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
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Transcript of Merz 28062012 - A Contribution to Design Foam

  • A contribution to design

    foam fractionation processes

    Zur Erlangung des akademischen Grades eines

    Dr.-Ing.

    von der Fakultt Bio- und Chemieingenieurwesen

    der Technischen Universitt Dortmund

    genehmigte Dissertation

    vorgelegt von

    Dipl.-Ing. Juliane Merz

    aus

    Linz am Rhein

    Tag der mndlichen Prfung: 28.06.2012

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

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

    Dortmund 2012

  • Ein Gelehrter in seinem Laboratorium ist nicht nur ein Techniker;

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

    Marie Curie

  • 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

  • ii

    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.

  • Zusammenfassung

    In biotechnologischen Prozessen ist die Bildung von Schumen grundstzlich unerwnscht,

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

    Tendenz zur Schaumbildung, meist ausgelst durch amphiphile beziehungsweise ober-

    flchenaktive Substanzen nicht zwangslufig negativ sein. Ein Aufreinigungsprozess der

    gerade diesen physikalischen Unterschied der Substanzgruppen zur Trennung ausnutzt ist

    die Zerschumung. Das Trennprinzip beruht dabei darauf, dass sich die oberflchenak-

    tiven Substanzen an eine Gas-Flssigkeits-Grenzflche 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 Zerschumung

    aus unbehandeltem Kulturberstand getrennt. Verschiedene Prozessgren, wie pH-Wert

    oder Temperatur und Kolonnengren, wie der Durchmesser oder die Lngen der Sule

    wurden variiert und im Hinblick auf Trenneffizienz und Schaumstabilitt systematisch

    untersucht. Dabei wurde statistische Versuchsplanung als systematischer Ansatz gewhlt

    um absatzweise und kontinuierliche Zerschumung zu untersuchen. Fr die kontinuierliche

    Zerschumung wurden die Betriebsweisen enriching mode und stripping mode nher

    untersucht. Fr hochverdnnte oder schwach oberflchenaktive Substanzen fhrte die

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

    tischen Versuchsplanung konnten Einflussgren und deren Wechselwirkungen identifiziert

    werden und die Zerschumung so angepasst werden, dass die gewnschte Ausbeute oder

    Anreicherung erlangt werden konnte. Zum weiteren Verstndnis der Vorgnge whrend

    der Zerschumung wurden Farbstoffexperimente durchgefhrt und die Oberflchenspan-

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

    Enzym Kutinase aus dem Kulturberstand 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 Zerschumung aufgrund der einfachen Durch-

  • iv

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

    intensivierung betrachtet werden. Um die Zerschumung jedoch industriell einsetzen zu

    knnen, ist eine Mastabsvergrerung notwendig. In dieser Arbeit wurden zwei tra-

    ditionelle Anstze zur Mastabsvergrerung der Zerschumung betrachtet, wobei der

    Labormastab nicht berschritten wurde. Zum einen wurde der Ansatz eines konstan-

    ten Verhltnisses von Lnge zu Breite und zum anderen der Ansatz konstanter Lnger

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

    und Breitenverhltnis fhrte zu einer verbesserten Trennleistung bei gleichbleibenden

    Schaumeigenschaften. Die Mastabsvergrerung mit dem Ansatz konstante Lnge und

    variierender Durchmesser der Sule hingegen verschlechterte die Trenneffizienz und die

    Schaumstruktur vernderte sich. Whrend dieser Versuche wurden zustzlich Profile der

    enzymatischen Aktivitt ber die Lnge der Sulen vermessen. Es konnten Zusammen-

    hnge zwischen der Gasleerrohrgeschwindigkeit und der Kolonnenlnge ermittelt werden,

    die fr die sptere Sulenkonfiguration von Bedeutung sind.

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

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

    tels Zerschumung 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 hnlich.

