Ana Pinto_Tese

128 Pages·2009·9.89 MB·Portuguese

Characterisation of mixing in order to develop a scale-
-down approach for a two-liquid phase bio-oxidation
reaction

Ana Catarina da Silva Damas Pinto

Dissertação para a obtenção do Grau de Mestre em
Engenharia Biológica

Júri

Presidente: Prof. Luís Joaquim Pina da Fonseca, Departamento de
Engenharia Química e Biológica (DEQB), IST

Orientação: Prof. Duarte Miguel de Franca Teixeira dos Prazeres,
Departamento de Engenharia Química e Biológica (DEQB),
IST
Dr. Frank Baganz, UCL Biochemical Engineering Department

Vogais: Prof. Pedro Carlos de Barros Fernandes, Centro de
Engenharia Biológica e Química do DEQB, IST

Dezembro 2009
Acknowledgements

I want to laud Instítuto Superior Técnico (IST) for everything that I am today. This school
give me so many life and academic skills that I almost can say that built me as a individual that is
very well prepared to face and be great at my prossifinal life.

I am truly grateful for the opportunity gave by IST Biological and Chemistry Engineering
Department (DEQB) to do an international experience by doing my Biological Engineering Master
Degree at the University College London (UCL) Biochemical Engineering Department in London.

At UCL, I met amazing people both in Roberts and Katz Building that welcomed me and
helped me into find solutions and reasons for the many project bottlenecks. However, I have to
truthfully thank all the support, interest and almost nursing that was given by Dr. Frank Baganz and
Mr. Christopher Grant. I have really to give my thanks to Mr. Christopher Grant that always helps
me both during the project and for reviewing my Master degree thesis.

Cannot describe how I am so thankful to the most important people in my life, my family.
They give me all the support to live in London, but most of all for all the kindness and support in the
hardest times that I faced.

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To my family

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Abstract

The biocatalysis oxidation of long chain alkanes is a key commercial target. The bio-
oxidation studied was the E.coli pGEc47∆J n-dodecane bioconversion into 1-dodecanol. It was
made the mixing characterisation in order to develop a scale-down approach for a two-liquid phase
bio-oxidation reaction. It involved high speed camera (HSC) imaging, perform bioconversions and
apply DoE software. From the HSC imaging it was identify an apparently good mixing in SFs and
the 24-DSW are the best for mixing but still show a mixing limitation. That can be overcome by
using surfactants or the Duetz system. The 24-SRW was considered inadequate. In all
bioconversions performed dodecanoic acid was present indicating over-expression of the plasmid.
The interface began to be analysed. The Triton X-100 and PPG addition reduced the liquid loss in
sacles. In SFs bioconversions using Triton X-100 demonstrate a 3fold bioconversion improvement
(0,8 g/L ) compared with other SFs and 2L-fermenter using n-octane. The use of Deutz
organic phase
system with the rigid silicone sandwich cover layer also is equal a good solution (0,3±0,3 g/L
organic
). The alternative use of Duetz System with gas permeable membrane with 24-DSW with half
phae
sphere without surfactants prove also a high bioconversion (0,2±0,2 g/L ). In conclusion, it
organic phase
was achieved a scale-down method which is considered a breakthrough and a platform for more in-
depth studies.

Keywords: Two-liquid phase systems, bio-oxidation reaction, Triton X-100; 1-dodecanol, DoE

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Resumo

A bio-oxidação de alkanos de longa cadeia tem elevada importância comercial. A bio-
oxidação estada foi a bioconversão de n-dodecane em 1-dodecanol por E.coli pGEc47∆J. Foi
efectuada a caracterização da mistura para desenvolver a miniaturização da bio-oxidação de um
sistema de duas fases líquidas. Para tal foi usado imagens de alta velocidade, fermentações e o
recurso ao software DoE. Das imagens de alta velocidade detectou-se uma boa mistura nos
erlenmeyers e o poço rectangular é o melhor apesar de apresentar ainda problemas na mistura, os
quais podem ser resolvidos através da adição de surfactantes e/ou da aplicação do Duetz system.
Os poços cilíndricos são considerados inadequados. Em todas as fermentações o ácido
dodecanóico estava presente evidenciando a sobre-expressão do plasmidio. A análise à interface
começou a ser feita. A adição de Triton X-100 e PPG implicaram a diminuição da perda do líquido
em todas as escalas. No erlenmeyer de 1L a adição do Triton X-100 aumentou em 3 vezes a
produção de 1-dodecanol (0,8 g/L ). A aplicação do Duetz system com a membrana de
fase orgânica
silicone rígida alcançou-se bons níveis de bio-oxidação (0,3±0,3 g/L ). A alteração no
fase orgânica
Duetz sytem ao adicionar a membrana permeável com fundo esférico provou alcançar elevada
bioconversão (0,2±0,2 g/L ). Em conclusão, foi alcançado um novo progresso para a
fase orgânica
miniaturização de sistemas de dois líquidos e criou-se uma plataforma de arranque para novos
desenvolvimentos.

