble size over the foam column or by forming a viscoelastic molecule filmaround the bubbles. For fast desorbing molecules, like low molecular weight surfactants,the shrinkage of the bubbles will not be affected. In case of proteins, which desorb slowly,the concentration of molecules in the film increases while the gas bubble shrinks. Thus,surface rheological properties, like the dilatational elasticity, are improved increasing thestability of the gas bubble [20].

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10 2 Theoretical part

2.3.4 Coalescence

Coalescence is the merging of two or more gas bubbles to one big gas bubble. Coalescenceis an irreversible process and occurs if bubbles collide with each other or with column walls.Additionally, coalescence can occur if holes in the molecule films exist due to contact withdisturbing molecules, incomplete adsorption or film thinning through drainage.

2.3.5 Foam structure

Drainage, disproportionation, and coalescence cause less liquid hold-up in the foam phase.As a result of the decreasing liquid content the geometry of the foam changes. When thebubbles leave the liquid they are spherical and separated through thick liquid films. Thebubbles do not interact with each other and can be regarded as independent. When theliquid content decreases the spherical foam turns into polyhedral foam (cf. Fig. 2.9). Thebubbles are polyhedral and separated by thin liquid films known as lamellae. Throughthe thin films the bubbles can interact with each other. Polyhedral foam can only beformed in the presence of surface-active substances, which stabilize the lamellas betweenthe bubbles [32]. For foam fractionation, drainage, disproportionation, and coalescencecan be used to efficiently concentrate the target molecule in the foam phase and removeexcess water and surface-inactive components.

Figure 2.9: Change of spherical to polyhedral foam (reprinted from [33])

2.4 Influencing parameters

According to former chapters, the ability of molecules to adsorb at an interface and thecomposition and arrangement of the molecules in the film determine whether a foam can

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2 Theoretical part 11

emerge or not. Additionally, the specificity of the adsorption is of main importance for theseparation efficiency of the foam fractionation process. Many factors affect the adsorptionprocess as well as foam stability and the interactions can be complex. For an efficientand robust process various parameters, such as pH value, gas flow rate, temperature, orcolumn geometry need to be considered.

2.5 Foaming process, devices and modes

The foaming process is carried out by supplying gas into a solution, which contains surface-active components. The surface-active molecules can adsorb to the gas-liquid interfaceand stabilize the rising bubbles. Bubbles leaving the surface of the feed solution carryboth adsorbed compounds and bulk liquid into the emerging foam column. Liquid drainsback along the lamellae of the bubbles and drags surface-inactive components with it tothe initial solution. The emerging foam leaves the column and is collapsed. The liquefiedfoam is called foamate and the depleted solution is called retentate. A typical batch foamfractionation column used in this study is depicted in figure 2.10.

Gas

Flow meter

Heating water

Heating water

Foamate

Porous frit

Enzyme solution

Vacuum pump

Figure 2.10: Typical batch foam fractionation device

The foam fractionation process can be operated batchwise or continuously. For these pro-cess variants several operation modes have been introduced by Lemlich [16]: For batchwisefoam fractionation simple mode and simple mode with recycle have been developed as de-picted in figure 2.11. For simple batch foam fractionation the feed solution containingthe surface-active molecules is introduced into the foam fractionation column and theemerging foam is removed continuously. In simple mode with recycle part of the liquefiedfoam is fed again to the batch foam fractionation column. Batch experiments allow firstinsights whether foam fractionation is feasible and efficient.

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12 2 Theoretical part

Gas

Flow meter

Heating water

Heating water

Foamate

Porous frit

Vacuum pump

Gas

Flow meter

Heating water

Heating water

Foamate*

Porous frit

Vacuum pump

*recycle

a) b)

Figure 2.11: Batch foam fractionation in a) simple mode or enriching mode, b) simplemode with recycle [16]

For continuous foam fractionation four modes of operation are available: simple modeor in this thesis named enriching mode, simple mode with recycle, stripping mode, andcombined mode [16]. The four modes of operation are depicted in figure 2.12.

Gas

Flow meter

Low-pressure

Foamate

Retentate

Feed

Porous frit

a)

Gas

Flow meter

Low-pressure

Foamate*

Retentate

Feed

Porous frit

*Recycle

b)

Gas

Flow meter

Low-pressure

Foamate

Feed

Retentate

Porous frit

c)

Gas

Flow meter

Low-pressure

Foamate*

Feed+ *Recycle

Retentate

Porous frit

d)

Figure 2.12: Continuous foam fractionation in a) simple mode or enriching mode, b) simplemode with recycle, c) stripping mode, and d) combined mode [16]

In simple mode or enriching mode the feed enters the column at the top of the liquidphase. Thus, the adsorption of surface-active molecules takes place in the liquid phase.The emerging foam can rise up the column and effects like drainage and disproportionationdetermine foam stability and the enrichment of the target molecule. In enriching modewith recycle, part of the liquefied foam is fed again into the foam column. This operationmode shall further increase the enrichment of the target molecule. In stripping mode thefeed enters the column somewhere in the foam column. Hence the main adsorption ofsurface active molecules takes place in the foam phase. Because the feed solution flowscounter current to the rising foam the degree of stripping or the recovery of the targetmolecule is improved. The enrichment, however, decrease because the permanent liquid

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2 Theoretical part 13

feed and with it the refilling of the bubble lamellae reduces effects like drainage anddisproportionation. The combined mode is the combination of enriching and strippingmode and shall favor both advantages of the processes, resulting in high recovery andhigh enrichment of the target molecule [16]. Continuous operation can be more efficientcompared to batchwise operations and is a potential starting point for scaling-up foamfractionation. Both batchwise and continuous operation can be operated as serial columnfoaming. The liquefied foam is the starting solution for the next column. Serial foamingshall enhance the enrichment and purification. A further development of serial columnfoaming is the multistage foaming system introduced by Darton et al. [34]. The systemconsists of several trays, each representing an independent foaming unit with glass frit togenerate and a paddle to destroy the foam. A schematic representation is displayed infigure 2.13.

Figure 2.13: Multistage foam fractionation column developed by Darton et al. [34]

Serial and multistage column foaming reduces the liquid content of the foam and allowshigher enrichment and purification because surface-inactive contaminants can be removedwith excess liquid. The disadvantages of these methods can be decreasing recovery anddenaturation in case of foaming enzymes.

2.6 Model system a

In the continuous search for new products or intermediates, natural selection has beenfound to be superior to combinatorial chemistry [35]. According to Schulz et al. [35], fordiscovering novel substances, such as secondary metabolites or enzymes the search should

aParts published in Colloids and Surfaces A: Physicochemical and Engineering Aspects 382 (2011), 81-87

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concentrate on organisms that inhabit novel biotopes, which is valid for miscellaneousfungi. It is supposed that fungi contain biocatalysts, which have novel biodegradativeand biosynthetic properties. According to Rajarathnam [36], there is an opportunity toadvance the production and extraction of flavors, antioxidants and active pharmaceuticalingredients from chemical inert lignin and Cellulose/Hemicellulose-mixtures via fungalhydrolases and oxidoreductases. In this thesis, several fungal species of Phanerochaetechrysosporium and Coprinopsis cinerea are screened for their ability to secrete esterasetype enzymes into the culture supernatant (chapter 3). Esterases and lipases are enzymetypes with high growth potential in industrial enzyme markets and can be used in severalapplication fields [37–39]. The fungi belong to the class of basidiomycetes. Phanerochaetechrysosporium is the most intensively studied white-rot fungus and was the first basi-diomycete genome to be sequenced [40]. Coprinopsis cinerea, formerly known as Coprinuscinereus, is easy to cultivate and extensive genetic and molecular analyses of this organismhave been performed [41]. The fungus has black spore prints and gills that liquefy as themushroom matures via self digestion (autodigestion). Because of this phenomenon it is aso-called inky cap mushroom [42]. The basidiomycete Coprinopsis cinerea 29-2 is found tobe the best producer of esterase type enzymes. The target enzyme secreted was identifiedas a cutinase. Cutinases belong to the class of serine esterases and to the superfamily ofα/β-hydrolases. They are hydrolytically active and naturally degrade cutin, the cuticularpolymer of higher plants. The cutin polyesters are composed of hydroxy and epoxy fattyacids. In spite of the fact, that cutinases show lipolytic activity, they differ from classicallipases as they do not exhibit interfacial activation [43]. They hydrolyze soluble esters aswell as emulsified triacylglycerols [44]. Due to that, cutinases show interesting propertiesfor industrial products and processes and can be considered as a link between esterasesand lipases. Potential applications include the dairy industry, house hold detergents, theoleochemical industry, the synthesis of tailored triglycerides, polymers, and surfactants,ingredients of personal-care products, and agrochemicals and pharmaceuticals with oneor more chiral centers [43]. The degradation of plastics represents another interestingapplication. For instance, a cutinase from Fusarium solani pisi partially hydrolyzedpolyethylene terephthalate (PET) and polycaprolactone to water-soluble products [45, 46].Esterase type enzymes also play a major role in the degradation of renewable materialsand industrial pollutants [47–50].

2.7 State of the art

The first who considered foam fractionation as a separation technique is Ostwald. Hesubmits a patent for registration on

Process for the vaporization of fluids with the purpose of enrichment, separa-

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2 Theoretical part 15

tion or drying of dissolved or emulsified materials through foam fractionation...,

which is issued by the German Federal Patent Office on the 16th of October 1920 [51].In the early stages of foam fractionation till now the method is used to remove metals,organic or inorganic agents [52–60]. Additionally, foam fractionation is used for wastewater treatment to purify industrial and communal slops [61–65]. Other authors describedfoaming as a principle for water treatment of fish farms and aquariums [65–67]. Themethod is well established in industrial scale and various contractors exist, which sell socalled foam fractionators or protein skimmers. Also a growing number of aquarists aresuccessfully using the technique to remove excess proteins from home aquariums. Otherindustrial applications beside waste water treatment are the separation of gluten andstarch [68, 69], or the enrichment of microorganisms [19, 70]. For these applications theconcentration of the target product in the foam phase is of main importance, however,for waste water treatment the target fraction is the depleted, clarified water. Otherfields of operation, where the target product is concentrated in the foam phase, are theenrichment and separation of natural compounds out of plant extracts, like rosemary,potatoes, or cannabis [10, 32, 71–75]. Foam fractionation is also used for the isolation ofproteins. Scientists study the separation of proteins out of raw plant juices [10, 76], fromurine [76], beer [77], or eggs [78]. A continuation to protein isolation is the separationand concentration of enzymes with foam fractionation. The challenge thereby is thepreservation of the catalytic activity. Ostwald and Mischke [76] describe the enrichmentof lipases and diastases. Uraizee and Narsimhan [15] describe that D-amino acid oxidase,tripeptide synthetase, and keto-enol tautomerase may be separated with negligible activityloss. Also enzymes like α-amylase, invertase, catalase, laccase, and lipase [12, 79–81] havebeen separated and enriched through foaming. It is also shown, that mixtures of proteinscan be selectively separated. For instance, catalase can be separated from its mixture withamylase [12], invertase from a mixture with amylase [81], urease from its mixture withcatalase [13], or β-casein from mixtures with BSA or lysozyme [14]. If the enzyme is toohydrophilic to adsorb at the gas-liquid interface, a collector may be added, which bindsto the enzyme and forms a surface-active complex [16]. This type of foaming is calledtweezers foaming and is used by Gerken et al. [30] to enrich laccase and horseradishperoxidase bound with cetryl trimethylammonium bromide (CTAB) and by Burapatanaet al. [82] to separate lysozyme and cellulase by binding with hydrophobic β-cyclodextrin.So far however, with few exceptions, the enzyme separations using foam fractionationare “proof of principles”. Commercial available enzymes are separated out of buffers orstandard media. Real protein concentrations, as they occur during fermentation, andcontaminants are hardly considered. Some examples of enzymes, which are separated outof crude starting materials, e.g. fermentation broths, are lipases, laccases, streptokinases,proteases, cellulases [9, 25, 79, 80, 83–86].

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Beside the search for foamable molecules, fundamentals, like drainage or coalescence be-havior are investigated [87–93], approaches to measure these effects are generated [94–97], and mathematical models are developed to describe these effects and the foamfractionation process [53, 88, 98–104]. Another big issue in the research of foam frac-tionation is the investigation of influencing parameters, such as pH value, gas flow rate,concentration of surface-active molecules, addition of salts, temperature, or feed flow ratefor continuous foam fractionation [12, 25, 71, 73, 86, 105–114]. In these studies, mostlyone to four parameters are varied as common “one-factor at a time” experiments. Theinfluence of each parameter can be determined, but systematic approaches or parameterinteractions are hardly considered. Studies, which deal with systematic approaches havebeen performed by Grieves and Wood [112], Wong et al. [115], Brown [116], who discussalso parameter interactions, and Aksay and Mazza and Oliveira et al., who use responsesurface methodology to optimize the separation process [117, 118]. In addition to thetraditional batchwise and continuous foam fractionation processes and their operationmodes, the feasibility of foam fractionation in coiled columns, multistage reactors, orserial foaming devices has been investigated by several authors [34, 119–125].

2.8 Motivation and content of the work

Foam fractionation has been used for more than 70 years to selectively separate andconcentrate enzymes or other surface-active molecules. To identify the real potential offoam fractionation as an enzyme separation technique, crude starting materials, such asfermentation broths, need to be investigated. In this study, a host system is chosen,which produces extracellular esterase type enzymes (see chapter 3), because esteraseshave several industrial application fields and a high growth potential on the global enzymemarkets [37–39]. After identifying a host system and studying the feasibility of the systemfor foaming, the enzyme concentrated in the foam phase is biochemically characterized.With the own fermentation system, real protein and contaminant concentrations, andbiological variability is studied in regard to separation efficiency.

Although many authors investigated foam fractionation, the process is not fully under-stood [83]. This may be due to the lack of understanding the complete adsorption processat the gas-liquid interface or the complex interactions in the separation medium foam.Additionally, almost all variables, like process parameters, such as pH value or gas flowrate, or design parameters, like the length of the foam column, influence the efficiencyof the foam fractionation process and complex interactions exist. According to chapter2.7, there is only a small number of scientist who considered the interactions of processparameters and only Brown [116] investigated column design parameters in this context.To understand the process of foam fractionation it is not sufficient to determine the effects

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of single parameters, but the interactions of the parameters need to be analyzed, too.

In this thesis, the impact of a wide range of process and column design parameters, con-sidered to influence adsorption behavior and foam stability, are investigated. The parame-ters investigated can be classified in foamability parameters and performance parameters.Foamability parameters like pH value, temperature, and concentration of surface-activemolecules determine the foaminess of the system. If the system is foamable the separa-tion efficiency of foam fractionation is influenced by performance parameters: gas flowrate, column design, and feed flow rate for continuous foam fractionation. For batchand continuous foam fractionation single effects and interactions of the foamability andperformance parameters are determined by means of Design of Experiments (DoE)(seechapter 4, 5). Additionally, for continuous foam fractionation the enriching and strippingmode are examined and the performance parameters are discussed in detail (see chapter6). Experiments, like surface tension measurements and dye experiments complete theinvestigations during this thesis. The focus of the work using DoE is the identification ofimpact parameters and interactions and thus, the understanding of the process. To showthe potential of foam fractionation, traditional purification strategies are compared withit. Furthermore, foam fractionation is investigated for the ability to be used in processintensification strategies (see chapter 6). However, to use foam fractionation in industry,a scale-up of the system is necessary. Thus, a scale-up methodology for foam fractiona-tion is proposed in chapter 7. However, the investigated and enlarged foam fractionationcolumns are still lab-scale equipment. Finally, the results obtained during the thesis aresummarized in a guideline. The guideline should help to adjust the foam fractionationprocess fast and efficiently to general enzyme systems (see chapter 8).

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3 Cutinase from C. cinerea: Screeningexperimentsa

In a diploma thesis prior to this thesis, different fungal strains were investigated for theirability to secrete esterolytically active enzymes into the culture media [126]. The cul-ture system with the highest esterolytic activity was chosen for screening experimentsto show the potential of foam fractionation as a purification technique. The strain Co-prinopsis cinerea 29-2 cultivated in mineral salt main culture medium showed the highestesterolytic activity and good foaming properties in the screening experiments. Thus, thesystem was used to investigate the influence of parameters like pH value of the culturesupernatant, gas flow rate, and addition of detergents on the separation efficiency of foamfractionation. In addition to foaming experiments, enzymes in the foam phase were bio-chemically characterized and the target esterolytically active enzyme was sequenced. Allfoam fractionation experiments as well as the biochemically characterization were partof the diploma thesis [126]. Based on this preliminary work the foaming system wascharacterized. During the later work the fermentation as well as the activity assay wereenhanced.

3.1 Material and methods

3.1.1 Chemicals

EDTA (≥ 99 %), gum Arabic (from acacia tree), kerosene (purum), and p-nitrophenylpalmitate (≥ 98 %) were obtained from Sigma-Aldrich (Seelze, Germany), acetic acid(p.a., 10 %), aluminium sulfate (Al2O12S3 · 13.4 − 14.5H2O, crist.), formaldehyde (p.a.,37 %), α-D(+)-glucose (C6H12O6 ·H2O), glutaraldehyde (50 %), hydrochloric acid (pure,37 %), isopropanol (HPLC grade), ortho-phosphoric acid (≥ 85 %), potassium dihydro-gen phosphate (≥ 99 %), di-potassium hydrogen phosphate (K2HPO4 · 3H2O, ≥ 99 %),silver nitrate (≥ 99.9 %), sodium carbonate (≥ 99 %), sodium deoxycholat (≥ 98 %),sodium hydroxide (≥ 99 %), sodium thiosulfate (Na2S2O3 · 5H2O, ≥ 98.5 %), Tris base(≥ 99 %), Tris hydrochloride (≥ 99 %), Tween 80 (ph-Eur.), and yeast extract from

aParts of the chapter are published in Separation and Purification Technology 69 (2009), 57-62

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20 3 Cutinase from C. cinerea: Screening experiments

Roth (Karlsruhe, Germany), agar-agar (Kobe), agarose (research grade), ammonium per-oxodisulfate (≥ 99 %), anode fluid 3, L-asparagine (C4H8N2O3 · H2O, ≥ 99 %), bovineserum albumin (lyophil.), calcium chloride (CaCl2 · 2H2O, ≥ 99 %), cathode fluid 10,Coomassie brilliant blue G250, dodecylsulfate sodium salt (≥ 99 %), disodium hydrogenphosphate (Na2HPO4 · 2H2O, ≥ 98 %), magnesium sulfate (MgSO4 · 7H2O, ≥ 99.5 %),TEMED (≥ 98.5 %) from Serva (Heidelberg, Germany), ammonium nitrate (p.a.), cop-per sulfate (CuSO4 · 5H2O, ≥ 99 %), ferric chloride (FeCl3 · 6H2O, ≥ 97 %), glycine(ph. Eur.), malt extract (96.5 - 98 %), sodium chloride, and zinc sulfate (ZnSO4 · 7H2O,≥ 99 %) from AppliChem (Darmstadt, Germany), and ethanol (96 %) from Kassner(Dortmund, Germany). Substances were used as received.

3.1.2 Strains

The fungal strains Phanerochaete chrysosporium 246.84 CBS, Phanerochaete chrysospo-rium 481.73 CBS, and Phanerochaete chrysosporium 316.75 CBS were obtained fromCentraalbureau voor Schimmelcultures in Utrecht, the Netherlands. The strains Co-prinopsis cinerea g003/USA and Coprinopsis cinerea 29-2 were obtained from the culturecollection of Friedrich Schiller University Jena.

3.1.3 Cultivation and enzyme production

Mycelia of P. chrysosporium (PCH 246.84 CBS, PCH 481.73 CBS, PCH 316.75 CBS)were maintained on 1.5 % agar plates with standard nutrient liquid (SNL) [127] and themycelia of Coprinopsis cinerea (29-2, g003/USA) on 2 % agar plates with malt extract[20 g L−1 malt extract]. Pre-cultures of all fungi were prepared from a mycelium block(1 cm2) as inoculum, which was excised from the agar plate and placed in a 300 mLErlenmeyer flask containing 100 mL pre-culture medium. Strains of P. chrysosporiumwere pre-cultivated in SNL medium [127] and strains of C. cinerea in malt extract medium[20 g L−1 malt extract]. After treatment with an Ultra Turrax homogenizer (IKA WerkeGmbH & CO. KG, Staufen) the fungal cultures were incubated at 150 rpm and 24 °C(Minitron, Infors HT, Bottmingen, Switzerland). The PCH strains and C. cinerea 29-2were pre-cultivated for three days, C. cinerea g003/USA for four days. For the productionof esterolytically acitve enzymes, different main culture media were tested in terms of highenzyme secretion. PCH strains were cultivated in SNL, SNL without glucose, and mineralsalt medium (MM) [128]. Media for Coprinopsis strains were malt extract medium, SNLwithout glucose, and MM. The main cultures were prepared in 500 mL Erlenmeyer flaskscontaining 250 mL medium, which was inoculated by transferring 20 mL of pre-cultureinto each flask. Additionally, 0.4 % (v/v) Tween 80 was added for inducing the secretion

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3 Cutinase from C. cinerea: Screening experiments 21

of lipolytic enzymes into the culture medium [8, 129]. The main cultures were incubatedat 150 rpm and 24 °C for seven days (Minitron, Infors HT, Bottmingen, Switzerland).For the foam fractionation experiments, the culture supernatants were separated from themycelia by filtration under reduced pressure. Media and equipment were autoclaved priorto use, and sterile techniques were applied throughout the procedures.

3.1.4 Protein quantification

Protein concentrations were determined according to Bradford [130] using bovine serumalbumin as standard.

3.1.5 Activity assay

Esterolytic activity was quantified according to the method of Winkler and Stuckmannusing p-nitrophenylpalmitate (pNpp) as substrate [131]. The substrate solution was pre-pared by mixing 10 mL isopropanol containing 30 mg pNpp with 90 mL of 0.05 MSoerensen phosphate buffer, pH 8 (buffer: solution A ((8.9 g L−1 Na2HPO4 · 2H2O),and solution B (6.8 g L−1 KH2PO4) were mixed in a ratio of 17:1) containing 207 mgsodium deoxycholate and 100 mg gum Arabic. 1 mL of freshly prepared substrate solutionwas mixed with 42 μL culture supernatant or water (blank) and incubated for 15 minutesat 37°C and 650 rpm in a Thermomixer comfort (Eppendorf AG, Hamburg, Germany).After incubation, the absorbance was measured at 410 nm. One enzyme unit U was de-fined as one μmol p-nitrophenol enzymatically released from the substrate per minute.Under the conditions described the extinction coefficient of p-nitrophenol was determinedto be ε410nm=18,495 L(mol · cm)−1. All samples were analyzed at least as a duplicate.

3.1.6 Electrophoresis

3.1.6.1 Semi-native SDS-PAGE and activity measurement

Sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed ona Mini-Twin system (1 mm, 8.6 cm x 7.7 cm) from Whatman Biometra GmbH (Goettin-gen, Germany). The SDS-PAGE analyses were performed using a 12 % polyacrylamideresolving gel. Gel, running buffer, and sample application buffer were prepared using only10 % of the SDS amount according to Laemmli [132]. The samples were concentratedusing Amicon� Ultra-15 and Microcon� centrifugal filter modules with a molecular weightcut off of 10 kDa and mixed with sample application buffer in a ratio of 1.25:1 (v/v). Elec-trophoresis was carried out at 4 °C. For estimation of molecular masses, marker proteins

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22 3 Cutinase from C. cinerea: Screening experiments

from 14.3 kDa to 200 kDa were used (Roti� Mark, Roth, Karlsruhe, Germany). Half of thegel was stained with colloidal Coomassie (83 % (v/v) dest. water, 10 % (v/v) ethanol, 5 %(w/v) aluminium sulfate, 2 % (v/v) ortho-phosphoric acid, and 2 spatula tops CoomassieG250). The other half of the gel was used for the determination of esterolytically ac-tive enzymes. According to the stained gel, bands were excised and incubated in 1 mLsubstrate solution (see 2.5) for 45 minutes at 37 °C and 650 rpm in a Thermomixer com-fort from Eppendorf AG (Hamburg, Germany). The extinction was measured at 410 nmagainst a gel sample without bands.

3.1.6.2 Isoelectric focusing (IEF) and activity measurement

IEF polyacrylamide gel electrophoresis was performed on a Multiphor II system (GEHealthcare München, Germany) using Servalyt� Precote� gels with an immobilized pHgradient from pH 3 to pH 10 (Serva Electrophoresis, Heidelberg, Germany). Isoelectricpoints were estimated using an IEF protein standard with pIs from 3 to 10 (Serva Elec-trophoresis, Heidelberg, Germany). Gels were silver stained according to the methodof Heukeshoven and Dernick [133]. For activity staining, a pNpp-agarose-overlay wasdeveloped. Agarose (2 %) was dissolved in 45 mL 0.05 M Soerensen buffer, pH 8, with103.5 mg sodium deoxycholate and 50 mg gum Arabic under boiling. After dissolving,5 mL isopropanol with 15 mg pNpp were added. After focusing, the IEF gel was incubatedon the pNpp-agarose-overlay at 37 °C till the bands became visible.