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

    fizienten bertragung herkmmlicher Enzymsysteme zur Zerschumung entwickelt. Um

    die Qualitt des Leitfadens zu prfen, wurden weiterhin ein Reinstoffsystem und nicht

    zerschumbare Enzyme untersucht.

  • 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 Grenzflchen, 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-

  • vi

    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 Zerschumung. 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, Nrnberg (2010)

  • 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

  • viii Contents

    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

  • Contents ix

    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 () . . . . . . . . . . . . . . . . . . 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 () . . . . . . . . . . . . . . . . . . . . . . 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

  • x Contents

    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

  • 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

  • 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 symbols Star value for Design of Experiments

    Difference symbol

    410nm Molar extinction coefficient at a wavelength of 410 nm

  • Nomenclature xiii

    Concentration of component at the interface

    i Concentration of component i at the interface

    Spreading pressure

    Surface tension of solution with surface active components

    0 Surface tension of pure solution

    Liquid hold up in the foam phase

  • 1 Introduction

    History of modern enzyme technology goes back to the 19th century where scientists likeBuchner and Khne 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 reportEnzymes 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])

  • 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 [815]. 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).

  • 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

  • 4 2 Theoretical part

    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 , which is defined as = 0 - (0 is the surface tension ofthe pure solution and the surface tension of the solution with surfactants) reduces thesurface tension and the free energy of the system respectively. Aveyard and Haydon [22]

  • 2 Theoretical part 5

    describe the spreading pressure 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: G = H - T S the process of surface adsorption isexergonic and with it spontaneous (S>0 and H

  • 6 2 Theoretical part

    surface excess can be displayed graphically with a Langmuir isotherm (Fig. 2.4) [24].A constant value of 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 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).

  • 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 kidthe 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

  • 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

  • 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].

  • 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

  • 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.

  • 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

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

  • 14 2 Theoretical part

    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 [3739]. 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 of/-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 [4750].

    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-

  • 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 [5260]. Additionally, foam fractionation is used for wastewater treatment to purify industrial and communal slops [6165]. Other authors describedfoaming as a principle for water treatment of fish farms and aquariums [6567]. 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, 7175]. 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 -amylase, invertase, catalase, laccase, and lipase [12, 7981] 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 -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 -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, 8386].

  • 16 2 Theoretical part

    Beside the search for foamable molecules, fundamentals, like drainage or coalescence be-havior are investigated [8793], approaches to measure these effects are generated [9497], and mathematical models are developed to describe these effects and the foamfractionation process [53, 88, 98104]. 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, 105114]. 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, 119125].

    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 [3739]. 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

  • 2 Theoretical part 17

    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).

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

  • 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 L1 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 L1 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

  • 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 L1 Na2HPO4 2H2O),and solution B (6.8 g L1 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 L culture supernatant or water (blank) and incubated for 15 minutesat 37C 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 mol p-nitrophenol enzymatically released from the substrate per minute.Under the conditions described the extinction coefficient of p-nitrophenol was determinedto be 410nm=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

  • 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 Mnchen, 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.

  • 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 m), 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).

  • 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 =AFAI

    (3.1)

    R =AF VFAI VI 100% (3.2)

    P =AF /CFAI/CI

    (3.3)

    AF and AI are the enzymatic activities [U L1] 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 L1] 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 L1 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.

  • 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 min1, 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

  • 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 min1, 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 min1, 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 min1. A good compromise between high recovery and high enrichment factor wasobserved at a gas flow rate of 20 mL min1 (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 min1. 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

  • 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 min1, 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).

  • 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).

  • 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 min1. The maximum enrichment was observed at an air flow rate of15 mL min1 (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.

  • 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.

  • 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 261 fractional factorial cube, a star and four center points,resulting in 48 experiments. The 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

  • 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 - -1 0 1

    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 min1 ] (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[ m ] (58)

    16 m), P3 (max. 40 m) and P2 (max. 100 m) 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 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 onR 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 Students 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].

  • 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