3.1.7 ESI-MS/MS

For ESI-MS/MS analyses, the relevant protein spot was excised from an SDS-PAGE gel,dried, and digested with trypsin. The resulting peptides were extracted and purifiedaccording to standard protocols at the Helmholtz centre for infection research, researchgroup biophysical analysis. A QTof II mass spectrometer (Micromass, Manchester, Eng-land) equipped with a nanospray ion source and gold coated capillaries (Protana, Odense,Denmark) was used for electrospray ionization (ESI) MS peptides. For collision-induceddissociation experiments, multiple charged parent ions were selectively transmitted fromthe quadrupole mass analyzer into collision cell (collision energy 25 - 30 eV for optimalfragmentation). The resulting daughter ions were separated by an orthogonal time-of-flight mass analyzer. The acquired MS-MS spectra were enhanced (Max. Ent. 3, Micro-mass) and used for the initio sequencing of tryptic peptides.

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3 Cutinase from C. cinerea: Screening experiments 23

Gas

Flow meter

Heating water

Heating water

Foamate

Porous frit

Enzyme solution

Vacuum pump

a)

Gas

Flow meter

Heating water

Heating water

Foamate

Porous frit

Enzyme solution

Vacuum pump

b)

Figure 3.1: Foaming devices used for foam fractionation experiments. a) experimentswithout Tween 80, b) experiments with Tween 80

3.1.8 Configuration of equipment and experimental procedure for

foam fractionation

The foaming device consisted of a liquid column (length = 15 cm, diameter = 3 cm) witha porous frit at the bottom (P3, pore diameter 16 - 40 μm), a foam column (length =24 cm, diameter = 1.6 cm or 3 cm), a horseshoe bend, and a receiving flask (Fig. 3.1 a)).For the foam fractionation experiments with Tween 80, an additional glass bowl wasplaced on top of the column (Fig. 3.1 b)). The experiments were carried out with 70 mLenzyme solution filled into the foaming column. Air was employed through a porous fritas a foaming gas. The emerging bubbles rose through the feed solution and surface-activecomponents could adsorb at the bubble surface. Due to adsorption the bubbles werestabilized and formed foam after leaving the liquid phase. The foam left the foam columnover the horseshoe bend and was destroyed under reduced pressure and collected in thereceiver. This foam breaking method is conveniently used in laboratory scale setups andwell documented in literature [17, 79]. For Tween 80 experiments, 0.2 % (v/v) Tween80 was added to the feed solution, filled into the liquid column and handled as describedbefore. Samples for protein and activity analyses were taken from the initial solution, theremaining sample solution after foaming (retentate), and the liquefied foam (foamate).

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24 3 Cutinase from C. cinerea: Screening experiments

3.1.9 Separation performance

To describe the separation performance of foam fractionation the enrichment factor (EC),the recovery of active enzyme (R), and the purification factor (P ) were defined as follows:

EC =AF

AI(3.1)

R =AF · VF

AI · VI· 100% (3.2)

P =AF /CF

AI/CI(3.3)

AF and AI are the enzymatic activities [U L−1] in the foamate and in the initial solution,respectively. VF and VI are the volumes [L] of the collapsed foam and the initial solution.CF and CI are the total protein concentrations [g L−1] in the foamate and the initialsolution measured according to section 3.1.4. All quantitative data represent averagevalues of at least duplicate analyses.

3.2 Results

3.2.1 Influence of medium composition on the production of

esterase type enzymes

Activity was monitored over a culture period of seven days. According to fig. 3.1 theproduction of extracellular esterolytic activity was strongly dependent on the carbonsource in the main culture medium. Highest hydrolytic activity of 700 U L−1 was ob-served for C. cinerea 29-2 after 6 days in mineral salt medium (Fig. 3.1d)). The culturesupernatants of C. cinerea 29-2 of day 6 in mineral salt medium were used for the foamfractionation experiments.

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3 Cutinase from C. cinerea: Screening experiments 25

0

10

20

30

40

50

0 2 4 6 8

Act

ivity

[UL-1

]

Cultivation time [d] (a) PCH 316.75 CBS

0

10

20

30

40

0 2 4 6 8

Act

ivity

[UL-1

]

Cultivation time [d] (b) PCH 481.73 CBS

0

10

20

30

40

50

0 2 4 6 8 Act

ivity

[UL-1

]

Cultivation time [d] (c) PCH 246.84 CBS

0 100 200 300 400 500 600 700 800

0 2 4 6 8

Act

ivity

[U L

-1]

Cultivation time [d] (d) C. cinerea 29-2

0

2

4

6

8

10

0 2 4 6 8

Act

ivity

[U L

-1]

Cultivation time [d] (e) C. cinerea g003/USA

Figure 3.1: Secretion of lipolytic activity by Phanerochaete chrysosporium: (a) - (c); andC. cinerea: (d)- (e) depending on carbon source. SNL medium with glucose and 0.4 %(v/v) (�), SNL medium without glucose and 0.4 % (v/v) Tween 80 (�), mineral saltmedium containing 0.4 % (v/v) Tween 80 (•), and malt extract medium with 0.4 %(v/v) Tween 80 (�)

3.2.2 Isolation and concentration of extracellular enzymes from

culture supernatant

The recoveries of enzymatic activity were examined in the foamate at varying pH values,column diameters, and gas flow rates. Furthermore, experiments with the addition ofTween 80 to the feed solution were carried out. The pH value of the feed solution wasadjusted from 3.0 to 8.5 with 1 M HCl or 1 M NaOH, respectively. All other parameterswere kept constant (40 mL air min−1, room temperature, and foaming period until nofoam could be generated). According to figure 3.2, the pH optimum was found to bepH = 7.0, where 80 % of the esterolytic activity was recovered into the foam phase. Foamfractionation experiments were run for approximately 15 minutes until no foam could be

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26 3 Cutinase from C. cinerea: Screening experiments

generated.

0

1

2

3

4

5

0

20

40

60

80

100

3.0 4.0 5.0 6.0 6.3 7.0 8.0 8.5

Enri

chm

ent f

acto

r [-]

Rec

over

y of

act

ivity

[%]

pH

Figure 3.2: Recovery of activity and enrichment factors depending on pH value(40 mL air min−1, room temperature, column diameter 1.6 cm, and foaming periodtill end of foam formation). Activity in foamate (�), activity in retentate (�), andenrichment factor (�)

To stabilize the foam for more than 15 minutes, and to further increase recovery andenrichment, 0.2 % (v/v) Tween 80 was added to the feed solution. Addition of thisdetergent caused foaming periods of more than 60 minutes. However, the recovery ofactivity decreased from 80 % to 6 %, and the enrichment factor from 4.3 to 1.1. Thesecond approach to increase the enrichment was the use of a bigger column diameterto increase drainage and coalescence. A foam fractionation experiment with a columndiameter of 3 cm, a gas flow rate of 40 mL min−1, pH = 6.3, room temperature, anda foaming period till end of foam formation was performed. In comparison with theexperiment in the column with 1.6 cm diameter (Fig. 3.2), the enrichment increasedto 5.0, the recovery, however, decreased to 44 %. The last parameter evaluated for thescreening experiments was the gas flow rate. The air flow was adjusted from 15 to 50mL min−1. A good compromise between high recovery and high enrichment factor wasobserved at a gas flow rate of 20 mL min−1 (Fig. 3.3), where a recovery of 79 % and anenrichment factor of 10.5 was determined. The highest enrichment factor of 25 with 60 %recovery was obtained at an air flow rate of 15 mL min−1. Throughout the experimentsa activity loss of ≤ 1 % was observed.

3.2.3 Characterization of enzymes in the foam phase

To characterize the enzymes concentrated in the foam phase and to visualize the selectivityof the foaming process, a "semi-native" SDS-PAGE was carried out. The hydrolyticactivity of the four protein bands indicated in figure 3.4 was determined. The entire

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3 Cutinase from C. cinerea: Screening experiments 27

0

5

10

15

20

25

30

0

20

40

60

80

100

15 20 30 40 50

Enri

chm

ent f

acto

r [-]

Rec

over

y of

act

ivity

[%]

Flow rate [mL min-1]

Figure 3.3: Recovery of activity and enrichment factors depending on the air flow rate (pH7.0, room temperature, column diameter 1.6 cm, and foaming period till end of foamformation). Activity in foamate �), activity in retentate (�), and enrichment factor(�)

hydrolytic activity of the foamate was attributed to the enzyme of band 3. The molecularweight of this enzyme was estimated to be 29.6 kDa. For further specification, an IEF gelelectrophoresis was performed. After focusing, the gel was cut, and one part was subjectedto silver staining, while the second part was activity stained with a pNpp-agarose overlay.The target enzyme was detected at a pI of 6.7 (data not shown).

Figure 3.4: Semi-native SDS-PAGE of initial solution, retentate and foamate (parametersof foam fractionation: pH 7.0, 15 mL air min−1, room temperature, Column diameter1.6 cm, and 15 minutes)

After in-gel tryptic digestion and peptide extraction, two peptides (LVGTVLFGYTKand LVTGGYSQGAALVAAALR) were sequenced by means of electrospray ionizationtandem MS at the Helmholtz centre for infection research, research group biophysicalanalysis (Fig. 3.5). Data bank searches showed significant homologies to cutinases ofFusarium oxysporum (EF613272) and Fusarium solani (M29759).

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28 3 Cutinase from C. cinerea: Screening experiments

Figure 3.5: Alignment of peptide 1 with the sequences of potential cutinases from C.cinerea (abbreviations represent accession nos. in the EMBL database) with ClustalW [134]

3.3 Discussion

3.3.1 Separation of active enzyme

Foam is a dispersion of gas and liquid, with the volumetric content of gas phase prepon-derating [135]. The two phases are separated by a thin interface region, to which am-phiphilic solutes may adsorb. Many authors postulated, that adsorption of an enzymeto an interface came along with denaturation and thus loss of their enzymatic activi-ty [19, 136]. In the present study, an extracellular cutinase was separated from culturesupernatants of C. cinerea by foam fractionation with negligible loss of enzymatic activity.

3.3.2 Influence of pH value

The hydrophobic properties and therewith the surface-activity and adsorption strength ofproteins is strongly affected by the pH value of the feed solution. A number of scientistsobserved and verified that enrichment and recovery by foam fractionation were best at theisoelectric point (pI) of the target protein [9, 12, 13, 114]. At this pH, the protein carrieszero net charge and the solubility in the aqueous phase is minimal. Furthermore, repulsiveforces between the molecules will be at a minimum. Due to that, the surface adsorptionof the target protein should be enhanced, yielding a high enrichment and purification ofthe protein by foam fractionation [12, 137]. Based on the different isoelectric points ofproteins, they may be fractionated selectively. The effect of pH was studied in the rangeof pH 3.0 to 8.5. The maximum enrichment and recovery were observed at pH 7, close toits pI of 6.7 (see section 3.2.3).

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3 Cutinase from C. cinerea: Screening experiments 29

3.3.3 Influence of gas flow rate

The gas flow rate was identified as another sensible parameter of the foaming process.High yields of activity with an adequate enrichment were achieved at a gas flow rateof 20 mL air min−1. The maximum enrichment was observed at an air flow rate of15 mL min−1 (Fig. 3.3). These results are in good agreement with the observationsof other scientists [9, 14, 71]. At low gas flow rates less liquid is transferred into the foamphase and the residence time of the bubbles in the liquid as well as in the foam is higher.Long residence times enhance drainage and coalescence (enrichment) and offer more timefor adsorption to the gas-liquid interface. High gas flow rates lead to a larger quantity ofgas bubbles, which result in a larger hydrophobic surface. The large surface offers morespace for enzyme adsorption and at the same time more bulk liquid with target and otherproteins is transported into the foam phase. In addition, the period in which the foamremains in the column is reduced, resulting in reduced drainage and coalescence effects.Thus, enrichment decreases and recovery increases with increasing gas flow rates and viceversa.

3.3.4 Influence of additives

Foam inducing additives may be added, if foam formation capacity is limited. Additivesmay be non-ionic, cationic, or anionic tensides. Also various salts have been used asadditives [73]. They reduce the solubility of the proteins and thus enhance their hy-drophobicity. As suggested by Linke [8], the non-ionic detergent Tween 80 was added toincrease the foam formation capacity. Furthermore, Tween 80 was used for the induc-tion of esterolytically active enzymes during fermentation. According to Lockwood et al.[11], the initial population of molecules at the interface depends on several parameters,such as concentration, diffusivity, molecular flexibility and hydrophobicity. Due to theirhigh molecular weights, proteins often slowly adsorb to interfaces. Surface active lowmolecular mass detergents, such as Tween 80 however, adsorb much faster [8, 138]. Thus,adsorption to the air-liquid interface is a time-dependent competition between detergentand protein [11]. If the residence time of the gas bubbles in the liquid phase is too shortfor the target protein to adsorb to the interface or to replace the detergent, the foamingprocess preferably enriches detergent molecules in the foam phase instead of the targetprotein. Considering the low recovery rates but the high foam formation of the detergentexperiment, it is assumed that Tween 80 was enriched into the foam phase instead of thecutinase.

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30 3 Cutinase from C. cinerea: Screening experiments

3.3.5 Comparison to alternative separation processes

Traditional processes for the purification of extracellular cutinases typically consist ofseveral precipitation and chromatography steps. These processes result in high purifi-cation factors, but are accompanied by great losses of enzymatic activity. Gindro andPezet and Sebastian and Kolattukudy, for instance, recovered only 3 % of active enzyme[139, 140]. In addition, these multistage processes are time-consuming, expensive, andrequire several additional filtration, dialysis and centrifugation steps [141, 142]. Withfoam fractionation 79 % of active enzyme in a single purification step was recovered withan enrichment factor of 10.5 within 15 minutes.

3.3.6 Biochemical characterization

By coupling semi-native SDS PAGE and mass spectrometric analyses to the foam frac-tionation procedure, a novel esterase type enzyme was characterized on molecular level.Homology searches with de novo sequenced peptides 1 and 2 returned cutinase typeenzymes from several ascomycetes as the best hits. Surprisingly, the amino acid dataof the peptides did not match the data from C. cinerea genome project and of a re-cently cloned polyesterase capable of suberin and cutin hydrolysis [143]. While limitedconsensus was observable with peptide 2 (Fig. 3.5), no significant homologies were iden-tified with peptide 1. These discrepancies may be caused by inter-strain variations andby the secretion of several isoenzymes.

3.4 Conclusion

Foam fractionation is an efficient method for recovering esterase type enzymes from culturesupernatants of C. cinerea. Beside the preservation of enzymatic activity, no solventsor other substances, which have to be separated in additional work-up steps, had tobe added during foam fractionation process. Further advantages of foam fractionationresult from its mechanical and operational simplicity and therefore, low investment andoperating costs. Nevertheless, various process parameters such as pH value, gas flow rate,temperature, volume ratio of foam to liquid can influence the separation efficiency of foamfractionation. To describe the influence and to gain better insight into the batch foamfractionation process these parameters were investigated statistically in chapter 4.

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4 Statistical investigation of batchfoam fractionationb

In this chapter, foam fractionation was used to purify an extracellular cutinase fromuntreated supernatant of submerged cultures of the basidiomycete Coprinopsis cinerea.Because the foam fractionation process is not completely understood [83], the impactof various process parameters, like pH value or the configuration of the foaming device,was analyzed. To determine the influence of these parameters with regard to separationefficiency, Design of Experiments (DoE) was used. The advantages compared to classical"one factor at a time experiments" were a reduced number of experimental runs and thepossibility to determine impact factors as well as factor interactions. With this approachthe desired information was gained in a minimum of time and costs, respectively.

4.1 Theory

Design of Experiments is a systematic approach to investigate a system or process. Theapproach is to develop an experimental plan in which the input variables (factors) arechanged specifically to observe changes in the output variables (responses). Beside the de-termination of impact factors and interactions, nonlinear relations between the factors andresponses can be identified if special designs are used. An experimental design which ful-fills this specification is the central composite design [144]. The parameters temperature,pH value, gas flow rate, starting volume, foam volume, and frit porosity were investigatedbecause they were considered to have influence on adsorption power and foam stabilityaccording to the explanations in chapter 3. Altogether 6 factors were varied on 5 levelsusing the central composite design (see Table 4.1).

The design consisted of a 26−1 fractional factorial cube, a star and four center points,resulting in 48 experiments. The α value was calculated to ±1.949 [144]. In considerationof the accuracy and adjustability of the used equipment the values for factors A, B andC could slightly differ from the one calculated (Thermostat: 0.1 steps; pH meter: ±0.05;flow meter: 5 mL steps). In case of the candle frits, the commercial sizes P4 (max.

bParts of the chapter are published in Colloids and Surfaces A: Physicochemical and Engineering Aspects382 (2011), 81 -87

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32 4 Statistical investigation of batch foam fractionation

Table 4.1: Factors (A-F) and levels investigated. In brackets: Calculated factor values,which differ from the experimentally used factor values

Factor -α -1 0 1 α

ATemperature 22.6

25.0 27.5 30.032.4

[ °C ] (22.63) (32.37)

BpH value 5.0

6.0 7.0 8.09.0

[ - ] (5.05) (8.95)

CGas flow rate 20

30 40 5060

[ mL min−1 ] (21.51) (59.49)

DFoam volume

65 6590

120 120[ mL ] (92.5)

EStarting volume

90 90 135 180 180[ mL ]

FPore diameter

16 1640

100 100[ μm ] (58)

16 μm), P3 (max. 40 μm) and P2 (max. 100 μm) were used. Other sizes were notavailable. Thus the calculated pore size for level 0 cannot be realized. Additionally, theused glass columns were handmade, thus explaining the difference in factor level 0 to thecalculated value. All experiments were carried out in random order. The enrichmentfactor (EC) and the recovery of active enzyme (R) were used to quantify the efficiency ofthe foam fractionation process and to present the responses in the Design of Experiments(see section 3.1.9).

Because the responses were dependent on more than one factor, a multiple linear regressionwas necessary [144]. The regression quantifies effects, which represent the relation betweenthe factors and the responses. For this purpose, the experimental plan had to be checkedfor orthogonality, because levels ±α for foam volume (D), starting volume (E) and porediameter (F) were face-centered [144]. It could be shown, that the plan was orthogonalin regard to all factors, even for those different from the calculated ones. A single factor,factor interaction or nonlinearity had significant influence on R or EC , if the effect exceededthe 95 % confidence interval and the noise value. The 95 % confidence interval wascalculated via standard deviation of the center points and Student’s distribution [144].The noise of the experiments can be estimated based on higher interactions. Because thefactors D, E and F were face-centered, the significance of their quadratic effects had tobe checked carefully. Additionally, the 95 % confidence interval for these quadratic effectshad to be enlarged [144].

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4 Statistical investigation of batch foam fractionation 33

Table 4.2: Experimental plan in systematic order and experimental data for the responsesR and EC . A: Temperature, B: pH value, C: Gas flow rate, D: Foam volume, E: Startingvolume, F: Pore diameter

No Factor A Factor B Factor C Factor D Factor E Factor F R EC

1 -1 -1 -1 -1 -1 -1 76.5 3.3

2 1 -1 -1 -1 -1 1 27.3 20.2

3 -1 1 -1 -1 -1 1 53.5 9.5

4 1 1 -1 -1 -1 -1 72.0 2.8

5 -1 -1 1 -1 -1 1 33.5 6.6

6 1 -1 1 -1 -1 -1 70.5 2.2

7 -1 1 1 -1 -1 -1 76.0 1.8

8 1 1 1 -1 -1 1 30.8 7.0

9 -1 -1 -1 1 -1 1 19.4 10.8

10 1 -1 -1 1 -1 -1 52.9 3.2

11 -1 1 -1 1 -1 -1 93.5 4.2

12 1 1 -1 1 -1 1 48.9 17.4

13 -1 -1 1 1 -1 -1 76.2 2.3

14 1 -1 1 1 -1 1 23.9 7.5

15 -1 1 1 1 -1 1 67.4 6.8

16 1 1 1 1 -1 -1 69.8 2.2

17 -1 -1 -1 -1 1 1 3.0 10.6

18 1 -1 -1 -1 1 -1 68.6 3.1

19 -1 1 -1 -1 1 -1 91.6 3.0

20 1 1 -1 -1 1 1 38.9 20.0

21 -1 -1 1 -1 1 -1 69.6 1.9

22 1 -1 1 -1 1 1 13.7 7.4

23 -1 1 1 -1 1 1 77.6 5.8

24 1 1 1 -1 1 -1 81.0 2.0

25 -1 -1 -1 1 1 -1 84.9 3.7

26 1 -1 -1 1 1 1 23.5 16.8

27 -1 1 -1 1 1 1 64.3 10.6

28 1 1 -1 1 1 -1 63.1 2.8

29 -1 -1 1 1 1 1 43.0 4.0

30 1 -1 1 1 1 -1 92.8 2.9

31 -1 1 1 1 1 -1 100.0 1.5

32 1 1 1 1 1 1 56.0 9.6

33 -1.949 0 0 0 0 0 84.9 3.7

34 1.949 0 0 0 0 0 86.2 4.3

35 0 -1.949 0 0 0 0 78.4 4.1

36 0 1.949 0 0 0 0 72.1 3.4

37 0 0 -1.949 0 0 0 75.2 10.8

38 0 0 1.949 0 0 0 80.3 2.7

39 0 0 0 -1 0 0 89.9 3.0

40 0 0 0 1 0 0 88.5 4.4

41 0 0 0 0 -1 0 87.5 4.6

42 0 0 0 0 1 0 88.2 4.5

43 0 0 0 0 0 -1 80.3 3.8

44 0 0 0 0 0 1 33.6 9.6

45 0 0 0 0 0 0 92.7 4.3

46 0 0 0 0 0 0 86.3 4.2

47 0 0 0 0 0 0 82.7 3.9

48 0 0 0 0 0 0 93.6 4.6

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34 4 Statistical investigation of batch foam fractionation

4.2 Materials and methods

In comparison to the cultivation procedure in chapter 3, section 3.1.3, day 3 was chosenfor harvesting the culture supernatant. Foam fractionation experiments (compare centerpoint experiments in Table 4.2 with Fig. 3.2) and SDS-PAGEs showed, that using culturesupernatant of day 3 or day 6 did not influence the performance. However, the fermenta-tion period could be reduced drastically. The esterolytic activity was quantified accordingto the method described in chapter 3.1.5. The batch foaming device (Fig. 3.1a)) with-out glass bowl was chosen for the batch foam fractionation experiments and the sameprocedure was applied.

4.2.1 Chemicals

EDTA (≥ 99 %), gum Arabic (from acacia tree), magnesium sulfate anhydrous (99 %),p-nitrophenyl palmitate (≥ 98 %), and sodium deoxycholate (≥ 98 %) were purchasedfrom Sigma-Aldrich (Seelze, Germany). Agar-agar (Kobe), ammonium nitrate (≥ 99 %),hydrochloric acid (37 %), calcium chloride (CaCl2 · 2H2O, ≥ 99 %), copper sulfate(CuSO4 ·5H2O, ≥ 99 %), ferric chloride (FeCl3 ·6H2O, ≥ 97 %), isopropanol (hplc grade),malt extract (96.5 - 98 %), potassium dihydrogen phosphate (≥ 99 %), di-potassiumhydrogen phosphate (K2HPO4 · 3H2O, ≥ 99 %), sodium chloride (≥ 99.8 %), sodiumhydroxide (≥ 99 %), di-sodium hydrogen phosphate (≥ 93 %), Tween 80 (ph. Eur.), andzinc sulfate (ZnSO4 · 7H2O, ≥ 99 %) were purchased from Roth (Karlsruhe, Germany).Substances were used as received.

4.3 Results

According to section 4.1, the effects for each single factor, factor interaction and non-linearity were calculated based on multiple linear regression. It could be verified, that theexperimental plan depicted in Table 4.1 was orthogonal respective to all factors and therewas no drift in the experiments. The effects calculated are depicted in Figure 4.1.

A positive effect of a factor X means, that an increase of factor X from level -1 to 1increases the response. For example, the increase of the pore diameter (F) from 16 μmto 100 μm enhanced the enrichment factor about 7.8 on average. In case that the changefrom level -1 to 1 causes a negative effect, the response decreased. For instance, the sameincrease in pore diameter (F) decreased the recovery of activity about 41.1 % on average(see Fig. 4.1a), 4.1b) ).

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4 Statistical investigation of batch foam fractionation 35

-5

-3

-1

1

3

5

7

9

A B C D E F AA BB CC DD EE FF AB AC AD AE AF BC BD BE BF CD CE CF DE DF EF

Effe

ct o

f EC

Factors

95% confidence interval Noise a)

-60

-50

-40

-30

-20

-10

0

10

20

A B C D E F AA BB CC DD EE FF AB AC AD AE AF BC BD BE BF CD CE CF DE DF EF

Effe

ct o

f R

Factors

95% confidence interval Noise b)

Figure 4.1: a) Effects for the response EC , b) Effects for the response R. Black columnsshow significant, gray columns insignificant effects. A: Temperature, B: pH value, C:Gas flow rate, D: Foam volume, E: Starting volume, F: Pore diameter

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36 4 Statistical investigation of batch foam fractionation

A significant quadratic effect indicates a nonlinearity of the response at the respectivefactor range. Sometimes quadratic effects can be significant even if the factor itself isnot. The explanation for this is, that the average response values for the factor levels -1and 1 cancel each other out. Thus, the calculated single effect does not exceed the 95 %confidence interval, but is significant for further considerations. An example can be seenin Figure 4.1b), where factor CC was significant for response R, while C was not.

A factor interaction XY is the average difference of the influence of factor X at high andlow levels of factor Y. If an effect like XY is significant, both factors are interdependent.Thus, the combination of X and Y has to be regarded for further considerations. Evenif factors X or Y of the interaction XY are not significant themselves, they have to beconsidered as significant due to their dependence on each other. An example is the effectBE, which was significant for the enrichment factor, while the single factor B was not.

When all effects were checked for significance, the regression models can be formed foreach response. The resulting models, which only contain significant effects, can be used forprediction and optimization of the foam fractionation process. However, these statementsare only valid in the range of the factors investigated. The resulting regression modelsare shown in equations (4.1) and (4.2).

EC = 2.44 + 1.05 · xA − 2.18 · xC + 3.92 · xF + 0.93

·xC · xC + 1.78 · xE · xE + 2.22 · xF · xF − 0.64

·xA · xC + 1.3 · xA · xF + 0.29 · xB · xE − 1.61

·xC · xF

(4.1)

R = 94.05− 5.85 · xA + 8.36 · xB − 20.56 · xF − 5.97

·xB · xB − 5.29 · xC · xC − 25.47 · xF · xF − 5.26

·xA · xB + 4.91 · xB · xF(4.2)

The quadratic effects DD and EE for response R and DD for EC were regarded as notsignificant (see section 4.1). By setting these factors into the regression models, thedifferences between experimental data and theoretical model grew. Additionally, thestability indexes of the models exceed 100 %. The assumption to dismiss these quadraticeffects was justified, because factor levels D and E were face-centered (see section 4.1).Thus, the confidence interval was enlarged for these factors [144]. Because the calculationof the widened confidence was not feasible in this study, the standard 95 % confidenceinterval was depicted in Figure 4.1 and the significance of the face-centered factors D, Eand F was checked logically in regard to the influence on foam fractionation and to the

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4 Statistical investigation of batch foam fractionation 37

regression model.

The stability indexes for the regression models were 99 % for EC and 85 % for R. Onthe basis of these models the optimal factor values for each response could be calculated.First, the interactions were determined in regard to optimal level combinations usingcontour plots (examples shown in Fig. 4.2). The contour plots were generated using theregression models for R or EC , whereas only the factors of the determined interaction werevariables and all other factors were held constant at the mean level. For interaction AC,for instance, the regression model of EC was only dependent on factor A (xA) and C (xC).The resulting contour lines were curves along which the regression model had constantvalues. Second, the levels of single factors, which were not involved in interactions wereset to optimize R or EC . It should be noted that the levels of factors D, E and F werevaried in the range of -1 to 1. Additionally, R should not exceed 100 % and the responseswere not allowed to become negative, especially if setting the optimal level combinationfor the response R, for instance, into the regression model of EC .

In Table 4.3 the optimized level combination for each response is depicted. Insignificantfactors were held constant at the average factor level (0).

Table 4.3: Calculated factor levels for the optimization of the responses R and EC

Factor Level Level value for R Level Level value for EC

xA -1 25.0 °C +α 32.4 °C

xB 0 7.0 +1 8.0

xC 0 40.0 mL min−1 -α 20.0 mL min−1

xD 0 90 mL 0 90 mL

xE 0 135 mL +1 180 mL

xF 0 40 μm +1 100 μm

Inserting the factor levels depicted in Table 4.3 into the regression models a maximalrecovery of 99 % active enzyme (EC = 1.4) or a maximal enrichment factor of 28.6(R = 13.6 %) was predicted. Experiments with these level combinations yielded an ac-tivity recovery of 99 % with an enrichment factor of 1.5 or a maximal enrichment factorof 28.2 with a recovery of 12 % active enzyme. Altogether, the results were in very goodagreement with the regression models. If both responses should be optimized at the sametime, a pareto plot and contour plots can be used. With the help of such plots the optimalcombination of recovery and enrichment can be adjusted for each requirement. Neverthe-less, it has to be pointed out, that the values used in the contour plots as well as in thepareto plot were only predicted due to the regression models and more than one level com-bination could yield the same enrichment or recovery. Considering the pareto plot (Fig.

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38 4 Statistical investigation of batch foam fractionation

2

2

2

2

4

4

44

6

6

66

8

88

10

1012

2

Temperature (A)

-1 0 1

Gas

flow

rate

(C)

-1

0

1

4,54,0

4,04,03,53,5

3,53,53,0

3,03,0 3,0

2,52,52,5

2,5

2,52,5

2,52,5

3,0 3,03,0

3,03,53,5

3,53,5

4,04,0

4,04,5

pH value (B)

-1 0 1

Star

ting

volu

me

(E)

-1

0

1

a)

4

4

4

4

4

22

2

2 2

6

6

6

68

8

810

10

10

12

1214

16

Gas flow rate (C)

-1 0 1

Pore

dia

met

er (F

)

-1

0

1

12

10

10

8

8

6

6

6

4

4

4

4

22

2

2

Temperature (A)

-1 0 1

Pore

dia

met

er (F

)

-1

0

1

b)6060

60

60

7575

75

90

90 90

90

90

90

90

45

45

75

30

75

75

60

15

pH value (B)

-1 0 1

Pore

dia

met

er (F

)

-1

0

1

1.949

1.949 1.949

1.9491.949

1.949

-1.949

-1.949 -1.949

-1.949-1.949-1.949

70

80

80

80 80 80 80

70 70 70 7060 60 60

90 9090

90

90

100

100

100

50

Temperature (A)

-1 0 1

pH v

alue

(B)

-1

0

1

1.949

1.949-1.949

-1.949

Figure 4.2: Contour plots for the factor interactions of a) EC and b) R

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4 Statistical investigation of batch foam fractionation 39

4.3) a recovery of 99 % with an achievable enrichment factor of 8.0 could be obtained. Togain these values the level combination: Temperature: -α, pH value: +α, gas flow rate:-α, foam volume and frit porosity: 0, starting volume: +1 need to be determined. Theassociated experiments yielded an enrichment factor of 8.2 with an activity recovery of 97%. Thus, a targeted optimization of both responses was possible.

0

5

10

15

20

25

30

0 20 40 60 80 100

Enri

chm

ent f

acto

r (E C

)

Recovery of active enzyme (R)

Figure 4.3: Pareto chart for the dependencies of R and EC

4.4 Discussion

For response R temperature, pH value, gas flow rate as well as pore diameter of thefrit were of main importance. The factors pH value (B), gas flow rate (C) and poresize of the frit (F) showed significant quadratic effects, implying nonlinear dependencies.Additionally, the factor pH value had significant interactions with temperature and fritpore size. The enrichment factor, however, was affected by effectively all investigatedfactors. Here the factors gas flow rate (C), starting volume (E) and pore diameter (F)have shown significant quadratic effects or rather nonlinear behavior. The temperature(A) had significant interactions with the gas flow rate (C) and the pore diameter (F),additionally the gas flow rate (C) interacted with the pore diameter (F), and the pHvalue (B) with the starting volume (E).

4.4.1 pH dependence (A)

The surface-activity or hydrophobicity, which is a measure of adsorption strength, isstrongly affected by the pH value of the bulk solution. Previous studies observed andverified that stable foams and high enrichments were obtained at the isoelectric point[9, 12, 13]. At pH = pI, the protein carries zero net charge and the solubility in theaqueous bulk solution is minimal. Thus, the surface adsorption of the desired protein

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40 4 Statistical investigation of batch foam fractionation

appears to be enhanced, yielding concentration and purification [12, 137]. Nonetheless,the enrichment was not significantly affected by pH, only the interaction BE (pH valuewith starting volume) was significant. The recovery of active enzyme, however, showedeven nonlinear behavior for the pH value. On the basis of the regression models theoptimal pH value for EC was calculated to pH = 8.0 and for R to pH = 7.0. Consideringthe contour plots (interaction BE in 4.2 a)) a pH value of pH = 7.0 resulted in the sameenrichment factor. Thus, the optimal pH value for recovery and enrichment was set topH = 7.0, close to the isoelectric point of the used enzyme cutinase (pI = 6.7).

4.4.2 Temperature dependence (B)

The impact of temperature on foam fractionation has rarely been discussed. Liu et. alfound no influence of the temperature on the foaming process [145]. Here, temperaturesignificantly influenced enrichment as well as recovery. High temperatures enhanced theenrichment and low temperatures the recovery of active enzyme. It can be expected, thatthe viscosity of the bulk solution decreases with higher temperatures. It is tempting toconclude, that the decrease of viscosity causes a decrease of foam stability and therewiththe decrease of liquid hold-up in the foam yielding a higher enrichment factor. At lowtemperatures the viscosity increases decreasing the drainage effects. Thus, the liquidhold-up is higher and adsorbed as well as free cutinase (bulk solution) are carried into thefoam phase resulting in a higher recovery.

4.4.3 Gas flow rate (C)

Gas flow rate was identified as another sensitive factor for foam fractionation. Gas flowrate together with column design limits the residence time of the bubbles. Thus, the timefor the adsorption process as well as for drainage of excess liquid is limited. Long residencetimes or rather low gas flow rates enhance drainage effects, resulting in drier foams, andoffering more time for adsorption to the gas-liquid interface. The disadvantage of low gasflow rates is the increase in process time. High gas flow rates lead to a larger quantity ofgas bubbles, offering a larger hydrophobic surface. At the same time the residence timeof the foam is reduced, resulting in decreasing drainage effects and more bulk liquid inthe foam phase. Thus, enrichment decreases and recovery increases with increasing gasflow rates and vice versa. For both responses the effect of gas flow rate was quadratic.The recovery of active enzyme had its maximum at a gas flow rate of 40 mL min−1. Theenrichment factor, however, had its maximum value at low gas flow rates.

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4 Statistical investigation of batch foam fractionation 41

4.4.4 Foam volume (D)

Brown et al. postulated, that enrichment factors increase as foam volume increases. Thisis a result of longer drainage times and increased coalescence. The lower liquid hold-upin the foam promotes higher protein concentrations, resulting in increased enrichmentfactors [14]. However, the foam volume had no significant effect. Thus, the foam volumewas set to the mean level (0) for the optimization of the responses.

4.4.5 Starting volume (E)

High starting volumes extend residence time of the bubbles in the bulk liquid. Thus,the concentration of surface-active molecules at the gas-liquid interface can approachequilibrium resulting in higher recovery and enrichment. In this case, the starting volumehad a significant positive quadratic effect on the enrichment. Considering the contourplots, the minimum of enrichment could be observed at the mean level (0). At levels �= 0the enrichment increased equally. In case of recovery of activity, the effect of the startingvolume was not significant but positive. Thus, the starting volume could be set to level+1 (180 mL) allowing more time for adsorption favoring enrichment and recovery.

4.4.6 Pore diameter (F)

Pore diameter or rather bubble size is one of the most important factors to control theefficiency of the foam fractionation process. Smaller bubbles increase the interfacial areaavailable for protein adsorption, but cause a higher liquid hold-up resulting in higherrecoveries. Bigger bubbles, however, have less available interfacial area, but cause lessliquid hold-up improving enrichment. Thus, considering the optimization of enrichmentand recovery at the same time, a compromise has to be accepted. Good results forenrichment as well as recovery were observed by setting the pore diameter to the mean level(16 μm). Additional to the pore size of the sparger type, the bubble size is also affectedby surfactants (including proteins), pH (see interaction BF in Fig. 4.1 b)), temperatureand gas flow rate (see interactions AF and CF in Fig. 4.1 a)).

4.5 Conclusion

It has been shown, that Design of Experiments can be used to determine the factorsinfluencing foam fractionation of cutinase. Basically, all factors investigated had an in-fluence on the enrichment and recovery of active enzyme. Only foam volume (D) did not

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42 4 Statistical investigation of batch foam fractionation

have an impact on the responses and starting volume (E) influenced the enrichment ofactive enzyme only. Based on the regression models, which were developed by multiplelinear regression, the contour plots and the pareto chart the enrichment factor and theactivity recovery were predicted and optimized. Thus, using Design of Experiments batchfoam fractionation can be tailored efficiently and fast to different separation requirements.Because the flow capacity can be increased using continuous foam fractionation the batchmode was adapted to continuous foam fractionation. In chapter 5 significant operatingparameters as determined in this chapter are investigated to compare their influence onbatch and continuous operation.

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5 Statistical investigation of continuousfoam fractionationc

To make foam fractionation competitive to other purification strategies it is necessaryto increase the flow capacity of the process. Therefore, the foam fractionation processwas operated continuously and several process parameters, like pH value or gas flow ratewere investigated systematically. In general, according to chapter 2 continuous foamfractionation can either be operated in stripping mode (Fig. 5.1a)) or in enriching mode(Fig. 5.1b)) [16]. In enriching mode, the feed enters the column in the liquid pool. Thus,drainage occurs in the foam over the whole foam column. Due to drainage the excessliquid between the bubbles flows back to the liquid pool of the column resulting in a higherconcentration of the target molecule [16]. However, the formation of stable foams is a basicrequirement. In stripping mode, the feed enters the column at the top of the foam columnand trickles down through the rising foam [16]. Due to this counter-current stream of foamand fresh feed, bulk surface-active molecules are present in the whole foam phase which canadsorb to free adsorption places and stabilizing the foam. Thus, stripping mode offers newalternatives for diluted and low surface-active systems, which do not form stable foamsfor continuous foam fractionation in enriching mode (Fig. 5.1b)). In literature, strippingmode has rarely been discussed. Talmon and Rubin as well as Kinoshita et al. [146, 147]studied the separation of dyes or metals using continuous foam fractionation in strippingmode. Oka et al. and Meikap et al. [119, 120] studied the feasibility of counter-currentfoam fractionation in coiled columns or multistage reactors, respectively. However, theaim of these studies was to prove feasibility and not the systematic determination ofimportant operating parameters. To control foam fractionation in stripping mode, theinfluencing parameters, their interactions, and nonlinearities need to be investigated. Todetermine the influence of parameters like pH value or feed flow rate on the separationefficiency systematically, Design of Experiments (DoE) was used. Such studies of foamfractionation in stripping mode with the feed port at the top of the foam column involvingDoE do not yet exist.

cParts of the chapter have been published in Separation and Purification Technology 82 (2011), 10-18

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44 5 Statistical investigation of continuous foam fractionation

Gas

Flow meter

Low-pressure

Foamate

Feed

Retentate

Porous frit

stripping mode

a)

Gas

Flow meter

Low-pressure

Foamate

Retentate

Feed

Porous frit

enriching mode

b)Figure 5.1: Continuous foam fractionation column in a) stripping mode and b) enriching

mode

5.1 Material and methods

The chemicals used were equal to the chemicals used in chapter 4. The production of theculture broth containing cutinase was carried out as described in section 4.2. the activityassay described in chapter 3 was modified to increase the . A Soerensen phosphate bufferof pH 7.7 by mixing solution A (8.9 g L−1 Na2HPO4 · 2H2O) and solution B (6.8 g L−1

KH2PO4) in a ratio of 7.4:1. A 1 mL aliquot of freshly prepared substrate solution wasmixed with 42 μL culture supernatant or water (blank) and incubated for 15 minutesat 26 °C and 650 rpm in a Thermomixer comfort (Eppendorf AG, Hamburg, Germany).After incubation, the absorbance was measured at 410 nm. One enzyme unit U wasdefined as one μmol p-nitrophenol enzymatically released from the substrate per minute.Under the conditions described, the extinction coefficient of p-nitrophenol was determinedto be ε410nm= 0.028 L (μmol cm)−1. All samples were analyzed at least as a duplicate.

5.1.1 Surface tension

Surface tension of the culture supernatants was determined using the ring-tensiometerK10ST (Krüss GmbH, Hamburg, Germany). Approximately 20 mL of pH and temperatureadjusted culture supernatant was filled into the test vessel and surface tension was mea-sured according to the method of Lecomte Du Noüy [148]. Temperature and pH were

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5 Statistical investigation of continuous foam fractionation 45

varied according to the levels of the experimental plan. All samples were analyzed atleast as a duplicate.

5.1.2 Configuration of equipment and experimental procedure for

foam fractionation

SetupFor continuous foam fractionation the device consisted of a liquid column for the feedsolution (length = 18 cm, diameter = 3 cm) with a porous glass frit at the bottom, a foamcolumn (length = 56 cm, diameter = 1.6 cm), a horseshoe bend, and a receiving flask. Afeed inlet (4.5 cm from top of the foam column) and one outlet (1.0 cm from the bottomof the column) for the depleted liquid were placed at the glass column (Fig. 5.1a)). Tomaintain a constant temperature during the experiments, the column was jacketed (tem-perature adjustment using thermostat; cooling/heating medium H2O). Additionally, thefeed solution was preheated to the desired temperature. The experiments were started bypumping enzyme solution into the foam fractionation column till a height of 18 cm fromthe bottom was reached, and the feed flow rate according to the experimental design wasadjusted. The liquid height was kept constant throughout the experiments by manuallyadjusting the speed of the peristaltic pump (Watson Marlow, Rommerskirchen, Germany)used to withdraw the depleted liquid from the bottom of the column. Air as foaming gaswas passed through the porous frit. The emerging foam left the column through a horse-shoe bend, was collapsed at reduced pressure, and collected in the receiving flask. Thisfoam breaking method is conveniently used in laboratory scale setups and well documentedin literature [17, 79]. Samples were taken for activity assays from the feed solution, thedepleted liquid (retentate), and the collapsed foam (foamate). The retentate and foamatesamples were taken after stationary operation had been achieved.

Steady state operationRegarding the experimental design, the combinations of the lowest and highest feedflow rate (5 mL min−1; 15 mL min−1) with the two lowest gas flow rates (28 mL min−1;40 mL min−1) were tested, while the other parameters were kept constant (pH 7.0, 25.0 °C,and a frit with a pore size of 40 μm (P3)). Each 5 to 10 minutes, a sample was taken fromthe flowing out retentate stream and analyzed over a period of up to 90 minutes. Steadystate operation was achieved when the enzymatic activity and the flow of the retentatestream were constant.

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46 5 Statistical investigation of continuous foam fractionation

5.1.3 Determination of liquid holdup (ϕ)

The liquid holdup in the foam column was measured to characterize the average liquidcontent of the foam phase. The liquid holdup ϕ should not be used for the optimiza-tion of the foam fractionation process but should give explanations for differences infoam structure and drainage behavior. In order to determine the liquid holdup, thewhole foam fractionation column including the horseshoe bend was emptied and the foamwas collapsed at the end of each experiment. The resulting liquid fraction was namedVtotalliquid. With this value, the liquid volume in the foam Vfoamliquid can be determinedby the following equation 5.1:

Vfoamliquid = Vtotalliquid − Vliquidcolumn (5.1)

The foam column volume (Vfoamcolumn) and liquid column volume (Vliquidcolumn) wereconstant during all experiments, as the same column was used and the liquid height waskept constant for each experiment. Thus, the liquid holdup ϕ can be defined as the ratioof liquid volume in the foam Vfoamliquid to the volume of the foam column Vfoamcolumn

(equation 5.2):

ϕ =Vfoamliquid

Vfoamcolumn(5.2)

5.1.4 Design of Experiments (DoE)

For the systematic investigation of continuous foam fractionation, a Design of Experimentswas used. An experimental plan was developed in which the input variables (factors) werechanged specifically to observe changes in the output variables (responses). Beside thedetermination of impact factors and interactions, nonlinear relations between the factorsand responses were identified using a central composite design [144]. The parameterspH value, temperature, feed flow rate, gas flow rate, and frit pore size were investigated.These parameters were considered to have influence on adsorption strength and foamstability. Altogether, 5 factors were varied on 5 levels using the central composite design(Table 5.1).

The design consisted of a 25−1 fractional factorial cube, a star and 15 center points,resulting in 41 experiments. The α value was calculated to ± 2.19 [144]. In considerationof the adjustability of the used equipment, the values for the factors feed and gas flow rate

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5 Statistical investigation of continuous foam fractionation 47

Table 5.1: Factors (A - E) and levels investigated. In brackets: Calculated factor values,which differ from the factor values used in the experiments

Factor -α -1 0 1 α

ApH value

4.8 6.0 7.0 8.0 9.2[ - ]

BTemperature

22.0 25.0 27.5 30.0 33.0[ °C ]

CFeed flow rate 5.0 7.74 10.0 12.29 15.0

[ mL min−1 ] (5.07) (7.78) (9.96) (12.22) (15.03)

DGas flow rate 30

40 50 6070

[ mL min−1 ] (28) (72)

FPore diameter

16 1640

100 100[ μm ] (58)

(D) slightly differed from the calculated data (pump: 0.54 mL min−1, flow meter: 5 mLsteps). In case of the candle frits, the commercial sizes P4 (16 μm), P3 (40 μm) and P2(100 μm) were used. Different sizes were not available. Thus, the calculated pore size forlevel 0 could not be realized.

The fermentation of C. cinerea for cutinase production was limited to 7 L per week. Thebroth was stored at 4 °C (stable for 7 days), as freezing decreased the enzymatic activityof cutinase to 60 %. To realize the experimental plan, 4 to 6 randomized experimentswere arranged in a block. For each block, three center points were performed. Due tothis experimental procedure, differences between the fermentation batches could be recog-nized and interpreted. Additionally, the standard deviation was calculated via the centerpoints. Thus, the natural variation of the feed composition was taken into account. Todescribe the efficiency and quality of the foam fractionation process, the enrichment fac-tor (EC), the recovery of active enzyme (R), and the liquid holdup (ϕ) were used andrepresented the responses in the Design of Experiments. The relative liquid holdup wasused to determine the influence of different process parameters on foam wetness only. Toquantify the effects representing the relations between the factors and the responses, amultiple linear regression was used [144]. Before starting the experimental investigation,the orthogonality of the experimental plan was checked, because the factor E, i.e. porediameter, was face-centered. The examination showed that the plan was orthogonal con-cerning all factors. An effect had a significant influence on the responses EC , R, or ϕ if itexceeded a confidence interval higher than the noise value. The confidence intervals werecalculated via standard deviation of the center points and Student’s distribution [144].The noise of the experiments was estimated by three way factor interactions.

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48 5 Statistical investigation of continuous foam fractionation

5.2 Results

5.2.1 Configuration of equipment

The variation of feed and gas flow rates resulted in a stable retentate enzyme activityafter approximately 40 minutes in each case, as can be seen in figure 5.2. As a precaution,all foam fractionation experiments were initially run for 60 minutes. This way, stationaryoperation was ensured. After 60 minutes, the receivers for foamate and retentate werechanged and the experiments were performed for approximately 10 minutes collectingfoamate and retentate.

0

10

20

30

40

50

0 20 40 60 80 100

Act

ivity

rete

ntat

e [U

L-1

]

Time [min]

Figure 5.2: Activity in the retentate over time. (� gas flow rate 28 mL min−1, feed flowrate 15 mL min−1; � gas flow rate 28 mL min−1, feed flow rate 5 mL min−1; • gas flowrate 40 mL min−1, feed flow rate 15 mL min−1; � gas flow rate 40 mL min−1, feed flowrate 5 mL min−1).

5.2.2 Design of Experiments

For completeness, the Design of Experiments and the data for the resulting responseswere listed in Table 5.2 in regular order. Experiments number 28 and 29 are missing,because the experiments failed.

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5 Statistical investigation of continuous foam fractionation 49

Table 5.2: Experimental plan in systematic order and experimental data for the responsesEC , R, and ϕ. A: pH value, B: Temperature, C: Feed flow rate, D: Gas flow rate, E:Pore diameter

No Factor A Factor B Factor C Factor D Factor E EC R ϕ

1 -1 -1 -1 -1 1 3.78 22.2 0.05

2 1 -1 -1 -1 -1 1.59 92.2 0.38

3 -1 1 -1 -1 -1 1.63 77.7 0.34

4 1 1 -1 -1 1 2.27 9.6 0.23

5 -1 -1 1 -1 -1 1.48 93.7 0.34

6 1 -1 1 -1 1 1.56 7.3 0.06

7 -1 1 1 -1 1 1.80 7.0 0.20

8 1 1 1 -1 -1 1.31 124.0 0.25

9 -1 -1 -1 1 -1 1.70 96.4 0.17

10 1 -1 -1 1 1 3.36 22.5 0.34

11 -1 1 -1 1 1 5.47 36.6 0.12

12 1 1 -1 1 -1 1.45 71.3 0.78

13 -1 -1 1 1 1 1.31 9.0 0.09

14 1 -1 1 1 -1 1.03 100.4 0.31

15 -1 1 1 1 -1 1.37 69.8 0.79

16 1 1 1 1 1 1.72 12.7 0.41

17 -2.1923 0 0 0 0 2.09 89.2 0.24

18 2.1923 0 0 0 0 3.29 82.2 0.20

19 0 -2.1923 0 0 0 2.03 101.4 0.28

20 0 2.1923 0 0 0 1.45 86.0 0.41

21 0 0 -2.1923 0 0 4.77 114.2 0.41

22 0 0 2.1923 0 0 1.86 76.6 0.86

23 0 0 0 -2.1923 0 1.63 64.5 0.22

24 0 0 0 2.1923 0 1.11 64.4 0.15

25 0 0 0 0 -1 2.39 97.7 0.17

26 0 0 0 0 1 3.11 13.4 0.10

27 0 0 0 0 0 1.40 79.1 0.18

30 0 0 0 0 0 2.44 90.4 0.30

31 0 0 0 0 0 2.36 99.3 0.19

32 0 0 0 0 0 2.66 100.0 0.17

33 0 0 0 0 0 2.41 76.4 0.18

34 0 0 0 0 0 2.46 67.6 0.25

35 0 0 0 0 0 2.50 62.4 0.16

36 0 0 0 0 0 1.72 86.0 0.28

37 0 0 0 0 0 2.79 100.0 0.20

38 0 0 0 0 0 2.29 100.0 0.44

39 0 0 0 0 0 1.51 96.2 0.34

40 0 0 0 0 0 1.32 91.8 0.25

41 0 0 0 0 0 1.70 89.3 0.41

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50 5 Statistical investigation of continuous foam fractionation

The change in the α value to ± 2.08 was considered to be neglectable (see Table 5.3,because of the accuracy and adjustability of the used equipment(Thermostat: 0.1 °Csteps; pump: 0.54 mL min−1; flow meter: 5 mL steps). The face-centered factor porediameter (F) was not affected by the change of α. Additional star experiments with thenew α value for factor pH (A) were carried out and the results did not differ from theones obtained during the experimental plan.

Table 5.3: Factor values for α = 2.08 for the Factors (A - D)

Factor -α α

ApH value

4.9 9.1[ - ]

BTemperature

22.3 32.7[ °C ]

CFeed flow rate

5.228 14.75[ mL min−1 ]

DGas flow rate

29 71[ mL min−1 ]

The effects of single factors, factor interactions and nonlinearities were calculated andare depicted in figure 5.3a) - c) regarding EC , R, and ϕ. A positive effect of a singlefactor X means, that an increase of factor X from level -1 to +1 increases the response.The increase of pore diameter (E) from 16 μm to 100 μm increased EC by a value of 1.2on average (Fig. 5.3a)). In case that the change from level -1 to +1 causes a negativeeffect, the response decreased. For instance, the same variation in pore diameter (E)decreased the recovery of active enzyme about 76 % on average (Fig. 5.3b)). Significantquadratic effects XX indicate a nonlinear behavior of the response in the respective factorrange. That means the change of the response value is not linear to the changes of thefactor levels made. Thus, the value of the response could achieve a local maximum orminimum or be an asymptotic value. Quadratic effects can be significant, while the singleeffect X does not necessarily need to be significant. For instance, the quadratic effectof the factor gas flow rate (DD) for R was significant, while the single factor D was not(Fig. 5.3b)). A factor interaction XY is the average difference in influence of factor Xat levels -1 and +1 of factor Y. If a factor interaction XY is significant, both factors areinterdependent. Thus, both factors have to be considered, although their single effectsmight not be significant. Factor interaction pH and temperature (AB) for the response EC ,for instance, was significant, while both single effects were not (Fig. 5.3a)). Significantinteractions are important in establishing process variables for the optimization of thefoam fractionation performance. As the factor E was face-centered (section 5.1.4), the

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5 Statistical investigation of continuous foam fractionation 51

-1.5

-1

-0.5

0

0.5

1

1.5

A B C D E AA BB CC DD EE AB AC AD AE BC BD BE CD CE DE

Effe

ct E

C

95% confidence interval Noise a)

-80

-60

-40

-20

0

20

A B C D E AA BB CC DD EE AB AC AD AE BC BD BE CD CE DE

Effe

ct R

95% confidence interval Noise b)

-0.3

-0.2

-0.1

0

0.1

0.2

A B C D E AA BB CC DD EE AB AC AD AE BC BD BE CD CE DE

Effe

ct

Factors

99% confidence interval Noise c)

Figure 5.3: Effects for responses a) EC , b) R and c) ϕ. Black columns show significant,gray columns insignificant effects. A: pH value, B: Temperature, C: Feed flow rate, D:Gas flow rate, E: Pore diameter

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52 5 Statistical investigation of continuous foam fractionation

significance of the related quadratic effect had to be considered carefully. A nonlinearrelation between factor pore diameter (E) and the response R was observed during theexperiments of the statistical plan. Thus, the quadratic effect of E for response R wasstated as significant. After identifying significant effects for each response, regressionmodels were formed (see equations 5.3, 5.4, 5.5).

EC = 2.15− 0.63 · xC + 0.58 · xE + 0.21 · xC · xC−0.20 · xD · xD − 0.17 · xA · xB + 0.22 · xA · xC−0.17 · xA · xE + 0.25 · xB · xD − 0.22 · xC · xD−0.46 · xC · xE + 0.18 · xD · xE

(5.3)

R = 91.64− 37.93 · xE − 7.81 · xD · xD − 32.98 · xE · xE+6.40 · xA · xE − 4.008 · xC · xD − 6.58 · xC · xE+5.28 · xD · xE

(5.4)

ϕ = 0.24 + 0.064 · xB + 0.041 · xC + 0.039 · xD−0.107 · xE + 0.078 · xC · xC − 0.09 · xA·xC + 0.042 · xA · xD + 0.061 · xB · xD

(5.5)

On the basis of the regression models, the performance of each experiment in the in-vestigated range could be predicted. An overview of all possible level combinations for

0

2

4

6

8

10

0 20 40 60 80 100

Enri

chm

ent f

acto

r (E C

)

Recovery of activity (R)

Figure 5.4: Possible combinations for responses EC and R based on the regression models

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5 Statistical investigation of continuous foam fractionation 53

recovery and enrichment factor on basis of the models is displayed in a pareto plot (Fig.5.4). The factor levels were chosen as discrete and not as continuous values, because notall factors were arbitrary. Figure 5.4 shows that recoveries of almost 99 % active enzyme(EC = 5.1) are possible. Also a maximal enrichment factor of 9.4 is predicted, albeitat lower recovery of 82 %. The level combinations predicted to aim maximal recoveryof active enzyme or maximal enrichment are depicted in Table 5.4. The correspondingexperiments with factor levels depicted in table 5.4 yielded an activity recovery of 98 %with an enrichment factor of 5.6, or a 9.8 fold concentration of active enzyme with arecovery of 79 %. These experimental values show, that an accurate prediction can beaccomplished by the developed models.

Table 5.4: Calculated factor levels for the maximal responses R and EC

Variables Level Value for maximal R Level Value for maximal EC

xA -2.19 4.8 -2.19 4.8

xB 2.19 33 2.19 33

xC -2.19 5 -2.19 5

xD 1 60 1 60

xE -1 16 1 100

To visualize the dependencies between factor interactions, contour plots were generatedusing the regression models for each response. In the model equations, only the factors ofthe interaction under evaluation were varied, which all other factors were held constantat the mean level (0). The contour lines are curves of constant values for the respectiveresponses. In figure 5.5, the contour plots for the responses R, EC and ϕ are shown. Notall factor interactions were significant for each response. Insignificant factor interactionswere displayed as blank spaces in figure 5.5.

In order to identify operation conditions, which show both high enrichment and high re-covery, the pareto plot can be used (Fig. 5.4). The highest enrichment (EC = 5.1) atmaximum recovery (R = 99 %) was found for the level combination depicted in Table 5.4.Beyond this, the contour plots (Fig. 5.5) were used to predict high recovery (R = 107 %)along with high enrichment (EC = 5.0) for the level combination: pH value: -1 (6.0), tem-perature: 0 (27.5 °C), feed flow rate: -2.19 (5 mL min−1), gas flow rate: 0 (50 mL min−1),pore diameter: 0 (40 μm). The corresponding experiment yielded 100 % recovery withan enrichment factor of 4.8. Also, further level combinations yielded maximum recovery,but were not depicted in figure 5.4. Altogether, the experimental results were in goodagreement with the regression models within the factor range investigated. Additionally,no loss of enzymatic activity was observed within the experimental plan.

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54 5 Statistical investigation of continuous foam fractionation

10202030303040 40 40 40

50 50 50 5060606060

70 70 70 7080 80 80 80909090

100100100

100

100

90

90

Feed flow rate-2 -1 0 1 2

Dia

met

er fr

it

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Feed flow rate-2 -1 0 1 2

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er fr

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95

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9090

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9090

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flow

rate

-2

-1

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2,5 2,5 2,52,53,0 3,0

3,03,0

3,53,5

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Feed

flow

rate

-2

-1

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0,2

0,4

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0,60,60,8

0,40,4

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0,2

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pH value-2 -1 0 1 2

Feed

flow

rate

-2

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2,0

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1,5

1,51,5

1,01,0

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1,51,0

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Temperature-2 -1 0 1 2

Gas

flow

rate

-2

-1

0

1

20,6

0,4

0,4

0,4

0,20,20,2

0,2

0,2

0,20,2

0,0

Temperature-2 -1 0 1 2

Gas

flow

rate

-2

-1

0

1

2

Enrichment factor (a) Recovery of activity (b) Liquid fraction (c)

2,0

2,0

2,02,0

2,0

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1,8

1,8

1,81,8

1,8

1,8

1,6

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2,2

2,22,4

2,4

2,42,62,6

1,6

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1,6

Gas flow rate-2 -1 0 1 2

Dia

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er fr

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CE

AC

BD

DE

Feed flow rate-2 -1 0 1 2

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it

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flow

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90 9090 90

9090

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flow

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Feed flow rate-2 -1 0 1 2

Gas

flow

rate

-2

-1

0

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CD

Feed flow rate-2 -1 0 1 2

Gas

flow

rate

-2

-1

0

1

2

Figure 5.5: Contour plots for the factor interactions of EC (a), R (b) and ϕ (c). Blankcontour plots resemble insignificant factor interactions

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5 Statistical investigation of continuous foam fractionation 55

5.2.3 Surface tension

The surface tension of the culture supernatants of C. cinerea was measured for eachcombination of the factors pH (A) and temperature (B) (Fig. 5.6). Figure (5.6) shows,that the surface tension is at a minimum at pH 6.0 or 7.0 depending on temperature.This is close to the isoelectric point of the protein cutinase investigated.

34

36

38

40

42

44

4 5 6 7 8 9 10

Surf

ace

tens

ion

[mN

m-1

]

pH value

Figure 5.6: Surface tension of C. cinerea culture supernatants at varying pH values andtemperatures (� 22.0°C, � 25.0°C, × 27.5°C, � 30.0°C, • 32.5°C

5.3 Discussion

5.3.1 Statistical analysis of single and quadratic effects

5.3.1.1 Recovery of activity (R)

As shown in figure 5.3b), the recovery of cutinase was significantly influenced by the porediameter of the frit (E). Additionally, gas flow rate (D) and pore diameter (E) showedsignificant quadratic effects, implying nonlinear dependencies. For high recovery it waspostulated, that small pore diameters or rather small bubbles were necessary [80, 110,111, 115, 116]. Small bubbles increase the interfacial area available for adsorption andcause high liquid hold-up promoting high enzyme removal from the feed solution and highrecovery. The single effect was negative agreeing with the trends noted before. But alsothe quadratic effect was significant and various interactions involving the pore diameteridentified. Thus, a generalization of a best pore diameter is difficult (see also section5.3.2). The gas flow rate (D) determines the residence time of the bubbles in the liquidas well as in the foam phase. The higher the gas flow rate, the shorter the residence timeand time for adsorption and drainage [109]. At the same time, high gas flow rates lead to

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56 5 Statistical investigation of continuous foam fractionation

a larger quantity of gas bubbles because more pores of the frit were streamed with gas.The larger quantity of bubbles increased the hydrophobic surface available for adsorption.Usually, high gas flow rates increase recovery [109, 116, 149]. However, the single effectwas not significant here. Just as for the frit pore size (E), the quadratic effect impliednonlinearities over the investigated factor range and several factor interactions existed.Thus, a general statement for a best value is difficult (section 5.3.2).

5.3.1.2 Enrichment factor (EC)

According to figure 5.3a), the enrichment factor was mainly affected by feed flow rate(C) and pore diameter (E) of the frit. Significant quadratic effects were observed forfeed (CC) and gas flow rate (DD). The feed flow rate generally determines the residencetime of the feed or rather the residence time of surface-active molecules in the foamfractionation column. In addition, the incoming mass of surface-active components de-pends on the feed flow. Thus, high feed flow rates lead to low enrichment factors, asthe higher mass of surface-active molecules stabilizes the foam, thus increasing the vol-ume of foamate collected. Despite the fact, that more surface-active molecules are takeninto the foam phase, adsorbed at the gas-liquid interface or in the lamellar liquid, theenrichment decreases. Low feed flow rates increase the enrichment because the incomingmass of surface-active molecules is lower and the stability of the foam decreases, leadingto lower liquid holdup. In this study, low levels of feed flow rate (C) increased the en-richment agreeing with the explanations given before and with observations from otherauthors [25, 116]. However, the quadratic effect implied that a low feed flow rate has notnecessarily been the optimum. In case of the pore diameter (E), big pores were favorablefor high enrichment factors. Large bubbles enhance the enrichment, because drainage isincreased decreasing the liquid holdup in the foam [25, 116]. The positive single effect offactor E confirmed this observation. As for the recovery of active enzyme, the gas flowrate (D) was only quadratically significant. In general, the enrichment factor decreasesas the gas flow rate (D) increases due to the decreased entrainment of the foam [25, 109].

5.3.1.3 Liquid holdup (ϕ)

The liquid holdup was affected by temperature (B), feed flow rate (C), gas flow rate (D)and pore diameter of the frit (E). Additionally, the feed flow rate (C) was quadraticallysignificant (Fig. 5.3c)). The single factors and their algebraic sign to minimize the liquidholdup were in line with common sense [110, 115]. Long time for drainage (low D), lessliquid loading of the foam and low mass of surface-active molecules (low C), big pores(high E) and high temperature (B) decreased the liquid holdup.

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5 Statistical investigation of continuous foam fractionation 57

5.3.2 Statistical analysis of factor interactions

5.3.2.1 Recovery of activity (R)

As mentioned before, various interactions with the frit pore size (E) were identified. It isbelieved, that the nature and concentration of system components, temperature, gas flowrate, and pH value are primary variables, which determine secondary variables, like bubblesize [103]. Thus, bubble size is not affected by the frit pore size of the sparger only, but alsoby pH (AE), feed flow rate or rather incoming mass of surface-active components (CE),and gas flow rate (DE) as seen in this study and observed by other scientists [110, 111, 115].All in all, interactions involving factor E could be explained with the theories given forthe single factors. The quadratic influence of factor E and D on recovery can be seen forinteraction CE and DE (Fig. 5.3 b)). Factor interaction CD was also consistent with theobservations discussed in section 5.3.1. Gas and feed flow rates determined the contacttime between gas bubbles and surface-active molecules. Additionally, the feed flow fixedthe mass of cutinase induced into the column. The quadratic effect of factor D indicatedthat a mean level of gas flow rate was necessary to recover cutinase efficiently. Anotherfactor interaction between pH value (A) and feed flow rate (C) existed. Generally, pHvalue affects the enzyme’s surface-activity and surface tension of the feed solution. Itis expected, that the adsorption strength at hydrophobic interfaces of the enzyme isenhanced at its isoelectric point (pI) as a result of both decreased repulsive forces andsolubility [12, 13, 25]. The minimum surface tension was observed at pH 6.0 - 7.0, close tothe isoelectric point of cutinase (Fig. 5.6). At low feed flow rates and pH factor levels ≤ 0,i. e. pH values ≤ 7.0, high recoveries were obtained validating the statement, that pHvalues close to the isoelectric point (low surface tension) were best. At pH values > 7.0the recovery decreased. This is a result of the increased surface tension, i.e. decreasedadsorption strength of cutinase (Fig. 5.7). However, stable and wet foams resulted fromthe experiments implying other surface-active substances stabilizing the foam. At highfeed flow rates the opposite effect was observed. At pH values ≤ 7.0 recovery decreaseddue to decreased residence time. For pH values > 7.0 it can be assumed, that the incomingmass of other surface-active molecules stabilized the foam sufficiently. Cutinase, however,was not transported entirely as adsorbed species but in the interstitial liquid as well ason the bubble surface.

5.3.2.2 Enrichment factor (EC)

For all factor interactions involving pore diameter (AE, CE, DE) a high factor levelof the pore size (E) enhanced enrichment. Additionally, low factor values of feed flowfavored high concentration of cutinase. These observations were consistent with literature

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58 5 Statistical investigation of continuous foam fractionation

Figure 5.7: Contour plot of factor interaction AC for response recovery (R) with surfacetension measurements for pH values at a temperature of 27.5°C

[25, 96, 116]. For the mean level of gas flow rate the best enrichment was observed,stating the significant quadratic effect as explained in section 5.3.1. For factor interactionAC, high enrichment was achieved for low feed flow rates (long residence time and lessliquid load) and pH values corresponding to low surface tensions (Fig. 5.6). Higher pHvalues or feed flow rates decreased enrichment. This is in agreement with the previouslydescribed effects of these factors. Considering factor interaction of temperature (B) andgas flow rate (D), the combination of either high or low levels was favorable for highenrichment. In general, high gas flow rates decreased the time for adsorption and drainage.Additionally, high temperature decreased foam stability but increased foam formation.Increased drain rates could be explained by reduction of bulk and surface viscosity andincreased evaporation of interstitial liquid. The increasing foam formation is likely to bea result of decreasing surface tension of the thin liquid lamellae improving foam bubblestability (Fig. 5.8) [112, 113, 150]. For high gas flow rates and high temperatures the highenrichment was a result of combined destabilizing effects of these parameters. Bubblescollapsed partly in the upper part of the foam fractionation column and the foam did notleave the column continuously. Cutinase rich liquid from the collapsed bubbles drainedback and stabilized upcoming bubbles enabling them to leave the column. Hence, dryand cutinase rich foam left the column batch-wise increasing the enrichment factor.

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5 Statistical investigation of continuous foam fractionation 59

Figure 5.8: Contour plot of factor interaction BD for response enrichment factor (EC)with surface tension measurements for temperatures consistent with level values at pH= 7.0

5.4 Conclusion

In this study, continuous foam fractionation in stripping mode was investigated. It couldbe shown, that Design of Experiments was a valuable tool to get better insights into thefoam fractionation process. Various factor interactions were determined and discussed.The regression models developed were in good agreement with the experimental data,and the optimization of the responses was successful. Experiments like surface tensionmeasurements completed the investigation and supported the argumentation. Beside thephysiochemical parameters, it could be shown, that also the mode of continuous operation,stripping or enriching mode, and the column dimensions influence the separation efficiencyof the process. Thus, column design parameters need to be discussed in more detail.Moreover, the foam fractionation process needs to be compared to traditional purificationstrategies and the potential must be evaluated.

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6 Statistical investigation of columndimensionsd

Traditionally, the downstream processing to purify enzymes consists of several processsteps combined in a so called multistage process as described in chapter 1. Becausethese multistage processes are time consuming and expensive, the improvement of thedownstream processing is an issue of interest. An improvement can be process inten-sification. According to Stankiewicz [152], process intensification is a development ofnew apparatuses or techniques, which substantially decrease equipment volume, energyconsumption, or waste formation and thus leading to cheaper and more sustainabletechnologies. Foam fractionation, a technique which can selectively separate enzymes,can be considered as an approach to intensify processes. Many studies show, that foamfractionation is a promising technique to separate enzymes [79–81, 105, 107]. However,various physiochemical and column design parameters affect the separation efficiency ofthe process. For a competitive foam fractionation process the process itself needs to beunderstood in more detail. In chapter 4 and chapter 5, the influence of various physiochem-ical parameters and the mode of operation were investigated and proofed to influence theseparation efficiency. Moreover, column design parameters, like the length of the liquid col-umn, the length of the foam column, and the column diameter are important parameters,which can influence the performance of foam fractionation [79, 86, 112, 115, 116, 153–155]. In this chapter, the influence of column design parameters in combination with gasand feed flow rates and pore size of the frit are investigated systematically to furtherenhance the foam fractionation process. The improved foam fractionation process is thencompared to classical purification strategies in terms of separation efficiency and abilityas process intensification technique.

6.1 Materials and methods

Materials and methods are equal to the ones described in chapter 5. The foaming devicewas slightly modified as can be seen in figure 6.1.

dPart of the results are obtained during the diploma thesis of Laura Reinecke [151]

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62 6 Statistical investigation of column dimensions

Gas

Flow meter

Heating water

Heating water

Foamate

Porous frit

Tempered feed reservoir

Residue retentate

Peristaltic pump

Peristaltic pump vacuum pump

Stripping mode

Enriching mode

Figure 6.1: Continuous foam fractionation column used for the experiments

The liquid height was adjusted to the experimental plan and kept constant by manuallyadjusting the speed of the peristaltic pump (Watson Marlow, Rommerskirchen, Germany)used to withdraw the depleted liquid from the bottom of the column. Precise dimensions ofthe columns used are depicted in figure 6.2. For each combination of length and diameterone column was made by the local glassblower.

1.6 cm 3.0 cm 4.4 cm

Stripping mode

Enriching mode

Diameter foam column

Center point

29 cm or 56 cm

12 cm or 24 cm

29 cm or 56 cm

12 cm or 24 cm

43 cm

18 cm

Figure 6.2: Continuous foam fractionation columns used in this study. The column inthe middle was used for the center points only. The columns with 1.6 cm and 4.4 cmdiameter were varied in foam and liquid column length according to the experimentalplan. For each combination one column was produced

Steady state operationThe time for steady state operation was determined both for stripping and enrichingmode. The most important point was the determination of the influence of different

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6 Statistical investigation of column dimensions 63

column dimensions for achieving steady state. The experiments were carried out to ensuresteady state conditions for all variable column setups and operating modes during theexperimental plan. Thus, comparable results could be collected. In chapter 5 it was shown,that increasing gas and feed flow rates decreased the time for achieving steady state.Thus, the lowest feed and gas flow rate regarding the experimental design investigatedwere chosen for the steady state determination (constant parameters: pH 7.0, 27.5 °C,40 mL air min−1, 7.7 mL feed min−1, and a frit pore size of 40 μum). Over a period of60 minutes samples were taken every 5 minutes from the retentate stream and enzymaticactivity assay was analyzed. Steady state operation existed when the enzymatic activityand the flow of the retentate stream were constant.

Feed positionThe influence of feed position was determined before starting the experimental design.According to Fig. 6.2, two feed inlet positions were evaluated: One at the top of theliquid phase (enriching mode) and one at the top of the foam phase (stripping mode).To determine, which operation mode was more efficient in terms of separation efficiency,experiments under similar conditions, but different feeding positions were carried out.The conditions chosen were pH 7.0, 27.5°C, 40 mL air min−1, 7.7 mL feed min−1, and afrit porosity of 40 μm.

6.2 Design of Experiments

For the systematic investigation of continuous foam fractionation, a Design of Experimentswas used. An experimental plan was developed in which the input variables (factors) werechanged specifically to observe changes in the output variables (responses). In this study,parameters gas flow rate (A), frit pore size (B), feed flow rate (C), length of liquid column(D), length of foam column (E), and diameter of foam column (F) were investigated.These parameters were considered to have influence on separation efficiency and foamstability. The process parameters (A) - (C) were already discussed in chapter 5, but needto be reinvestigated in this context, especially in terms of foam stability. Altogether, 6factors were varied on 3 levels using a 26−1 fractional factorial design. Factors and levelvalues are depicted in Table 6.1.

As described in chapter 4 and chapter 5 commercially available candle frits with porositiesof 16 μm, 40 μm and 100 μm were used and thus differ to the calculated pore size forlevel 0. However, as shown in chapter 4, this does not affect the validity of the plan.The glass columns were made by the local glassblower and could slightly differ in theirdimensions (< 0.5 mm). The experiments were randomized and 4 to 6 experiments werearranged in a block. For each block three center points were performed. Due to this

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64 6 Statistical investigation of column dimensions

Table 6.1: Factors (A - F) and levels investigated. In brackets: [Calculated factor value,which differ from the factor value used in the experiments]

Factor -1 0 1

AGas flow rate 40 50 60

[ mL min−1 ]

BPore diameter

16 40 100[ μm ] (58)

CFeed flow rate

7.74 10.0 12.29[ mL min−1 ] (7.78) (9.96) (12.22)

DLength of liquid column (Lliquid 12 18 24

[ cm ]

FLength foam column (Lfoam) 29 42.5 56

[ cm ]

FDiameter foam column (Dfoam) 1.6 3.0 4.4

[ cm ]

experimental procedure, differences between the fermentation batches could be recognized.Additionally, the standard deviation was calculated via the center points. To describe theefficiency and quality of the foam fractionation process, the enrichment factor (EC) andthe recovery of active enzyme (R) were used and represented the responses in the Design ofExperiments (see equations (3.1), (3.2)). To quantify the effects representing the relationsbetween the factors and the responses, a multiple linear regression was used [144]. Aneffect had a significant influence on the responses R or EC if exceeding a confidenceinterval which was higher than the noise value. In case that the noise value exceeded theconfidence intervals, the noise value was taken as measure for significance. The confidenceintervals were calculated via standard deviation of the 18 center points (experiments 33- 55 in Table 6.3) and Student’s distribution [144]. The noise of the experiments wasestimated by higher factor interactions.

6.3 Results

6.3.1 Surface tension

The surface tension of different dilutions of the culture supernatant of C. cinerea wasmeasured. The supernatant was diluted with water and adjusted to pH = 7.0 and 27.5 °C.Figure 6.3 shows that the surface tension reaches an approximately constant value at broth

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6 Statistical investigation of column dimensions 65

fractions above 60 %. The constant value indicates that the concentration of surface-activemolecules exceeds a critical value, the so called critical micelle concentration (CMC).

30

40

50

60

70

0 20 40 60 80 100

Surf

ace

tens

ion

[mN

m-1

]

Broth fraction [%]

Figure 6.3: Surface tension of C. cinerea culture supernatants for different dilutions(pH = 7.0 and 27.5 °C)

Above the CMC, it is possible that the cutinase will aggregate to micelles, which mayinteract less with the bubbles [12, 108]. Thus, foam fractionation experiments in columnsDfoam= 1.6 cm, Dfoam= 3.0 cm and Dfoam= 4.4 cm and stripping mode were carried outunder constant conditions (pH 7.0, 27.5°C, 40 mL air min−1, 7.7 mL feed min−1, and afrit porosity of 40 μm) but different diluted feed solutions. For feed solutions with 67 %supernatant in water the enrichment could be increased compared to experiments of pureculture supernatant (Table 6.2). Thus, each fermentation batch was diluted prior tofoaming. Water was used to dilute the culture supernatant because it did not affect theenzymatic activity and was available. Thus, no extra steps were necessary to preparedilution medium and it was comparatively cost-efficient.

Table 6.2: Comparison of foam fractionation experiments for non-diluted and dilutedculture supernatant and different column diameter. In brackets: enrichment factorsrecalculated for non-diluted feed solution

Column diameter 100 % culture supernatant 67 % culture supernatant

1.6 cm R 87 84

EC 2.4 6.1 (4.1)

3.0 cm R 82 79

EC 8.8 26.8 (17.9)

4.4 cm R 79 75

EC 17.1 44.4 (29.6)

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66 6 Statistical investigation of column dimensions

6.3.2 Configuration of equipment

Steady state operationSteady state operation is necessary for collecting reliable data of the different columnsetups. For the column with 1.6 cm foam column diameter, steady state operation wasreached faster in enriching mode than in stripping mode. Additionally, it seemed thatfoam height as well as liquid height did not affect the time for achieving steady state forboth operation modes. Figure 6.4 also shows that retentate activity in steady state islower for stripping mode compared to enriching mode.

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Enzy

mat

ic a

ctiv

ity in

re

tent

ate

[U L

-1]

Time [min]

Figure 6.4: Retentate activity vs. time for columns with Dfoam= 1.6 cm and differentliquid and foam column lengths. (� stripping mode, Lliquid= 24 cm, Lfoam= 56 cm;• stripping mode, Lliquid= 12 cm, Lfoam= 29 cm; � enriching mode, Lliquid= 24 cm,Lfoam= 56 cm; ◦ enriching mode, Lliquid= 12 cm, Lfoam= 29 cm)

Columns with Dfoam= 1.6 cm achieved steady state after 50 minutes at the latest, as canbe seen in Fig. 6.4. In the column with a foam column diameter of 3.0 cm (center pointexperiments) steady state operation was reached after 50 minutes in stripping mode and35 minutes for enriching mode. For the column with Dfoam= 4.4 cm, stripping modecould be investigated only because in enriching mode the foam was instable and collapsedin the column. Thus, no foamate could be collected. Even higher feed or gas flow ratescould not stabilize the foam. Steady state operation in stripping mode was achieved after30 minutes. It can be seen, that the increase in column diameter decreased the time forachieving steady state. As a precaution, all further foam fractionation experiments wereinitially run for 60 minutes. This way, stationary operation was ensured.

Feed positionThe influence of the feed position was determined prior to setting up the experimental de-sign. According to the results of steady state experiments, the column with Dfoam= 1.6 cmand Dfoam= 3.0 cm could be investigated in enriching and stripping mode. Columns

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6 Statistical investigation of column dimensions 67

with Dfoam= 4.4 cm could only be operated in stripping mode. For columns with Dfoam

= 1.6 cm, stripping mode lead to higher recoveries and enrichment factors compared toenriching mode. In order to explain these results, experiments with each possible columnlength combination (constant Dfoam= 1.6 cm) were operated. Figure 6.5 shows that foreach experimental setup, stripping mode lead to in higher recovery and higher enrichmentof active enzyme than enriching mode.

0

1

2

3

4

5

0

10

20

30

40

50

60

70

80

90

100

12-29 12-29 12-59 12-59 24-29 24-29 24-56 24-56

Enri

chm

ent [

-]

Rec

over

y of

act

ivity

[%]

Figure 6.5: Comparison of stripping and enriching mode for columns with Dfoam= 1.6 cmand variable foam and liquid column lengths. X-axis: Length liquid column - lengthfoam column; Black columns display the recovery R for stripping mode, gray columnsdisplay the recovery R for enriching mode, ◦ enrichment factor (standard deviationsadopted from DoE)

Experiments with higher gas flow rates lead to similar results, while an increase in feedflow rate decreased the differences between enriching and stripping mode. For columnswith Dfoam= 3.0 cm the experiment in stripping mode yielded a recovery of 85 % withan enrichment factor of 2.7. The experiment in enriching mode achieved a recovery of82 % with 2-fold enrichment. Thus, the results of stripping and enriching mode werealmost equal. To understand these results, the flow regime in a column with Dfoam=1.6 cm during stripping mode was investigated in more detail. After achieving steadystate operation feed solution dyed with patent blue V (1 mg mL−1 stock solution) wasintroduced at the stripping mode feed position (see Fig. 6.6a)). The dye is not surface-active and was thus only present in the bulk liquid phase. It was observed, that thestained feed solution was not distributed homogeneously inside the column. A part of thedyed solution was flowing to the liquid pool another part rose up with the counter currentfoam flow. Furthermore, it could be seen that dye was present in the liquid pool beforethe whole foam was dyed. Thus, channeling occurred in the foam column. After a certaintime, undyed feed solution was introduced again. It was observed that the dyed liquidfrom the liquid pool was steadily entrained into the foam phase. This suggested that acircular flow regime was present during the experiment. Thus, it was assumed that duringthe experimental plan under comparable conditions (Dfoam= 1.6 cm) the flow regime wascircular.

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68 6 Statistical investigation of column dimensions

a) b)

Feed with dye Feed with dye Feed without dye Feed without dye

Figure 6.6: Different flow regimes investigated via foam fractionation with dye. a) Circularflow in column Dfoam= 1.6 cm, b) Plug flow in column Dfoam= 4.4 cm

Similarly, the flow regime of the columns with Dfoam= 3.0 cm and Dfoam= 4.4 cm wasinvestigated (see Fig. 6.6b) for column with Dfoam= 4.4 cm). It was observed that thestained feed solution was distributed nearly homogeneously inside the column. The feedwas not entrained and no channeling could be observed. This suggested that a plug flowregime was present during the experiment. Even higher gas or feed flow rates caused nocircular flow. Thus, it was assumed that during the experimental plan under comparableconditions a plug flow was present in the columns with Dfoam= 3.0 and Dfoam= 4.4 cm. Tomake sure that the flow regime was caused by the geometry of the columns and not by thetest system used, another system was investigated. Fermentation broths with the targetenzyme PLA2 from recombinant Aspergillus niger were tested. The same flow regimes asfor the cutinase system were obtained. The stripping mode in column Dfoam= 1.6 cm wasmore efficient than enriching mode (stripping: R = 100 % EC = 2.1; enriching mode: R

= 87 % EC = 1.3). Due to these results stripping mode was chosen as the most suitablemode of operation. The foam was more stabilized and higher recoveries and enrichmentscould be achieved.

6.3.3 Design of Experiments

According to the last section, stripping mode was chosen as the mode of operation for theDesign of Experiments. The effects of single factors and factor interactions were calculatedand are depicted in Fig. 6.7 regarding responses EC and R. The corresponding data ispresented in Table 6.3.

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6 Statistical investigation of column dimensions 69

Table 6.3: Experimental plan in systematic order and obtained experimental results forresponses R and EC . A: Gas flow rate, B: Pore diameter, C: Feed flow rate, D: Lengthof liquid column, E: Length of foam column, F: Diameter of foam column [151]

No A B C D E F EC R

1 -1 -1 -1 -1 -1 -1 11.1 14.8

2 1 -1 -1 -1 -1 1 45.2 18.1

3 -1 1 -1 -1 -1 1 47.1 94.2

4 1 1 -1 -1 -1 -1 2.8 100

5 -1 -1 1 -1 -1 1 37.4 15.2

6 1 -1 1 -1 -1 -1 8.2 46,5

7 -1 1 1 -1 -1 -1 2.6 100

8 1 1 1 -1 -1 1 15.9 92.7

9 -1 -1 -1 1 -1 1 41.8 27.9

10 1 -1 -1 1 -1 -1 4 24.3

11 -1 1 -1 1 -1 -1 1 80.1

12 1 1 -1 1 -1 1 35.3 70.6

13 -1 -1 1 1 -1 -1 10.9 27.7

14 1 -1 1 1 -1 1 37.1 15.4

15 -1 1 1 1 -1 1 24.1 84.5

16 1 1 1 1 -1 -1 1.7 99.3

17 -1 -1 -1 -1 1 1 18.3 5.7

18 1 -1 -1 -1 1 -1 10.2 56.9

19 -1 1 -1 -1 1 -1 2.4 95.1

20 1 1 -1 -1 1 1 38.3 71.5

21 -1 -1 1 -1 1 -1 12.8 32

22 1 -1 1 -1 1 1 5.4 1.8

23 -1 1 1 -1 1 1 8.5 84.7

24 1 1 1 -1 1 -1 2.5 97.2

25 -1 -1 -1 1 1 -1 13.1 22.6

26 1 -1 -1 1 1 1 13.1 10.4

27 -1 1 -1 1 1 1 48.2 70.7

28 1 1 -1 1 1 -1 7 97.7

29 -1 -1 1 1 1 1 14.6 7.8

30 1 -1 1 1 1 -1 9.1 45.6

31 -1 1 1 1 1 -1 1.7 81.3

32 1 1 1 1 1 1 5.3 97.6

33 0 0 0 0 0 0 20.2 74.4

34 0 0 0 0 0 0 19.3 71.3

35 0 0 0 0 0 0 19.1 70.4

36 0 0 0 0 0 0 18.1 81.2

37 0 0 0 0 0 0 20.1 63.3

38 0 0 0 0 0 0 19.9 73.5

39 0 0 0 0 0 0 16.3 65.1

40 0 0 0 0 0 0 19.1 72.4

41 0 0 0 0 0 0 16.4 65.8

42 0 0 0 0 0 0 13.1 82.6

43 0 0 0 0 0 0 17.4 71.6

44 0 0 0 0 0 0 16.5 70.9

45 0 0 0 0 0 0 15.8 74.7

46 0 0 0 0 0 0 15.2 75.4

47 0 0 0 0 0 0 15.1 79.3

48 0 0 0 0 0 0 19.2 66.8

49 0 0 0 0 0 0 19.8 68.8

50 0 0 0 0 0 0 20.3 70.6

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70 6 Statistical investigation of column dimensions

-10

-5

0

5

10

15

20

A B C D E F AB AC AD AE AF BC BD BE BF CD CE CF DE DF EF

Effe

ct o

f EC

Factors

99.9% confidence interval Noise a)

-20

-10

0

10

20

30

40

50

60

70

A B C D E F AB AC AD AE AF BC BD BE BF CD CE CF DE DF EF

Effe

ct o

f R

Factors

99.9% confidence interval Noise b)

Figure 6.7: Effects for the responses a) EC , b) R. Black columns show significant, graycolumns insignificant effects. A: Gas flow rate, B: Pore diameter, C: Feed flow rate, D:length of liquid column, E: Length of foam column, F: Diameter of foam column

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6 Statistical investigation of column dimensions 71

A positive effect of a single factor X means, that an increase of factor X from level -1 to+1 increases the response. The increase of the foam column diameter (F) from 1.6 cmto 4.4 cm enhanced EC about 20.9 on average (Fig. 6.7a)). In case that the changefrom level -1 to +1 causes a negative effect, the response decreased. For instance, thesame variation in foam column diameter (F) decreased the recovery of active enzymeabout 16 % on average (Fig. 6.7b)). A factor interaction XY is the average differencein influence of factor X, at levels -1 and +1 of factor Y. If a factor interaction XY issignificant, both factors are interdependent. Thus, both factors have to be considered,although their single effects might not be significant. According to figure 6.7a), all effectswere significant, which exceed the 99.9 % confidence interval. Thus, the enrichment factorwas mainly affected by the foam column diameter (F), gas flow rate (A), pore size of thefrit (B), feed flow rate (C), and foam column length (E). Additionally, various interactionslike foam column length (E) and foam column diameter (F) existed. As shown in figure6.7b), the effects of active enzyme recovery were significant if exceeding the noise value.Thus, the recovery of active enzyme was mainly affected by frit pore size (B), gas flow rate(A) and foam column diameter (F). Additionally, two factor interactions existed betweengas flow rate (A) and foam column diameter (F) and for foam column length (E) and foamcolumn diameter (F). After identifying significant single effects and factor interactions theregression models were formed as shown in equations 6.1 and 6.2.

EC = 17.15− 1.7 · xA − 1.48 · xB − 4.4 · xC − 3.62 · xE+10.45 · xF − 1.07 · xA · xF − 3.07 · xB · xC+2.57 · xB · xE + 2.1 · xB · xF − 1.25 · xC · xE−4.28 · xC · xF − 4.64 · xE · xF

(6.1)

R = 61.7 + 3.2 · xA + 32.6 · xB − 7.9 · xF − 3.9 · xA · xF−3.2 · xE · xF

(6.2)

With the help of the regression models, the results of each experiment in the investigatedparameter range could be predicted. An overview of the results for all level combinationsfor recovery and enrichment factor is displayed in the pareto plot (Fig. 6.8). The figureshows that the calculations predict recoveries of 98 % active enzyme with enrichmentfactors of 11.7. A maximal enrichment factor of 47.1 is predicted with a recovery of 90.4 %for the level combination: gas flow rate: -1 (40 mL min−1), pore diameter: 1 (16 μm),feed flow rate: -1 (7.7 mL min−1), Lliquid: 1 (24 cm), Lfoam: -1 (29 cm), and Dfoam:

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72 6 Statistical investigation of column dimensions

0 5

10 15 20 25 30 35 40 45 50

0 20 40 60 80 100

Enri

chm

ent f

acto

r (E C

)

Recovery of activity (R)

Figure 6.8: Possible combinations for responses R and EC calculated based on the regres-sion models

1 (4.4 cm). This level combination was part of the experimental plan and representsexperiment number 3 in Tab. 6.3. The corresponding experiment showed an enrichmentof 47.1 with a recovery of 94.2 % active enzyme.

The maximal recovery (R = 98 %, EC = 11.7) is predicted for the level combination: gasflow rate: -1 (40 mL min−1), pore diameter: 1 (16 μm), feed flow rate: -1 (7.7 mL min−1),Lliquid: 1 (24 cm), Lfoam: 1 (56 cm), and Dfoam: -1 (1.6 cm). This level combination wasnot part of in the experimental design. Thus, an additional experiment was carried out.The corresponding experiment yielded a recovery of 98 % with an enrichment factor of12.6. The experimental results show that an accurate prediction can be accomplished bythe developed models within the factor range investigated.

6.4 Discussion

6.4.1 Configuration of equipment

For foam fractionation processes circular flow is only very little documented in technicalliterature. In our study, the use of columns with Dfoam= 1.6 cm and Dliquid= 3.0 cm incombination with stripping mode caused a circular flow regime with back-mixing, whichenhanced the separation efficiency (Fig. 6.5). During stripping mode, the feed was intro-duced at the top of the foam phase and flowed counter current to the rising foam. Thus, theadsorption of surface-active substances took place in the whole foam fractionation columnstabilizing the foam over the whole column. Additionally, liquid was steadily entrainedfrom the liquid pool into the foam phase, further increasing the residence time of the feedsolution in the foam fractionation column. Thus, the contact time between surface-active

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6 Statistical investigation of column dimensions 73

molecules and gas bubbles was higher, explaining the lower retentate activity in strip-ping mode compared to enriching experiments for the small column (Fig. 6.4). Becausethe liquid pool was more depleted in stripping mode, less surface-active molecules wereavailable to stabilize the emerging bubbles. Hence, the bubbles leaving the liquid poolin stripping mode were coarser and the liquid hold-up in the foam lower, respectively.Second, channeling was observed during the dye experiment allowing zones without con-stant feed charge. These zones without constant feed charge can be considered as localenrichment zones. Thus, the foam in stripping mode was drier and more surface-activemolecules were recovered explaining the higher enrichment factors achieved in strippingmode compared to enriching mode. The fact that enriching mode led faster to steadystate operation than stripping mode can be explained likewise (Fig. 6.4). The high reten-tion time of the feed solution and the strong back mixing decreased the organization ofthe foam in stripping mode. In enriching mode the adsorption of surface-active moleculesmainly took part in the liquid pool and the foam phase was only affected by drainage andcoalescence. Thus, steady state could be achieved faster in enriching mode because thesystem was not disorganized. For columns with Dfoam= 3.0 cm and Dfoam= 4.4 cm theresidence time of bubbles and enzyme is determined mainly by feed and gas flow rate. Forincreasing foam column diameter, the time for achieving steady state decreased, probablyresulting from the change of flow regime. For plug flow, the time for constant enzymaticretentate activity and retentate volume was shorter than for the circular flow, where liquidwas steadily entrained into the foam phase and channeling occurred. Obviously, the cir-cular flow regime, caused by the diminution of foam to liquid column, seemed to be moreefficient, the handling and controlling is difficult and the process is hardly scalable. Theincrease in foam column diameter led to a plug flow regime, which is easier to handleand to scale-up. Additionally, the increase in foam column diameter resulted in a moreefficient separation. Thus, in terms of scalability and controlling the plug flow regime isfavored.

6.4.2 Discussion of single effects

The liquid column length (D) was not significant. Because all experiments were runin stripping mode, the adsorption of surface-active substance mainly took place in thefoam column. Thus, the residence time of surface-active molecules in the liquid pool aswell as the volume of the liquid pool did not have an effect on the separation efficiency.The column diameter (F) was indicated as an important parameter for enhancing theenrichment of active cutinase. The recovery, however, decreased with increasing the foamcolumn diameter (F). Generally, an increase in column diameter increased the drainagerate because the supporting effect of the column wall decreased [13] and the velocity ofthe foam decreased increasing time for drainage. Thus, the liquid hold-up in the foam

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74 6 Statistical investigation of column dimensions

decreased yielding higher enrichments but decreased recoveries. In this chapter, the effectof the column diameter (F) could be influenced by the flow regime. In columns withDfoam= 1.6 cm, circular flow and in Dfoam= 4.4 cm plug flow was present. In litera-ture, plug flow is postulated to increase the mass transfer [153]. Additionally, Metznerand Brown [156] showed that a plug flow with maximal local circular flow (called by theauthors as local turbulence) achieved higher mass transfer rates than circular flow regimes.Altogether, similar recoveries for experiments in foam columns of Dfoam= 1.6 cm andDfoam= 4.4 cm were achieved (see Table 6.3). The enrichment factor was considerablyhigher for columns with Dfoam= 4.4 cm. Thus, the increased mass transfer caused in-creased recovery and the increasing cross section enhanced enrichment. Another sensitiveparameter was the frit pore size (B). Small pore diameters, or rather small bubbles, in-creased the interfacial area available for adsorption and caused high liquid hold-up promo-ting high enzyme removal from the feed solution and high recovery [80, 110, 111, 115, 116].In return, large bubbles enhanced the enrichment of enzyme, because the foam becamecoarser, drainage rates increased and the liquid hold-up decreased [25, 116]. The lastparameter affecting both enrichment and recovery was the gas flow rate (A). Generally,the gas flow rate determined residence time of the bubbles in the liquid as well as in thefoam phase. High gas flow rates decreased the residence time and thus time for adsorp-tion and drainage [109]. Higher gas flow rates led to a larger quantity of gas bubbles.Thus, more hydrophobic surface was available for adsorption. Usually, high gas flow ratesincreased recovery [109, 116, 149] and decreased enrichment of active enzyme due to thedecreased entrainment [25, 109]. Furthermore, the enrichment of cutinase was affectedby feed flow rate (C) and foam height (E) in the column. Feed flow rate (C) determinesthe incoming mass of surface-active molecules as well as their residence time in the foamfractionation column. Low feed flow rates increased the enrichment, because the foamis less moisturized and the lower incoming mass of surface-active molecules forms lessstable foam. Due to the stability decrease, the drainage rate increased resulting in higherenrichment. The negative effect of feed flow rate (C) on enrichment was in agreementwith the explanation given above and with literature [25, 109, 115, 116]. The effect of gasflow rate (A) and feed flow rate (C) could also be influenced by the flow regime. For plugflow the residence time of the bubbles or surface-active components was mainly affectedby feed and gas flow rate. For circular flow, residence time was influenced by the degreeof back-mixing. However, this could not be quantitatively determined. Thus, the factorinteractions with the column cross section (F), which could distinguish the flow pattern,had to be considered carefully. The last parameter affecting the enrichment factor was thefoam height (E) of the column. In literature, it was postulated that increasing foam heightincreased enrichment and decreases recovery due to lower liquid hold-up, caused by longerresidence time [13, 25, 109, 115, 116, 156]. In this study, the effect of foam height (E) wasnegative, favoring a short foam column for high enrichment. This can be explained only

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6 Statistical investigation of column dimensions 75

when the factor interactions involving factor foam height (E) are considered.

6.4.3 Discussion of factor interactions

The most significant factor interaction for response enrichment factor was the interactionof foam height (E) and foam column diameter (F). For columns with for Dfoam= 1.6 cmenrichment increased with increasing foam height as postulated by Brown et al. [14].Experiments with long foam columns and big diameter in combination with big pore sizesyielded low enrichment and low recovery. The foam was instable and very small foamatevolumes left the column. It is assumed, that enzyme deposited at the glass column and thehorseshoe bend. This resulted in low enrichment, although very dry foam left the column.The combination of big diameter and short foam column, which was independent of fritpore size (B), showed the best results. Hence, a short foam column is favored as seen inthe interpretation of the single effects. For the response recovery a short and small foamcolumn is favored as described in literature [25, 109]. As mentioned before, the factors gas(A) and feed flow rate (C) might be influenced by the flow regime present in the differentcolumns. For enrichment as well as for recovery, the factor interaction between gas flowrate (A) and column diameter (F) were found. On closer examination, the flow regimeseemed not to be the determining aspect in this case. High enrichment was achieved forlow gas flow rates and wide foam columns. High recovery was favored by high gas flowrates and narrow foam columns. These observations were in line with literature presentedbefore [13, 14, 25, 156]. For the enrichment factor, the interaction between feed flow rate(C) and foam column diameter (F) was the second most significant factor interaction.In this case, the flow regime seemed to influence the behavior of the foam fractionationprocess. For plug flow as mostly experienced in columns with Dfoam= 4.4 cm, the increasein feed flow rate caused decreasing enrichment and increasing recovery. This reflects theopinion described in literature [25, 109, 115, 116]. For circular flow, the increase in feedflow rate increased the recovery of active enzyme, while the enrichment remained constant.The increase in recovery could be explained by the increase of incoming surface-activemolecules. Thus, more protein could adsorb to the bubble surface. An increase in feedflow rate decreased the enrichment of cutinase for the narrow column drastically. It canbe assumed, that the circular flow regime with channeling, i. e. differences in liquid feeddistribution and resulting differences in bubble size, was enhanced. This attenuated thehigher liquid loading. This phenomenon was also observed by Haas and Johnson [153].The remaining factor interactions of pore size (B) with feed flow rate (C), length of foamcolumn (E), and diameter of foam column (F), and the factor interaction between feedflow rate (C) and length of foam column (E) were considered as linked interactions. Thestability of the foam is mainly affected by the bubble size, which is not only determinedby the pore size of the sparger, but also by feed flow rate (BC) [110, 111, 115], the length

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76 6 Statistical investigation of column dimensions

and diameter of the foam column (BE, BF). After consideration of all factor interactions,a low feed flow rate, short foam columns and big pores promoted the enrichment of activecutinase into the foam phase.

6.5 Foam fractionation as process intensification

Traditional purification strategies of cutinase in lab-scale normally consist of multistageprocesses with precipitation and chromatography/adsorption steps involving DEAE -cellulose columns, Sepharose columns, cation exchange, and hydrophobic interaction chro-matography [139, 140, 157]. In Table 6.4, the traditional processes and the improved foamfractionation are displayed.

Table 6.4: Traditional purification strategies vs. foam fractionation. Crude culture super-natant or crude extract were chosen as starting point for downstream processing.

Process Performance Intermediate steps Totalsteps

Foam fractionation

R = 94 %

EC = 47.1 pH adjustment 2

P = 18.8

Gindro and Pezet1 R = 32 % Dialysis, concentration, 9

P = 14.4 filtration, pooling/concentration

Sebastian and Kolattukudy2 R = 51 % Dialysis, centrifugation, 7

P = 8.0 washing, pooling/concentration1 [140], 2 [139]

The disadvantages of the traditional strategies are the several additional dialysis, equi-libration, filtration, and concentration steps. In addition, the multistage processes areoften associated with great losses of enzymatic activity as can be seen in Table 6.4. Foamfractionation can substitute several process steps and preserves the enzymatic activity.Additionally, foam fractionation concentrates the target molecule simultaneously, whereasthe traditional strategies dilute their target fractions and need additional concentrationsteps. Other studies described the purification of cutinase with micellar reversed phasesystems or aqueous two-phase systems (ATPS) as alternatives to the first precipitation,dialysis or concentration steps [141, 158]. The liquid-liquid extraction and back-extractionwith sodiumdi(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles of diluted fermenta-tion media containing cutinase yield in 52 % activity recovery and a purification factorof 10.2. According to Fernandes et al. [158], the ATPS technique yielded a recovery of96 % active cutinase from culture supernatant with an enrichment factor of 3.8 and a

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6 Statistical investigation of column dimensions 77

Table 6.5: Comparison of experimental results. High recovery and high enrichment ofactive cutinase was favored for the different studies

Operation mode Recovery of active Enrichment factorenzyme (R) (EC)

Batch foam fractionation 97 % 8.2(chapter 4)

Continuous foam fractionation with constant 98 % 5.6column geometry (chapter 5)

Continuous foam fractionation with variable 94 % 47.1 (P = 18.8)column geometry

purification factor of 8.0 in one step. In an optimized two-step extraction an overall yieldof 93 % active cutinase with an enrichment factor of 2.1 and a purification factor of 29was obtained. However, the extraction techniques need additives and solvents and involveintermediate steps like centrifugation and equilibration steps. Foam fractionation, how-ever, can yield the same performance with the lack of additives and solvents. Thus, costsand waste streams are reduced and the process is more sustainable. A new approach toenhance the efficiency of downstream processing is the use of protein engineering. Lien-queo et al. [159], studied the influence of tryptophan tags fused to the cutinase on a twostep purification process involving cationic bed adsorption (EBA) followed by hydrophobicinteraction chromatography (HIC). The highest overall recovery was obtained for the wildtype cutinase (94 %), the highest purification factors by the use of the tagged cutinases(P = 15.2 with R = 67 % or P = 35.9 with R = 44 %). The enrichment factors rangefrom 6.4 to 17.3 for the tagged cutinases and 18.8 for the wild type cutinase. However, thetagged proteins only increased the selectivity of the process, the two-step process itself isa good alternative to the traditional methods. Anyhow, considering foam fractionationthese results can be achieved using one step only. In comparison to the other named al-ternatives no additional steps like equilibration or centrifugation are necessary. Anotheradvantage of foam fractionation is the avoidance of solvents and additives which need tobe removed for further downstream processing or product application.

Due to the investigation of the column design parameters, the process was understoodin more detail and conclusion could be drawn for further scale-up experiments. It wasshown, that physiochemical and process parameters alone offer a limited optimizationpotential. Due to the variation in column design, the enrichment factor increased six- toeight-fold at almost constant recovery (c.f. Table 6.5).

Comparing the separation efficiency of foam fractionation with the named processes orprocess steps, foam fractionation has the ability to be an efficient alternative. In one step

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78 6 Statistical investigation of column dimensions

the cutinase was selectively recovered without activity loss and concentrated in the foamphase and process streams were reduced. Because no additives or solvents were used anafter treatment is not necessary. Thus, foam fractionation combines operational simplicityand high separation efficiency and hence, can be considered as process intensification forrecovering cutinase.

6.6 Conclusion

In this chapter, continuous foam fractionation was demonstrated as technique for processintensification. To improve the performance of the separation process, column design para-meters were studied. After optimization within the factor range investigated, the over-all separation efficiency could be enhanced compared to previous studies. Additionally,the foam fractionation process could be understood in more detail. With the knowledgegained and the improved separation process, foam fractionation was compared with tradi-tional and newly developed purification strategies. Based on the separation efficiency, thesimplicity of the process, and the lack of additional process steps, solvents, or additives,foam fractionation could be a potential process to substitute several steps in traditionalmultistage purification strategies. Thus, according to the definition of Stankiewicz [152],the application of foam fractionation for the purification of cutinase from culture super-natant can be considered as a tool for process intensification. Beside the identification ofinfluencing parameters, like pH value, temperature or column geometry, the scalability ofthe system needs to be investigated to make foam fractionation a competitive separationtechnique.

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7 A contribution to the scale-up offoam fractionation

To introduce foam fractionation as a new unit operation a scale-up is necessary. Ingeneral, foam fractionation can be scaled up via increasing the dimensions of the columnor by continuous operation. Additionally, bundles of columns can be used to increasethe throughput while preserving the stability of the system. Until now, the scale-up offoam fractionation has rarely been discussed. Tanner et al. and Tanner and Prokop[69, 71] described the scale-up as easy if considering all influencing parameters on foamfractionation. Because almost all parameters investigated were significant on foam frac-tionation and the dependencies between the parameters were not linear (various inter-actions) (cf. chapter 5, 6), for each new scale, the influence of the parameters has tobe analyzed again. Crofcheck and Gillette [160] investigated the scale-up for a semi-batch foam fractionation system. They varied different parameters like gas flow rate andcolumn dimensions and determined conditions for optimal performance as the authorssuggested before. Additionally, relationships between the parameters investigated andscale-up characteristics, like height to diameter ratio (H/D) were regarded. A generalscale-up characteristic was not found. Other authors [10, 80, 161] also deal with theenlargement of foam fractionation columns. However, the columns are set to a certaindimension without explaining the enlargement. Uraizee and Narsimhan [149] studied theinfluence of changing liquid height and foam height to coalescence and adsorption beha-vior, but no scale-up strategy was developed. In this chapter, the following methodologyis used to enlarge foam fractionation columns within lab-scale: First, an operating rangefor stable foam fractionation is investigated. Therefore, gas and feed superficial velocityare varied for the initial column and an operating range is defined. Second, activity pro-files in the initial column are measured by varying gas superficial velocity in the operatingrange defined. It can be shown that the column length needed for a specific recovery isinverse proportional to the gas superficial velocity. Thus, the column length, the feed andgas superficial velocity are determined for stable and efficient foam fractionation in twosteps. Finally, the initial column is enlarged. Scale-up approaches like constant heightto diameter (H/D) ratio and constant height to varying diameter, an approach derivedfrom moving bed chromatography, are discussed in terms of feasibility and separationefficiency. The approach from moving bed chromatography is chosen because Talmonand Rubin [147] compared foam fractionation with chromatography and showed that this

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80 7 A contribution to the scale-up of foam fractionation

technique was applicable for pulse as well as for continuous experiments. To carry thisapproach on, chromatography is replaced to moving bed chromatography, whereas themoving solid particles represent the moving foam phase.

Because the target enzyme cutinase is separated out of crude culture supernatant ofthe basidiomycete Coprinopsis cinerea an approach to standardize the broth for foamfractionation experiments is developed. The experiments presented in this chapter areoperated continuously in stripping mode. This mode of operation has proven to stabilizethe foam fractionation process, especially for bigger columns (see chapter 6).

7.1 Materials and methods

7.1.1 Feed preparation

The supernatant was preheated to 27.5 °C and adjusted to a pH of 7.0. The surfacetension of the supernatant was adjusted by diluting the supernatant with water. Thesurface tension was measured according to the method of Lecomte Du Noüy [148] usingthe ring-tensiometer K10ST (Krüss GmbH, Hamburg, Germany).

7.1.2 Configuration of equipment and experimental procedure

Three different foam fractionation columns were used (dimensions see Table 7.1). ColumnD30 and D44 have the same height to diameter ratio, whereas columns D44 and columnD54 have different diameters at constant length.

Table 7.1: Dimensions of foam fractionation columns

Total length [mm] Diameter [mm]

Column D30 610 30

Column D44 895 44

Column D54 895 54

Column D30 and Column D44 were equipped with 4 sampling points, column D54 withone sampling point. The sampling points were equally positioned over the length of thecolumns. Additionally, a feed inlet and an outlet for the depleted liquid were placed atthe foam fractionation column (Fig. 7.1). With a porous glass frit (16 - 40 μm pores) thefoaming gas air was dispersed in the liquid. To maintain a constant temperature duringthe experiments, the column was jacketed (temperature adjustment using thermostat;

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7 A contribution to the scale-up of foam fractionation 81

cooling/heating medium H2O). The experiments were carried out as described in chapter6. After achieving steady state samples from a sampling point were taken by opening thescrew cap. After taking one sample from a sampling point the next sample can be takenfirst after achieving steady state operation again. Samples from the feed solution, thedepleted liquid (retentate), the sampling points, and the collapsed foam (foamate) weretaken in steady state operation and used for the activity assay according to the method ofchapter 5. To describe the separation performance the enrichment (EC) and the recoveryof active enzyme (R) were used (see chapter 3)

Gas

Flow meter

Foamate

Porous frit

Tempered feed

Retentate

Peristaltic pump

Peristaltic pump

vacuum pump

Sampling

points

1

2

3

4

Heating water

Heating water

Figure 7.1: Continuous foam fractionation column with sampling points

7.2 Results and Discussion

7.2.1 Dependency between surface tension of feed and

performance of foam fractionation

According to Britten and Lavoie and Charm et al. [12, 108], the concentration of surface-active molecules is of main importance for efficient separation using foam fractionation.Because culture supernatant of C. cinerea is used, the concentration of a single surface-active molecule is hardly determinable and the composition of the supernatant can varyfrom batch to batch. Thus, a standardization of the culture supernatant for maintaining

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82 7 A contribution to the scale-up of foam fractionation

equal foaming behavior is important. Because the surface tension is related to the con-centration of surface-active molecules the surface tension was adjusted to a certain valueto achieve more even foam properties. Therefore, different dilutions of the culture super-natants were prepared. The surface tension of each dilution was measured and foamfractionation experiments under equal conditions (pH 7.0, 27.5°C, feed superficial velocity2.8 cm min−1, gas superficial velocity 4.2 cm min−1, and column D30 with 200 mm liquidheight) were performed.

0

2

4

6

8

10

12

14

0

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20

25

44 46 48 50 52 54 56

Enri

chm

ent f

acto

r [-]

Rec

over

y of

act

ivity

[%]

Surface tension [mN m-1]

a)

0.06

0.05

0.04

0.03

0.02

0.01

0.0 44 46 48 50 52 54 56

Foam

ate

to fe

ed ra

tio

[mL/

mL]

Surface tension [mN m-1]

b)

Figure 7.2: a) Foam fractionation: • Recovery of activity, � enrichment factor for differentdiluted supernatants and surface tensions. Data calculated in relation to undilutedsupernatant; b) Corresponding foamate to feed ratio

In figure 7.2 a) the recovery and enrichment as functions of the surface tensions are pre-sented. The results for recovery and enrichment are recalculated for nondiluted initialsolution to get comparable values. Fractions of foamate and retentate were collectedover 10 minutes after achieving stationary operation. The recovery related to undilutedsupernatant decreased with increasing dilution, because a high concentration of surface-active substances led to a stable and wet foam. Thus, adsorbed as well as free cutinase

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7 A contribution to the scale-up of foam fractionation 83

in the lamellar liquid was entrained into the foam phase. With increasing dilution thefoam got less stable and the liquid hold-up in the foam decreased. Thus, only adsorbedmolecules were carried into the foam phase decreasing the recovery. The enrichment factorsteadily increased as a consequence of decreasing liquid hold-up in the foamate. Figure7.2 b) presents the corresponding foamate to feed ratio for the foam fractionation experi-ments plotted against the surface tension. The foamate volume decreased with increasingdilution and surface tension. Considering the performance of the foam fractionation ex-periments and the foam stability of the system, it can be said that the best operationfor foam fractionation was between 47 mN m−1 and 53 mN m−1. Foams of feeds withsurface tension above 53 mN m−1 were instable and partly collapsed in the foam frac-tionation device. Foams of feed solutions with a surface tension below 47 mN m−1 weretoo stable and no real separation (low EC) was achieved. Thus, a surface tension of50 ± 1 mN m−1 was assumed to be adequate for foam fractionation experiments. For allfollowing experiments, the feed surface tension was adjusted to 50 ± 1 mN m−1. Hence,foaming behavior, foam stability and separation efficiency are ensured to be independentof the quality of the fermentation batch. Due to the standardization, foam fractionationexperiments under equal conditions but different fermentation batches led to deviationsof less than 5 % for recovery as well as for enrichment.

7.2.2 Operating range of column (D30)

For foam fractionation the amount of feed and gas applied to the column has to bedetermined. Column D30 was exemplarily used to define the operating range of the foamfractionation process for different feed and gas flow rates.

Operating range as a function of gas superficial velocityThe gas superficial velocity was varied from 4.2 cm min−1 to 21.7 cm min−1 and all otherparameters were kept constant: pH 7.0, 27.5°C, feed superficial velocity 2.8 cm min−1,and column D30 with 200 mm liquid height). In figure 7.3 recovery and enrichment forthe experiments and the corresponding foamate to feed ratios are presented. Fractions offoamate and retentate were collected over 10 minutes after achieving stationary operationby changing the collecting vessels for retentate and foamate.

Up to a gas superficial velocity of 14.2 cm min−1 recovery and enrichment showed constantvalues. Additionally, the foamate volume was constant in this gas flow range. The contacttime of a single bubble with the enzyme decreased with higher gas superficial velocitiesleading to less stabilized foam. Thus, the liquid hold-up in the foam decreased and bubblesize increased with increasing column height (cf. Fig. 7.4a)-b)). On the other hand,higher gas velocities led to a higher amount of bubbles per time. Thus, the availablebubble surface for adsorption increased leading to higher recoveries. Additionally, the

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84 7 A contribution to the scale-up of foam fractionation

0

10

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30

40

0

20

40

60

80

100

0 5 10 15 20 25

Enri

chm

ent f

acto

r [-]

Rec

over

y of

act

ivity

[%]

Gas superficial velocity [cm s-1]

a)

0.0

0.02

0.04

0.06

0.08

0 5 10 15 20 25

Foam

ate

to fe

ed ra

tio

[mL/

mL]

Gas superficial velocity [cm s-1]

b)

Figure 7.3: a) Recovery and enrichment for foam fractionation experiments in column D30

at varying gas superficial velocities. • Recovery of activity, � enrichment factor. b)Corresponding foamate to feed ratio for varying gas superficial velocities

foam flow rate increased so that the separation efficiency and the foamate to feed ratiowere constant although the foam structure changed (cf. Fig. 7.4a)-b)).

For gas superficial velocities above 19.8 cm min−1 the foam became more instable andfor a gas superficial velocity of 21.7 cm min−1 the foam left the column batch-wise only.The contact time between surface-active molecules and bubbles was too short for efficientstabilization and the foam collapsed in the column. The collapsed foam drained backand stabilized upcoming bubbles. This phenomenon reran till a certain amount of foamwas stable enough to leave the column resulting in batch-wise foam overflow. These foamamounts were composed of relatively small bubbles entraining a high amount of feedsolution directly into the foamate collecting vessel (Fig. 7.4). The foamate volume wasdoubled increasing the recovery to 98 % and decreasing the enrichment to a value of 16(Fig. 7.3a)).

Operating range as function of feed superficial velocity

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7 A contribution to the scale-up of foam fractionation 85

Figure 7.4: Picture of foam fractionation experiments with gas superficial velocityscreening: a) 4.2 cm min−1 and b) 14.2 cm min−1, c) 21.7 cm min−1 (pH 7.0, 27.5°C,feed superficial velocity 2.8 cm min−1, and column D30 with 200 mm liquid height)

With increasing feed superficial velocity the concentration of surface-active moleculesper cross section area increases. For the feed screening a gas superficial velocity of14.2 cm min−1 was chosen. With this gas superficial velocity a continuous foam over-flow could be expected during the experiments. Because lower superficial gas velocitiescreated enough bubble surface to recover cutinase at constant superficial feed velocity, itcan be assumed that for a gas superficial velocity of 14.2 cm min−1 higher amounts ofsurface-active molecules can be handled, too (cf. Fig. 7.3a)). The feed superficial velocitywas varied from 1.4 cm min−1 to 5.0 cm min−1 at constant parameters (pH 7.0, 27.5°C,gas superficial velocity 14.2 cm min−1, and column D30 with 200 mm liquid height). Theresults of the foam fractionation experiments and the foamate to feed ratios are presentedin figure 7.5. Fractions of foamate and retentate were collected over 10 minutes afterachieving stationary operation by changing the collecting vessels for retentate and foa-mate.

As expected, for the feed superficial velocity lower than used for the gas flow screeningexperiments the foam was more destabilized as a consequence of a decreased amountof surface-active molecules. Thus, the liquid hold-up in the foam decreased, decreasingfoamate volume and recovery of enzyme and highly increasing enrichment. For higher feedsuperficial velocities a constant recovery of around 90 % could be achieved. Enrichment,however, decreased with increasing feed flow. With increasing feed flow more surface-active molecules adsorbed to the bubble surface. Thus, the foam stabilized and effectslike drainage and coalescence in the foam phase decreased. As a result, a high amountof feed liquid was entrained into the foamate decreasing enrichment and keeping recovery

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86 7 A contribution to the scale-up of foam fractionation

0

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0 1 2 3 4 5 6

Feed superficial velocity [cm s-1]

Enri

chm

ent f

acto

r [-]

Rec

over

y of

act

ivity

[%]

a)

0.0

0.02

0.04

0.06

0.08

0 1 2 3 4 5 6

Foam

ate

to fe

ed ra

tio

[mL/

mL]

Feed superficial velocity [cm s-1]

b)

Figure 7.5: a) Recovery and enrichment for foam fractionation experiments in column D30

at varying feed superficial velocities. • Recovery of activity, � enrichment factor. b)Corresponding foamate to feed ratio for varying feed superficial velocities

constant (foamate flow increased linearly with increasing feed superficial velocity (Fig.7.5b)). The same trend of decreasing enrichment and similar recoveries was observed forexperiments with varying feed dilution (cf. Fig. 7.2a)). To gain more knowledge if thiseffect is based on the feed liquid amount or the amount of surface-active molecules in thefeed, a supplementary experiment was carried out. In comparison to the experiment witha surface tension of 50.1 mN m−1 in figure 7.2a) an experiment with a surface tensionof 46.2 mN m−1 (doubled feed activity) and halved feed superficial velocity was run.Thus, per time the same amount of surface-active molecules entered the column but theliquid amount of the feed varied. A recovery of 18.8 % and an enrichment factor of 10.5were obtained in comparison to 14 % recovery and an enrichment factor of 7.7 for theexperiment with a surface tension of 50.1 mN m−1 (see Fig. 7.2a)). The next step wasthe comparison of the 50.1 mN m−1 experiment with the experiment with 46.2 mN m−1

(doubled feed activity) and constant feed superficial velocity (cf. Fig. 7.2a)). Thus, the

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7 A contribution to the scale-up of foam fractionation 87

liquid amount in the feed per time was equal and the feed activity varied. Accordingto figure 7.2a) a recovery of 18.0 % with an enrichment factor of 4.0 was obtained forthe 46.2 mN m−1 and equal feed superficial velocity experiment. Therefore, both amountof surface-active molecules and amount of liquid containing the surface-active moleculesinfluenced the separation efficiency. For efficient separation the amount of surface-activemolecules in relation to the available surface need to be determined and the amountof feed liquid should be low for strengthening drainage and to facilitate the access ofsurface-active molecules to the bubble surface.

7.2.3 Activity profiles of column (D30)

As shown in the previous section, foam fractionation experiments can result in the sameseparation performance, even though the stability of the system and the mechanismscausing the separation are different. To get a better insight into the foam fractionationprocess activity distribution over the column length was investigated. In stationary opera-tion samples from the sampling points were taken and analyzed. As examples, the activityprofiles of the gas superficial velocity experiments for 4.2 cm min−1, 14.2 cm min−1, and21.7 cm min−1 are presented in figure 7.6 exemplarily.

0

10

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50

60

70

80

90

0 100 200 300 400 500

Enzy

mat

ic a

ctiv

ity [U

L-1

]

Stripping length [mm]

Feed solution

Figure 7.6: Activity profiles of column D30 at varying gas superficial velocities (pH 7.0,27.5°C, surface tension 50 ± 1 mN m−1, feed superficial velocity 2.8 cm min−1, andstripping length 370 mm). � gas superficial velocity of 4.2 cm min−1, � gas superficialvelocity of 14.2 cm min−1, and • gas superficial velocity of 21.7 cm min−1

The sampling points were placed every 100 mm beneath the feed port. The total strippinglength was the length from the feed port to the liquid phase and had the value 370 mm forcolumn D30. Sampling point number 4 was located in the liquid phase and the enzymatic

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88 7 A contribution to the scale-up of foam fractionation

activity therefore represented the enzymatic activity of the retentate for the experimentsinvestigated. Thus, for the experiments the whole recovery process took place in the foamphase. For a gas superficial velocity of 21.7 cm min−1 the enzymatic activity decreaseddrastically over the column length (see Fig. 7.6). For a stripping length of 200 mm thefeed was depleted about 93 %. Thus, the amount of surface-active cutinase in the feedliquid was not able to stabilize the bubbles over the whole foam column or strippinglength. The foam collapsed in the column resulting in a batch-wise foam overflow (seeFig. 7.4c)). An additional experiment was carried out to investigate if a stripping lengthof 200 mm was enough to recover approx. 90 % of the cutinase. The experiment wasrun under equal conditions but only a stripping length of 200 mm. A recovery of 93 %and an enrichment factor of 15 were obtained. Thus, only 35 % of the available strippinglength was necessary to almost completely recover cutinase (see Fig 7.3a)). Based onthe activity profiles, for a gas superficial velocity of 14.2 cm min−1 a stripping length ofapprox. 300 mm and for a gas superficial velocity of 4.2 cm min−1 a stripping length of 340mm was sufficient to deplete the feed about 90 %. Due to the increase in gas superficialvelocity the available interfacial area increased. Because the amount of surface-activemolecules was equal for all experiments the stripping length or foam column could beshortened. By means of the three activity profiles and the additional experiment withshortened stripping length, the dependency between gas superficial velocity and strippinglength necessary for efficient separation was linear. Nevertheless, the experiments withgas superficial velocities above 14.2 cm min−1 yielded in instable systems. Thus, forgeneral statements stable systems had to be investigated. Hence, activity profiles for gassuperficial velocities of 7.1 cm min−1 and 11.3 cm min−1 were measured. Gas superficialvelocities less than 4.2 cm min−1 were not analyzed because it was assumed that thebubble surface generated or the stripping length available was not efficient for recovering90 % of the enzyme. The excess of surface-active molecules would be discharged withthe retentate and the recovery will decrease. In figure 7.7a) the slopes of the linear partof the activity profiles are displayed for gas superficial velocities between 4.2 cm min−1

and 14.2 cm min−1. For these gas flows stable foams were generated and continuousfoam overflows were observed. In figure 7.7b) the corresponding stripping lengths neededfor 90 % depletion of the feed solution calculated via the slopes of the activity profilesare depicted. It can be stated, that the stripping length needed to recover 90 % of thefeed activity under the conditions chosen was a linear function of gas superficial velocity.Additionally, it was shown that the depletion of the feed solution was a linear functionof stripping length (see Fig. 7.6). Thus, the loading capacities of the bubbles could bedetermined more precisely, because the activity in the lamellar liquid could be measuredor calculated. Due to the inverse proportional dependency of stripping length and gassuperficial velocity the foam fractionation column for stripping mode could be designed.Obvious other authors [109, 112] defined the liquid pool height as an impact parameter,

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7 A contribution to the scale-up of foam fractionation 89

however, the enzymatic activities for sampling points below the liquid level were equalto the enzymatic activity in the retentate. Thus, the liquid pool height was a parameterfree of choice and the stripping length could be calculated. With the experiments done,the feed and gas superficial velocity and the length of foam and liquid column could bedetermined for efficient and stable foam fractionation.

a) b)

R² = 0.9928

-0.32

-0.31

-0.3

-0.29 0 5 10 15

Slop

e

Gas superficial velocity [ cm min-1]

R² = 0.9891

290

300

310

320

330

340

350

0 5 10 15 St

ripp

ing

leng

th [m

m]

Gas superficial velocity [cm min-1]

Figure 7.7: a) Slope of the linear activity profiles from sampling point 1 to 3 at varyinggas superficial velocity. b) Stripping lengths calculated via the slopes of the activityprofiles to gain a feed depletion of 90 %

7.2.4 Scale-up approaches

In the previous sections some general statements on the operating range of column D30

were determined. Too stable foams as well as instable systems were not promising forefficient separation. The structure of the foam was mainly influenced by the amount ofsurface-active molecules and interface provided. Two different scale-up approaches shallbe investigated. First, the general approach of keeping the ratio of height to diameterconstant and second, a scale-up approach derived from chromatography was discussed.For both scale-up approaches, the ratio of the feed and gas superficial velocity were equalto the respective experiment in column D30 to ensure similar foam structure and stability.

The first scale-up approach investigated was the constant ratio of height to diameter.For these experiments, the experiment in column D30 with a gas superficial velocity of14.2 cm min−1 and a feed superficial velocity of 2.8 cm min−1 was compared to foamfractionation experiments in column D44 and D54 with equal superficial velocities andequal ratio of foam to liquid height (H/D ratio). In figure 7.8 the activity profiles ofcolumn D30 and column D44 are depicted. For column D44 the sampling points wereplaced every 160 mm below the feed port. Sampling point 4 was located in the liquidpool, and the enzymatic activity was equal to the retentate activity. The stripping lengthof column D44 was 550 mm. For the foam fractionation experiment in column D44 a

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90 7 A contribution to the scale-up of foam fractionation

recovery of 87 % and an enrichment factor of 44.5 could be obtained (D30: recovery:91 %, enrichment factor 24.6).

0

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30

40

50

60

70

0 20 40 60 80 100 120 140

Enzy

mat

ic a

ctiv

ity [U

mL-1

]

Percent of stripping length [%]

Figure 7.8: Activity profiles of column D30 and D44 for the scale-up approach of constantH/D ratio. � Enzymatic activity profile in column D30, � enzymatic activity profile incolumn D44

The recoveries as well as the activity profiles over the columns were similar. The enrich-ment factor, however, strongly increased in column D44. Additionally, the bubble sizeslightly increased with column height. This may be an effect of the decreasing supportingeffect of the column wall in column D44 compared to column D30 [13]. As a result, thedrainage rate increased, decreasing the liquid hold-up in the foam and increasing bubblesize and enrichment, respectively. The scale-up experiment in column D54 (strippinglength 675 mm) using constant H/D ratio yielded a recovery of 95 % and an enrichmentfactor of 52. Compared to column D44 the supporting effect of the column was even lowerexplaining the increase in enrichment. The approach of constant H/D ratio combined withconstant ratio of gas and feed superficial velocity yielded in a similar foaming behavior.The enrichment factor was improved due to the increase in column diameter and therecovery was kept constant. Thus, the approach is proved to be efficient for scaling-upfoam fractionation. Nevertheless, the approach is limited in the choice of column diameter,because at a certain diameter the foam will become instable and the system, too.

The second scale-up approach investigated derived from chromatography, especially mov-ing bed chromatography. According to Antia [162], the ratio of liquid to solid flows has tobe constant for scale-up. Additionally, as for other forms of chromatography the columnlength and the particle size of the bed must be kept constant. The scale-up is then simplydone by increasing the column diameter and increasing all liquid flows in proportion tothe cross-sectional area [162]. In case of foam fractionation the feed superficial velocitymust be held constant for all columns investigated. Based on the fact, that the adsorptionbed must be equal for all column sizes the parameters of the experiment with the mosthomogeneous foam were chosen. Thus, the experiment in column D30 with a feed superfi-cial velocity of 2.8 cm min−1 and a gas superficial velocity 4.2 cm min−1 was chosen for the

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7 A contribution to the scale-up of foam fractionation 91

scale-up experiments (cf. Fig. 7.4). The columns D44 and D54 were investigated, whereasthe gas superficial velocity and the stripping length were kept constant (370 mm accordingto the experiment in column D30). The results of the foam fractionation experiments aresummarized in Table 7.2.

Table 7.2: Separation efficiency of scale-up experiments in column D30, D44, and D54

Column Recovery of activity [%] Enrichment factor [-]

D30 90 24.7

D44 73 56.5

D54 51 58.7

With increasing column diameter recovery decreased and enrichment increased. For foamfractionation experiment in column D44 homogeneously distributed foam existed, but onaverage with bigger bubbles compared to the foam fractionation experiment in columnD30. Due to the bigger column diameter and decreasing supporting wall effect bubble sizeand enrichment increased. Retentate activity as well as the activity at sampling port 3,which was 110 mm below the liquid level, was five times higher than retentate activity forthe experiment in column D30 under equal conditions. Thus, the whole recovery processtook place in the foam phase and the stripping length seemed to be too short for completerecovery under the conditions chosen. In column D54 a continuous foam overflow existed,but the bubble size increased with increasing column height. Thus, a uniform bubble bedwas not realized. The bubble size increased significantly near the feed port. It can beassumed, that feed molecules were not homogeneously distributed over the column so thatthe bubbles were not efficiently stabilized to resist the mechanical stress in the column.Additionally, the stripping length was not long enough for efficient recovery. Higher gassuperficial velocity needed to be applied to the systems D44 and D54 generating morebubble surface so as to recover more enzymes. The approach of constant gas superficialvelocity does not seem to be not suitable to scale-up foam fractionation. Altogether, thescale-up approach on the basis of moving bed chromatography needs to be investigatedfurther and the gas flow needs to be adjusted to the requirements.

7.3 Conclusion

In this chapter, a methodology for enlarging foam fractionation was executed. First,experiments in an initial column were carried out to identify good operating ranges (sur-face tension, gas and feed superficial velocities) for stable and efficient foam fractionation.Second, activity profiles were analyzed, where inverse proportional linear dependency

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92 7 A contribution to the scale-up of foam fractionation

between stripping length and gas superficial velocity were found. As a result, the foamfractionation column could be designed for efficient separation. On the basis of the knowl-edge gained, the foam fractionation process was „scaled-up” within lab-scale. Traditionalscale-up approaches like constant height to diameter (H/D) ratio and varying diameter atconstant length were discussed in terms of separation efficiency and foam stability. Theapproach of constant H/D ratio led to a similar foaming behavior in all columns inves-tigated. The enrichment factor, however, increased with increasing column size. Thus,the approach seems to be applicable for scaling-up foam fractionation, if considering thefoam stability carefully.

For the scale-up approach derived from chromatography, the foaming behavior was notsimilar for the columns investigated. The foam in column D54 differed strongly to theexpected homogeneous foam structure. The recovery decreased and the enrichment factorincreased with increasing column size. Thus, the approach with the conditions chosen wasnot applicable for scaling-up foam fractionation. However, experiments should be done,in which the gas superficial velocity is adjusted to the different column sizes to generatemore gas-liquid interface increasing the recovery.

Altogether, on the basis of the three steps methodology appropriate gas and feed superfi-cial velocities were chosen, the column lengths could be adjusted to different requirementsand the process could successfully be enlarged.

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8 Design guideline for foamfractionation processes

In this thesis, foam fractionation was investigated to recover and concentrate the enzymecutinase out of culture supernatant of the basidiomycete Coprinopsis cinerea. For batchand continuous foam fractionation various process parameters, like pH value or tempera-ture, and column design parameters, such as length or diameter of the foaming columnwere systematically investigated with regard to separation efficiency and foam stability.Impact parameters and parameter interactions were identified using Design of Experiment.Thus, it was possible to tailor the foam fractionation process to desired yields and/or en-richments. For continuous foam fractionation the operation modes enriching and strippingwere examined, whereas stripping mode was found to be more efficient. Experiments withdyes and surface tension measurements further increased the understanding of the foamfractionation process. By means of the experiments knowledge has been gained which inthis chapter is summarized in a guideline to simplify screening of enzyme systems and toadjust foam fractionation to user requests. During the guideline development, not onlythe separation and enrichment of cutinase out of culture supernatant was investigated, butalso PLA2 out of culture broth, and bromelain as pure component system. Additionally, 5non-foamable enzymes out of culture supernatant or culture broth were tested to confirmthe guideline developed.

8.1 Equipment

Before screening of enzyme candidates and first foam fractionation experiments start, theequipment listed should be available:

− Surface tension measurement device

− Standard foam fractionation column: diameter of 3.0 cm and length of 50 cm. To usethe column for continuous foaming a retentate port at the bottom of the column andtwo feed inlet ports 10.0 cm and 49.0 cm away from the bottom should be available

− Foam breaker

− Flow meter for the adjustment of gas flow

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− Frits: Pore size 10 μm to 16 μm, pore size 16 μm to 40 μm, and pore size 40 μm to100 μm (or comparable gas distributors)

− Volume increasing fixtures which can be placed on top of the standard foam frac-tionation column: column with diameter of foam column (15 cm, 30 cm) or glassbowl (100 mL, 300 mL, 500 mL)

For continuous foam fractionation:

− Two pumps with an operating range of 2 mL min−1 to 200 mL min−1

8.2 Design guideline for foam fractionation

In figure 8.1 the guideline developed is depicted. In the following all steps of the guidelineand troubleshooting are described targeting at a fast and efficient adjustment of foamfractionation to certain test systems.

1st Step: Test systemThe workflow can be applied to pure component systems, extracellular enzymes in culturesupernatant and culture broth of different microbial sources and different foamability. Incase of foaming culture broths, the flotation of biomass needs to be considered. Dependingon size and weight, the amount of floated biomass can vary and the possibility exists thatthe whole foaming device can be blocked. The blocking tendency of the culture brothneeds to be considered in step 5. To collect cell-free foamate and to facilitate the use offurther separation techniques after foam fractionation, a filter centrifuge can be used todestroy the foam and to separate the biomass. If the use of culture broth is not manda-tory, the use of culture supernatant is recommended.

2nd Step: Determination of foamabilityUnder the assumption, that the target surface-active molecule mainly affects a changein surface tension, surface tension profiles at varying pH values can provide informationwhether a system can be foamed or not. If a surface-active substance is present in thefeed solution, a minimum in surface tension for a certain pH can be observed. If thesystem does not contain a surface-active agent, the surface tension will either decreasewith increasing pH due to the denaturation of enzymes present in the sample or at leastwill stay constant.

Thus, a screening can be performed without a single foam fractionation experiment. Forthe pH/surface tension profile measurements samples of pure component solved in wateror buffer in concentrations as defined for its application, found in literature, or at least

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8 Design guideline for foam fractionation processes 95

(a) Part I

Figure 8.1: Design guideline for foam fractionation processes part 1

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96 8 Design guideline for foam fractionation processes

(b) Part II

Figure 8.1: Design guideline for foam fractionation processes

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8 Design guideline for foam fractionation processes 97

(c) Part III

Figure 8.1: Design guideline for foam fractionation processes

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98 8 Design guideline for foam fractionation processes

(d) Part IV

Figure 8.1: Design guideline for foam fractionation processes

below the solubility limit, or samples of culture supernatant or broth as derived afterfermentation are adjusted to different pH values, commonly from pH = 3.0 to pH = 11.0.The surface tension should be measured against air at room temperature at least threetimes to determine standard deviations. If the enzyme denaturates in contact with air, thesurface tension is measured against nitrogen. For all pH adjusted samples the activity ofthe target enzyme needs to be checked to identify changes in stability. Additionally, acidsand bases in adequate molarity should be used to avoid the dilution of samples duringpH adjustment. Dependent on enzyme stability or activity, precipitation behavior, andenzyme application the pH range can be specifically decreased or increased. In case theminimal value(s) of surface-tension is/are on the border of the pH range investigated thepH range should be enlarged.

In case several minima are observed in the pH range investigated (possible for culturesupernatant or broth samples) the pH value yielding minimal surface tension correlatingto the target surface-active component can be selected due to the nearness to the isoelectricpoint of the target component. In case no information is available, all minima should bechecked in the following steps (step 3 - 5).

If no minimum in pH/surface tension profile is observed, the system is in all probabilitynot foamable (check buffer influence on the surface tension measurements for pure com-ponent systems). Nevertheless, a direct screening experiment (step 5) with pH = pI ofthe target component should be performed to be sure whether the system is foamable ornot (dashed line in Fig. 8.1). In case foam is emerging and fulfills the requirements instep 5, the investigation should hearken back to step 3 of the guideline(dashed line in Fig.8.1). If no foam is emerging the system is not foamable.

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3rd Step: Determination of temperature dependency

If a minimum in the pH/surface tension profile exists, the temperature dependence can beinvestigated by surface tension profiles, too. Samples of pure component system (concen-tration as for step 2), culture supernatant, and culture broth as derived after fermentationare adjusted to the pH value yielding minimal surface tension (according to the pH/surfacetension profiles of step 2) and to different temperatures. The temperature is commonlyvaried in steps of 5 °C or 2.5 °C from 20 °C to the temperature to which the enzymefulfills the customer and/or application requirements. Temperatures below 15 °C nega-tively affect the foamability of the systems investigated. In case the system is temperaturedependent a minimum in surface tension can be determined (consider standard deviation).The temperature range should be enlarged, in case the minimal value(s) of surface-tensionis/are on the border of the temperature range. Additionally, the long-term stability ofthe enzyme, especially for higher temperatures should be checked.

If no clear minimum is observable, the foam fractionation process should be operated atroom temperature (20 °C - 25°C) to save energy costs. If the energy costs (heating orcooling) are too high to adjust the foaming system to a certain temperature, a batchscreening experiment at room temperature should be operated under equal conditionsas the experiment with the temperature yielding in minimal surface tension (T/surfacetension profile). Depending on the separation efficiency (R, EC) a cost calculation cansupport the selection of the temperature.

4th Step: Determination of concentration dependencyThe last surface tension profile investigated conduces to the determination of the criticalmicelle concentration and can be used to standardize the feed solution to ensure equalfoaming behavior and separation efficiency independent from the quality of the fermenta-tion batch. In case of pure component systems standardization is not necessary. Samplesof culture supernatant and culture broth are adjusted to pH and temperature obtainedfrom step 2 and 3 and are differently diluted (see Fig. 8.2). In case of pure componentsystems the initial concentration should be varied in a range equivalent to the applicationrequirement, or according to literature. The initial concentration has to go far below thesolubility limit, to avoid precipitation because of the concentration during foam fractiona-tion. Similar to culture supernatant or broth samples pH value and temperature areadjusted as obtained from step 2 and 3.

In case the concentration/surface tension profile reaches a plateau the critical micelleconcentration is achieved or exceeded, respectively. Concentrations above the criticalmicelle concentration can limit the foamability of the enzyme system. Thus, the inves-tigation of lower concentrated sample solutions is recommended. Concentrations in the

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100 8 Design guideline for foam fractionation processes

30

35

40

45

50

55

Surf

ace

tens

ion

[mN

m-1

]

Concentration

Figure 8.2: Concentration/surface tension profile with classification of concentrationranges

transition area of the concentration/surface tension profile (see Fig. 8.2) show the bestfoaming properties. Nevertheless, the initial feed solution (concentration above CMC)needs to be considered to compare the foaming properties to the sample solution withconcentrations in the transition area of the concentration/surface tension profiles. This isnecessary as in some cases the CMC does not affect the foamability and thus a dilutionand increased feed volume can be avoided.

In case a critical micelle concentration is not reached for fermentation samples or purecomponent system samples, concentrations higher than those of the linear range of theconcentration/surface tension profile should be considered for further experiments (seeFig. 8.2). If that is not possible, because the concentration/surface tension profile isonly linear, the concentration range of the pure component system should be increased(depending on costumer and application requirements) and initial culture supernatant orbroth should be used for experiments in step 5.

5th Step: Batch screening experimentsFor the first batch screening experiments the standard foam fractionation column (cf.

section 8.1 with a frit of 16 μm to 40 μm pore size and a gas flow rate of 50 mL min−1(airor nitrogen for oxygen sensitive enzymes) should be used. 100 mL of the feed solu-tion (culture supernatant, broth, or pure component system) with concentrations in thetransition area of the concentration/surface tension profile or as described in step 4 forexceptional cases (step 4, Fig. 8.2) should be adjusted to pH and temperature obtained

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8 Design guideline for foam fractionation processes 101

from the minima in the surface tension profiles determined in step 2 and 3 and filled intothe foam fractionation column.

As mentioned in step 4, for culture supernatant and broth the surface tension can beused to standardize the feed solution to ensure equal foaming behavior and separationefficiency independent from the quality of the fermentation batch. The standard valueof surface tension or concentration is the value, where continuous foam overflow existsand a separation occurs. In case the separation efficiency (EC , R) of foam fractionationexperiments at varying concentrations do not differ, cost calculations should support theselection of the standard surface tension value.

In case the critical micelle concentration is exceeded in the concentration/surface ten-sion profile (Fig. 8.2) a foam fractionation experiment under the same conditions, butwith initial culture supernatant or broth, or pure component systems with concentrationsabove the CMC should be operated. Thus, differences in foaming behavior and sepa-ration efficiency compared to experiments with concentrations below the critical micelleconcentration can be observed. In case the exceedance of the CMC does not affect theseparation efficiency and a continuous foam overflow exists the standard value for surfacetension equates the surface tension value for the CMC.

All experiments should be ended if no continuous foam overflow exists, but definitely after60 minutes to shorten time for the screening experiments. An explicit determination offoaming time is carried out in step 6, where the improvement of batch foam fractionationis described. If no or little foam or froth is emerging during the screening experimentsconsider the troubleshooting for batch screening experiments.

As a result of the batch screening experiments the foamability parameters pH value andtemperature obtained from step 2 and 3 and standard surface tension are determined toyield a stable foaming system. Additionally, the performance parameters gas flow rate,frit pore size, and liquid volume used for the batch screening experiment or changed dueto troubleshooting to yield continuous foam overflow are defined. For further experimentslike the improvement of batch foam fractionation or the transfer to continuous foam frac-tionation the foamability and performance parameters defined in this step for stable foamfractionation represent the standard experimental batch conditions.

Troubleshooting for batch screening experiments

a) In case no or little foam (< 10 cm foam height) is emerging, even if using initial culturesupernatant or broth, or the maximal initial concentration for the pure componentsystem, batch screening experiments under the same conditions but with pH values± 1.0 from the minimum achieved in step 2 should be investigated. If still no foam

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is emerging, it can be assumed that the surface-activity of the target molecule is notefficient enough to generate stable foams.

b) If the foam is rising up to a certain height (≥ 10 cm foam height) and stays constant,the surface tension needs to be adjusted to a lower value to increase foamability. Incase the foam height is still constant and initial culture supernatant or broth is usedor the maximal initial concentration for the pure component system is reached thefollowing parameters should be successively changed until a continuous foam overflowexists. First, the gas flow rate should be increased in steps of 10 mL min−1. Second,a frit with a pore size of 10 μm to 16 μm should be tested at varying gas flow rates(starting at the gas flow rate set in step 5 and higher; steps of 10 mL min−1). Thenthe liquid volume should be increased (maximal liquid volume 50 % of total foamfractionation column volume). Finally, another column with a decreased diametercompared to the standard column (see section 8.1) should be used. First experimentsin the new column should be operated under the conditions gas flow rate, pore size,foaming time, and ratio of foam to liquid volume as described in step 5. If this is notleading to a continuous foam overflow, it can be assumed, that the surface-activity ofthe target is not high enough to form stable foams.

c) Is the foam too wet and almost the whole volume of foamate is equal to the volumeof feed solution (formation of froth instead of foam) the surface tension value of thefeed solution should be adjusted to higher values to form less stable foam. In caseall surface tension values or concentrations investigated lead to froth formation thefollowing performance parameters should be successively changed till foam is genera-ted. First, a frit with a pore size of 40 μm to 100 μm (or comparable) should beinvestigated at varying gas flow rates (starting at the standard gas flow rate (step 5)and lower; steps of 10 mL min−1). Second, the liquid volume should be decreased(minimal liquid volume 20 % of total column volume). Finally, another column withan increased diameter compared to the standard column (see section 8.1) should beused. First experiments in the new column should be operated under the standardexperimental conditions gas flow rate, pore size, foaming time, and ratio of foam toliquid volume as described in step 5. As long as froth is emerging the performanceparameters described in this troubleshooting passage can be changed over and overagain.

6th Step: Improvement of batch foam fractionationTo influence the separation efficiency of foam fractionation in batch mode performanceparameters like gas flow rate and column design are now regarded. Depending on theseparation requirements the guideline is categorized in improving recovery (R) (6a)),enrichment (EC) (6b)), or recovery and enrichment simultaneously (R, EC) (6c)). In case

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continuous foam fractionation is the operation mode favored switch immediately to step 7.

6a) Step: Increasing recovery (R)Based on the standard experimental batch conditions yielding a stable foaming system(step 5) and a foaming time of 60 minutes or less (end of continuous foam overflow), thefollowing performance parameters should be successively changed until the target recoveryis reached and no froth emerges. First, the gas flow rate VGas should be increased in stepsof 10 mL min−1. Second, a frit with a pore size of DPore= 10 μm to 16 μm (or comparable)should be investigated at varying gas flow rates (starting at the standard gas flow rate(step 5) and higher; steps of 10 mL min−1). Third, the liquid volume (Vliquidcolumn) shouldbe increased to maximal 50 % of total foam fractionation column volume. In case the foamfractionation experiments are discontinued after 60 minutes the foaming time (tfoaming)should be extended. However, this may have significant effect on separation costs. Finally,another column with a decreased diameter (DColumn) compared to the standard column(see section 8.1) should be used. The first experiments in the new column should beoperated under the standard experimental batch conditions gas flow rate, pore size, andratio of foam to liquid volume as derived from step 5. As long as a continuous foam over-flow exists, no froth is generated, and the target recovery is not reached the performanceparameters in the sequence presented can be changed over and over again (see dotted lineFig. 8.1).

6b) Step: Increasing enrichment (EC)Based on the standard experimental batch conditions yielding a stable foaming system(step 5) and a foaming time of 60 minutes or less (end of continuous foam overflow), thefollowing performance parameters should be successively changed until the target enrich-ment is reached and a continuous foam overflow exists. First, the gas flow rate VGas shouldbe decreased in steps of 10 mL min−1. Second, a frit with a pore size of DPore= 40 μmto 100 μm (or comparable) should be investigated at varying gas flow rates (starting atthe standard gas flow rate (step 5) and lower; steps of 10 mL min−1). Third, volumeincreasing fixtures (VFixtures) should be added to the standard column (see section 8.1),whereas glass bowls are more efficient than column elongations. Then the foaming time(tfoaming) should be decreased in steps of 5 to 15 minutes. Finally, another column with anincreased diameter (DColumn) compared to the standard column (see section 8.1) shouldbe used. The first experiments in the new column should be operated under the standardexperimental batch conditions gas flow rate, pore size, foaming time, and ratio of foam toliquid volume as derived from step 5. As long as a continuous foam overflow exists and thetarget enrichment is not reached the performance parameters in the sequence presentedcan be changed over and over again (see dotted line Fig. 8.1).

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6c) Step: Improvement of recovery (R) and enrichment (EC)To improve enrichment and recovery simultaneously a compromise for one response needsto be taken. In case high recovery and adequate enrichment are required the starting pointfor simultaneous improvement is the experiment in which highest recovery is obtained.Regarding the boundary conditions that the recovery required is achieved, the enrichmentcan be steadily improved by executing step 6b) of the guideline. In case high enrichmentis the main target, the starting point is the experiment in which highest enrichment isachieved. Under the boundary conditions that the enrichment required is obtained, therecovery can be steadily improved by executing step 6a) of the guideline.

However, it should be noted that not every combination of enrichment and recovery willbe possible.

7th Step: Continuous foam fractionationFor first continuous foam fractionation experiments the standard experimental batchconditions derived from step 5 should be used and the supplementary performance para-meter feed flow rate should be set to 10.0 mL min−1. To select the mode of operation,namely enrichment or stripping mode, the foam stability of the test system needs to beconsidered. If the foam is flimsy, dry, and the bubbles big during the standard batchexperiment (compare Fig. 8.3a)) stripping mode can be very efficient for stable foamfractionation. If the foam is strong, wet, and the bubbles small during batch operation(see Fig. 8.3b)) enriching mode can be interesting to yield good separation performance.However, depending on the target, the differences of stripping and enriching mode shouldbe tested for every system. Regardless of which operation mode is chosen a continuousfoam overflow is necessary during the experiments. In case no continuous foam overflowexists or froth is emerging consider the troubleshooting for continuous foam fractionation.

a) b)

Figure 8.3: a) Dry foam with big bubbles (polyhedral foam), b) Wet foam with smallbubbles (spherical foam)

Analog to step 5 standard experimental conditions can be defined. Thereby, the foama-bility parameters are kept constant and the performance parameters gas flow rate, feedflow rate, frit pore size, feed position, and column set-up equivalent to the first continuous

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foam fractionation experiment or changed due to troubleshooting leading to stable foamfractionation represent the standard experimental continuous conditions. On the basis ofthe standard experimental continuous conditions stationary operation can be investigatedin step 8.

Troubleshooting for continuous foam fractionation

a) If froth is formed instead of foam the following performance parameters should besuccessively changed until foam is formed. First, the gas flow rate should be decreasedin steps of 10 mL min−1. Second, the feed flow rate should be decreased in steps of2.0 mL min−1. Third, the pore diameter should be increased (40 μm to 100 μm orcomparable) at varying gas flow rates (starting at the standard gas flow rate (step 5)and lower; steps of 10 mL min−1). Finally, another column with an increased diametercompared to the standard column (see section 8.1) should be used. First experimentsin the new column should be operated under the standard experimental conditionsgas flow rate, pore size, foaming time, and ratio of foam to liquid volume as describedin step 5. As long as froth is emerging the performance parameters described in thistroubleshooting passage can be changed over and over again.

b) If the foam is weak and no continuous foam overflow exists, the following parametersshould be successively changed until a stable continuous foam is formed. First, in-crease gas flow rate (steps of 10 mL min−1). Second, increase feed flow rate (steps of2.0 mL min−1). Third, decrease pore diameter (10 μm to 16 μm or comparable) incombination with gas flow rate screening (starting at the gas flow rate set in step 5and higher; steps of 10 mL min−1). Finally, change the column to one with smallerdiameter. First experiments in the new column should be operated under the standardexperimental conditions gas flow rate, pore size, foaming time, and ratio of foam toliquid volume as described in step 5. As long as no continuous foam overflow existsthe performance parameters described in this troubleshooting passage can be changedover and over again.

8th Step: Determination of stationary operationSteady state operation is achieved, when the retentate activity and the liquid heightin the column (at constant pump performance) are constant. To determine stationaryoperation, experiments under standard experimental continuous conditions are operatedfor approx. 100 minutes. Every 10 minutes a sample of the retentate is collected and theactivity of the target enzyme analyzed. Additionally, the pump performance is adjustedto keep the liquid height constant. In case stationary operation is not reached in time thefoaming time needs to be extended. With respect to further continuous foam fractionation

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experiments some general statements are depicted how the time to achieve steady stateoperation is influenced:

a) Changing gas and feed flow rates do not significantly influence time for achievingsteady state.

b) With increasing column diameter the time to reach steady state operation decreases.

c) With increasing column length the time to reach steady state operation increases.

9th Step: Improving continuous foam fractionationIn general, for each mode of operation the separation efficiency (R, EC) can be influencedby performance parameter. Depending on the separation requirements the guideline iscategorized in improving recovery (R) (8a)), enrichment (EC) (8b)), or recovery andenrichment simultaneously (R, EC) (8c)).

9a) Step: Increasing recovery (R)Based on the standard experimental conditions for continuous foaming yielding a stablefoaming system (step 7) the following performance parameters should be successivelychanged until the target recovery is reached and no froth emerges. First, the gas flow rateVGas should be increased in steps of 10 mL min−1. Second, the feed flow rate VFeed shouldbe increased in steps of 2.0 mL min−1. Third, a frit with a pore size of DPore= 10 μmto 16 μm (or comparable) should be tested. If necessary, gas flow rate and feed flowrate should be varied in steps of 10 mL min−1 and 2.0 mL min−1, respectively. Then, theliquid volume (Vliquidcolumn) should be increased to maximal 50 % of total column volume.Finally, another column with a decreased diameter (DColumn) compared to the standardcolumn (see section 8.1) should be used. The first experiments in the new column shouldbe operated under the standard experimental conditions for continuous operation as de-rived from step 7. As long as a continuous foam overflow exists, no froth is generated, andthe target recovery is not reached the performance parameters in the sequence presentedcan be changed over and over again (see chain line Fig. 8.1).

9b) Step: Increasing enrichment (EC)Based on the standard experimental conditions for continuous operation yielding a stablefoaming system (step 7) the following performance parameters should be successivelychanged until the target enrichment is reached and a continuous foam overflow exists.First, the gas flow rate VGas should be decreased in steps of 10 mL min−1. Second, thefeed flow rate VFeed should be decreased in steps of 2.0 mL min−1. Third, a frit witha pore size of DPore= 40 μm to 100 μm (or comparable) should be tested. If necessary,

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gas flow rate and feed flow rate should be varied in steps of 10 mL min−1 and 2.0 mLmin−1, respectively. Then, volume increasing fixtures (VFixtures) should be added to thestandard column (see section 8.1), whereas glass bowls are more efficient than columnelongations. Finally, another column with an increased diameter (DColumn) compared tothe standard column (see section 8.1) should be used. The first experiments in the newcolumn should be operated under the standard experimental conditions for continuousoperation as derived from step 7. As long as a continuous foam overflow exists and thetarget enrichment is not reached the performance parameters in the sequence presentedcan be changed over and over again (see chain line Fig. 8.1).

9c) Step: Improvement of recovery (R) and enrichment (EC)Analog to step 6c) to simultaneously improve recovery and enrichment a compromise forone response needs to be taken. In case the recovery should be high in value and theenrichment should be increased the starting point for simultaneous improvement is theexperiment in which highest recovery is obtained. Under the boundary condition that therecovery required is achieved, the enrichment can be steadily improved by executing step9b) of the guideline. In case high enrichment is the main target, the starting point is theexperiment in which highest enrichment is achieved. Under the boundary condition thatthe enrichment required is obtained, the recovery can be steadily improved by executingstep 9a) of the guideline.

However, not every combination of enrichment and recovery might be possible.

10th Step: Scale-upTo enlarge a foam fractionation column the approach of constant height to diameter andthe use of equal feed and gas superficial velocities should be used as described in chapter 7.Based on an experiment, in which continuous foam overflow exists, the scale-up approachshould be tested. It should be noted that the enrichment steadily increases with increasingcolumn dimensions. Thus, the experiment selected to be enlarged can be a one withhigher recovery. The scale-up strategy can be repeated as long as a continuous foamoverflow exists. In case a further enlargement of the column dimensions yield in instablefoam (no continuous foam overflow) the capacity can still be increased by operating foamfractionation columns in parallel.

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9 Conclusion and Outlook

Beginning with the screening of an adequate host system, producing foamable esterasetype enzyme, and first foam fractionation experiments batch foam fractionation was inves-tigated in more detail using Design of Experiments. Furthermore, the foam fractionationsystem was adapted to continuous foaming and systematically investigated in severaloperation modes. Continuous foam fractionation in stripping mode was then further im-proved by investigating different column designs. Compared to traditional purificationstrategies involving adsorption, ultrafiltration, and dialysis steps, foam fractionation wasshown to be an alternative process step, which combines separation, concentration, andpurification in one unit operation. Thus, foam fractionation was demonstrated as a tool forprocess intensification. By means of the statistical investigation of batch and continuousfoam fractionation foamability parameters like pH value or temperature and performanceparameters such as the gas flow rate or column design were identified. With the know-ledge gained the foam fractionation process was steadily improved and a guideline wasdeveloped to adjust foam fractionation fast and efficiently to general enzyme systems.

Nevertheless, before foam fractionation can be competitive to traditional unit operationsa scale-up of the process is necessary. In this work, the capacity of foam fractionation wassteadily increased. Starting from a 70 mL scale in batch mode, the continuous operationof foam fractionation was implemented and feed flow rate and column dimensions wereincreased steadily. Finally, a scale-up methodology was introduced. In two steps gas andfeed superficial velocity and the column length could be determined for an efficient andstable foam fractionation process. Then, two traditional scale-up approaches were tested.The approach of constant height to diameter was efficient to enlarge foam fractionation.The separation efficiency increased due to decreasing impact of wall effects during en-largement. However, the column dimensions investigated were still lab-scale equipment,but a general insight in possible scale-up strategies could be given.

The main conclusion of the present thesis is, that, although foam fractionation is a com-parably old technique, the process is not fully understood yet. As shown in this thesisand in the steadily increasing research of other scientists, various parameters affect theseparation efficiency and stability. To introduce foam fractionation as a competitive sepa-ration technique a generalization of the process is necessary. As mentioned in the abstractand chapter 6 the separation of the industrial enzyme PLA2 with foam fractionation wasinvestigated in parallel to the cutinase system (data not shown). Additionally, several

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110 9 Conclusion and Outlook

non-foamable enzymes and a pure component system containing bromelain were inves-tigated within the scope of the guideline development. Thus, the guideline represents afirst approach for a generalization of the foam fractionation process.

To understand foam fractionation in detail, the concentration of surface-active moleculesin the lamellar liquid needs to be measurable. Until now, the concentration or activityof the target substance in the lamellar liquid is an approximated value. For calculatingthe activity in the lamellar liquid, the foam fractionation equipment has to be modifiedand methods to determine drainage rate, coalescence behavior, and bubble size distribu-tion over the column length have to be developed and implemented. Based on this, keyparameters like surface excess of the bubbles will be accessible. In the end, the overalltarget should be the mathematical description of the foam fractionation process and withit the prediction and control of the process performance. Thus, scale-up of foam fraction-ation could be supported, which is a requirement to make foam fractionation competitiveto traditional separation techniques. Because the scale-up of foam fractionation is limiteddue to the stability of the separation medium foam the capacity can also be increased byoperating foam fractionation columns in parallel. Thus, can be realized in several stand-alone foam fractionation columns or to a cluster combined foam fractionation columns,which share foam breaker and liquid reservoir.

Beside the macromolecular view on foam fractionation, also the micromolecular mecha-nisms should be investigated [163]. It is important to know, how proteins or enzymesbehave during the adsorption process. Do they partially unfold or do they only orient atthe interface. To get to this information, techniques like circular dichroism spectroscopy(CD), infrared reflection absorption spectroscopy (IRRAS), and attenuated total reflec-tion Fourier transform infrared spectroscopy (ATR-FTIR) can be used to analyze thestructure changes during the adsorption to an interface or after adsorption at an inter-face. Thus, reversible or irreversible structural changes can be determined. Using theseanalytical techniques, the understanding of adsorption mechanisms will increase signifi-cantly and they can offer the opportunity to identify the requirements of a protein toadsorb at an interface. Thus, the foamability of proteins or enzymes could be predicted.The knowledge could be used to identify possible candidates, which can be recovered andpurified with foam fractionation.

Besides the identification and testing of foamable proteins, enzymes, or other substances,the idea of “in-situ” foam fractionation should be considered. There are three main ap-proaches to realize in-situ foam fractionation. First, the foam can be suppressed untilthe day of harvesting. Second, the emerging foam, containing target and other molecules,can be recycled back to the reactor. The third approach is the use of a reactor cascadewhere cultivation/production reactors are coupled with foam fractionation reactors. Forcontinuous fermentation a continuous „in-situ” product separation could be realized via

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9 Conclusion and Outlook 111

foam fractionation. However, these strategies need to be investigated and evaluated viacost calculations.

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List of Figures

1.1 Global industrial enzymes market: Recent development and forecast . . . . 1

2.1 Schematic presentation of adsorptive bubble separation methods . . . . . 32.2 Schematic representation of surface-active molecules oriented at a gas-

liquid interface. • hydrophilic part, - hydrophobic part . . . . . . . . . . . 42.3 Dependency of surface tension at varying surfactant concentration (• hy-

drophilic part, - hydrophobic part) . . . . . . . . . . . . . . . . . . . . . . 52.4 Dependency of surface excess Γ and the concentration of surface-active

components in the bulk solution . . . . . . . . . . . . . . . . . . . . . . . . 62.5 Schematic adsorption behavior of surface-active molecules in dependence of

their hydrophobicity. Adsorption strength increases with increasing num-ber of hydrophobic parts oriented to the outer surface of the protein . . . . 6

2.6 Schematic depiction of the kinetic pathways for protein adsorption. P1:direct deposition of soluble protein onto available surface; P2: “piggyback”deposition of soluble protein onto cluster species i; P3: accretion/incorpo-ration of adsorbed protein onto cluster species i to form cluster species i+1.kia: association rate, kid: disassociation rate . . . . . . . . . . . . . . . . . 7

2.7 Cryo-SEM picture of a Plateau border . . . . . . . . . . . . . . . . . . . . 82.8 A lamella under influence of drainage. The lamella is stretched and regions

with high and low surface tension are formed. If no Marangoni effect retardsthe flow, the lamella is thinning till it ruptures. If Marangoni effect retardsdrainage, part of the drained lamellar liquid is dragged with the stretchingsurfactant film and the lamella is temporally stable . . . . . . . . . . . . . 9

2.9 Change of spherical to polyhedral foam . . . . . . . . . . . . . . . . . . . . 102.10 Typical batch foam fractionation device . . . . . . . . . . . . . . . . . . . . 112.11 Batch foam fractionation in a) simple mode or enriching mode, b) simple

mode with recycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.12 Continuous foam fractionation in a) simple mode or enriching mode, b)

simple mode with recycle, c) stripping mode, and d) combined mode . . . . 122.13 Multistage foam fractionation column developed by Darton et al. . . . . . 13

3.1 Foaming devices used for foam fractionation experiments. a) experimentswithout Tween 80, b) experiments with Tween 80 . . . . . . . . . . . . . . 23

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114 List of Figures

3.1 Secretion of lipolytic activity by Phanerochaete chrysosporium: (a) - (c);and C. cinerea: (d)- (e) depending on carbon source. SNL medium withglucose and 0.4 % (v/v) (�), SNL medium without glucose and 0.4 % (v/v)Tween 80 (�), mineral salt medium containing 0.4 % (v/v) Tween 80 (•),and malt extract medium with 0.4 % (v/v) Tween 80 (�) . . . . . . . . . . 25

3.2 Recovery of activity and enrichment factors depending on pH value (40 mL air min−1,room temperature, column diameter 1.6 cm, and foaming period till end offoam formation). Activity in foamate (�), activity in retentate (�), andenrichment factor (�) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.3 Recovery of activity and enrichment factors depending on the air flow rate(pH 7.0, room temperature, column diameter 1.6 cm, and foaming periodtill end of foam formation). Activity in foamate �), activity in retentate(�), and enrichment factor (�) . . . . . . . . . . . . . . . . . . . . . . . . 27

3.4 Semi-native SDS-PAGE of initial solution, retentate and foamate (param-eters of foam fractionation: pH 7.0, 15 mL air min−1, room temperature,Column diameter 1.6 cm, and 15 minutes) . . . . . . . . . . . . . . . . . . 27

3.5 Alignment of peptide 1 with the sequences of potential cutinases from C.cinerea (abbreviations represent accession nos. in the EMBL database)with Clustal W . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

4.1 a) Effects for the response EC , b) Effects for the response R. Black columnsshow significant, gray columns insignificant effects. A: Temperature, B:pH value, C: Gas flow rate, D: Foam volume, E: Starting volume, F: Porediameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

4.2 Contour plots for the factor interactions of a) EC and b) R . . . . . . . . . 38

4.3 Pareto chart for the dependencies of R and EC . . . . . . . . . . . . . . . . 39

5.1 Continuous foam fractionation column in a) stripping mode and b) enrich-ing mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.2 Activity in the retentate over time. (� gas flow rate 28 mL min−1, feedflow rate 15 mL min−1; � gas flow rate 28 mL min−1, feed flow rate 5 mLmin−1; • gas flow rate 40 mL min−1, feed flow rate 15 mL min−1; � gasflow rate 40 mL min−1, feed flow rate 5 mL min−1). . . . . . . . . . . . . . 48

5.3 Effects for responses a) EC , b) R and c) ϕ. Black columns show significant,gray columns insignificant effects. A: pH value, B: Temperature, C: Feedflow rate, D: Gas flow rate, E: Pore diameter . . . . . . . . . . . . . . . . . 51

5.4 Possible combinations for responses EC and R based on the regression models 52

5.5 Contour plots for the factor interactions of EC (a), R (b) and ϕ (c). Blankcontour plots resemble insignificant factor interactions . . . . . . . . . . . . 54

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List of Figures 115

5.6 Surface tension of C. cinerea culture supernatants at varying pH valuesand temperatures (� 22.0°C, � 25.0°C, × 27.5°C, � 30.0°C, • 32.5°C . . . 55

5.7 Contour plot of factor interaction AC for response recovery (R) with surfacetension measurements for pH values at a temperature of 27.5°C . . . . . . 58

5.8 Contour plot of factor interaction BD for response enrichment factor (EC)with surface tension measurements for temperatures consistent with levelvalues at pH = 7.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6.1 Continuous foam fractionation column used for the experiments . . . . . . 62

6.2 Continuous foam fractionation columns used in this study. The columnin the middle was used for the center points only. The columns with 1.6cm and 4.4 cm diameter were varied in foam and liquid column lengthaccording to the experimental plan. For each combination one column wasproduced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

6.3 Surface tension of C. cinerea culture supernatants for different dilutions(pH = 7.0 and 27.5 °C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6.4 Retentate activity vs. time for columns with Dfoam= 1.6 cm and differ-ent liquid and foam column lengths. (� stripping mode, Lliquid= 24 cm,Lfoam= 56 cm; • stripping mode, Lliquid= 12 cm, Lfoam= 29 cm; � enrich-ing mode, Lliquid= 24 cm, Lfoam= 56 cm; ◦ enriching mode, Lliquid= 12 cm,Lfoam= 29 cm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

6.5 Comparison of stripping and enriching mode for columns with Dfoam= 1.6 cmand variable foam and liquid column lengths. X-axis: Length liquid column- length foam column; Black columns display the recovery R for strippingmode, gray columns display the recovery R for enriching mode, ◦ enrich-ment factor (standard deviations adopted from DoE) . . . . . . . . . . . . 67

6.6 Different flow regimes investigated via foam fractionation with dye. a) Cir-cular flow in column Dfoam= 1.6 cm, b) Plug flow in column Dfoam= 4.4 cm 68

6.7 Effects for the responses a) EC , b) R. Black columns show significant,gray columns insignificant effects. A: Gas flow rate, B: Pore diameter, C:Feed flow rate, D: length of liquid column, E: Length of foam column, F:Diameter of foam column . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

6.8 Possible combinations for responses R and EC calculated based on theregression models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

7.1 Continuous foam fractionation column with sampling points . . . . . . . . 81

7.2 a) Foam fractionation: • Recovery of activity, � enrichment factor fordifferent diluted supernatants and surface tensions. Data calculated inrelation to undiluted supernatant; b) Corresponding foamate to feed ratio . 82

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116 List of Figures

7.3 a) Recovery and enrichment for foam fractionation experiments in columnD30 at varying gas superficial velocities. • Recovery of activity, � en-richment factor. b) Corresponding foamate to feed ratio for varying gassuperficial velocities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

7.4 Picture of foam fractionation experiments with gas superficial velocityscreening: a) 4.2 cm min−1 and b) 14.2 cm min−1, c) 21.7 cm min−1 (pH7.0, 27.5°C, feed superficial velocity 2.8 cm min−1, and column D30 with200 mm liquid height) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

7.5 a) Recovery and enrichment for foam fractionation experiments in columnD30 at varying feed superficial velocities. • Recovery of activity, � en-richment factor. b) Corresponding foamate to feed ratio for varying feedsuperficial velocities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

7.6 Activity profiles of column D30 at varying gas superficial velocities (pH 7.0,27.5°C, surface tension 50 ± 1 mN m−1, feed superficial velocity 2.8 cm min−1,and stripping length 370 mm). � gas superficial velocity of 4.2 cm min−1,� gas superficial velocity of 14.2 cm min−1, and • gas superficial velocityof 21.7 cm min−1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

7.7 a) Slope of the linear activity profiles from sampling point 1 to 3 at varyinggas superficial velocity. b) Stripping lengths calculated via the slopes ofthe activity profiles to gain a feed depletion of 90 % . . . . . . . . . . . . . 89

7.8 Activity profiles of column D30 and D44 for the scale-up approach of con-stant H/D ratio. � Enzymatic activity profile in column D30, � enzymaticactivity profile in column D44 . . . . . . . . . . . . . . . . . . . . . . . . . 90

8.1 Design guideline for foam fractionation processes part 1 . . . . . . . . . . . 958.1 Design guideline for foam fractionation processes . . . . . . . . . . . . . . . 968.1 Design guideline for foam fractionation processes . . . . . . . . . . . . . . . 978.1 Design guideline for foam fractionation processes . . . . . . . . . . . . . . . 988.2 Concentration/surface tension profile with classification of concentration

ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008.3 a) Dry foam with big bubbles (polyhedral foam), b) Wet foam with small

bubbles (spherical foam) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

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List of Tables

4.1 Factors (A-F) and levels investigated. In brackets: Calculated factor values,which differ from the experimentally used factor values . . . . . . . . . . . 32

4.2 Experimental plan in systematic order and experimental data for the re-sponses R and EC . A: Temperature, B: pH value, C: Gas flow rate, D:Foam volume, E: Starting volume, F: Pore diameter . . . . . . . . . . . . . 33

4.3 Calculated factor levels for the optimization of the responses R and EC . . 37

5.1 Factors (A - E) and levels investigated. In brackets: Calculated factorvalues, which differ from the factor values used in the experiments . . . . . 47

5.2 Experimental plan in systematic order and experimental data for the re-sponses EC , R, and ϕ. A: pH value, B: Temperature, C: Feed flow rate, D:Gas flow rate, E: Pore diameter . . . . . . . . . . . . . . . . . . . . . . . . 49

5.3 Factor values for α = 2.08 for the Factors (A - D) . . . . . . . . . . . . . . 505.4 Calculated factor levels for the maximal responses R and EC . . . . . . . . 53

6.1 Factors (A - F) and levels investigated. In brackets: [Calculated factorvalue, which differ from the factor value used in the experiments] . . . . . 64

6.2 Comparison of foam fractionation experiments for non-diluted and dilutedculture supernatant and different column diameter. In brackets: enrich-ment factors recalculated for non-diluted feed solution . . . . . . . . . . . . 65

6.3 Experimental plan in systematic order and obtained experimental resultsfor responses R and EC . A: Gas flow rate, B: Pore diameter, C: Feed flowrate, D: Length of liquid column, E: Length of foam column, F: Diameterof foam column [151] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6.4 Traditional purification strategies vs. foam fractionation. Crude culturesuper-natant or crude extract were chosen as starting point for downstreamprocessing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

6.5 Comparison of experimental results. High recovery and high enrichment ofactive cutinase was favored for the different studies . . . . . . . . . . . . . 77

7.1 Dimensions of foam fractionation columns . . . . . . . . . . . . . . . . . . 807.2 Separation efficiency of scale-up experiments in column D30, D44, and D54 . 91

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Curriculum Vitae

Personal Details

Name Juliane Merz

Date of birth November 16, 1983

Place of birth Linz am Rhein, Germany

E-mail [email protected]

Professional Experience

since 01/2012 Head of the “Innovative Downstream processing”-groupLaboratory of Plant and Process DesignTechnische Universität Dortmund

since 12/2008 PhD. student and scientific assistantLaboratory of Plant and Process DesignTechnische Universität Dortmund

11/2010 – 2/2011 Research projectDSM Food Specialities, Delft, The Netherlands

8/2007 – 1/2008 Internship in the field of protein purificationDSM Food Specialities, Delft, The Netherlands

1 – 8/2007 Research assistantLaboratory of Process Dynamics and Operations

1 – 8/2007 Internship in the field of manufacturingWirtgen GmbH, Windhagen

Education

10/2003 – 9/2008 Study of biochemical engineeringTechnische Universität DortmundDepartment of Biochemical and Chemical Engineering

8/1994 – 3/2003 Secondary schoolStaatl. Gymnasium Neustadt/Wied

8/1990 – 6/1994 Primary schoolGrundschule Neustadt/Wied