Palavras-chave: Sistemas de dois líquidos, bio-oxidação, Triton X-100; 1-dodecanol, DoE

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Table of Contents

Acknowledgements .......................................................................................................................................... 1
Abstract ............................................................................................................................................................. 3
Resumo .............................................................................................................................................................. 4
List of Figures ................................................................................................................................................... 9
List of Tables ................................................................................................................................................... 13
Abbreviations .................................................................................................................................................. 17
Introduction ..................................................................................................................................................... 18
Literature Review ............................................................................................................................................ 19
Two-Liquid Biocatalytic Systems .............................................................................................................. 19
Fundamentals ............................................................................................................................................... 20
Two-liquid phase biocatalysis process ......................................................................................................... 22
Two-liquid phase biocatalysis classification .................................................................................................. 22
Homogeneous mixture of the organic and aqueous phase .......................................................................... 22
Two-liquid phases mixture ............................................................................................................................ 23
Biocatalysts dissolved in organic phase: inverse micelles and covalently modified enzymes ...................... 24
Biocatalysts suspended in organic phase with low water quantity: immobilised biocatalysts. ...................... 25
Two-liquid phase biocatalysis process reactors ............................................................................................ 25
Downstream of two-liquid phase biocatalysis process .................................................................................. 25
Scale-up of a two-liquid phase biocatalysis .................................................................................................. 26
Safety Considerations .................................................................................................................................. 27
Mass transfer in two-liquid phase biocatalysis process ................................................................................ 27
Substrate concentration profiles of the two-liquid phase biocatalysis ........................................................... 28
Options to overcome mass transfer limitations ............................................................................................. 29
Use of Surfactants ........................................................................................................................................ 30
Scale-Down Process and mixing conditions in minibioreators ............................................................. 31
Types of Miniature bioreactors ..................................................................................................................... 32
Miniature shaken bioreactor systems: .......................................................................................................... 33
Shake flasks (SFs) ....................................................................................................................................... 33
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Microtitre plates (MTPs) ............................................................................................................................... 34
Miniature stirred bioreactor systems (MSBRs) and Miniature bubble column reactors (MBCR) ................... 36
Bacterial metabolism of long-chain n-alkanes ......................................................................................... 37
Genetic and Enzymology of Alkane Hydroxylation System .......................................................................... 40
Alkane Uptake and Solubilisation ................................................................................................................. 43
Physiology of Alkane-Degrading Pseudomonas ........................................................................................... 45
Whole Cell and Immobilise Enzyme and Oxygenases limitations ................................................................. 46
Hosts for Alkane Hydroxylation..................................................................................................................... 47
Economics .................................................................................................................................................. 50
Two-liquid phase biocatalysis processes examples ............................................................................... 51
Material and Methods ..................................................................................................................................... 53
Chemicals ..................................................................................................................................................... 53
Solutions ....................................................................................................................................................... 53
Trace mineral Stock: ..................................................................................................................................... 53
Medium [Wubbolts and at. al. 1996] ............................................................................................................. 54
Software ....................................................................................................................................................... 54
Devices ......................................................................................................................................................... 55
Methods ........................................................................................................................................................ 56
E.coli pGEc47∆J cell culture ......................................................................................................................... 56
Sample preparation ...................................................................................................................................... 56
Determination of cell density......................................................................................................................... 57
Determination of 1-dodecanol and dodecanoic acid concentrations............................................................. 57
Visualisation of liquid hydrodynamics by high speed video camera ............................................................. 57
Sterilisation and use of sandwich covers for deepwell microtitre plates (MTPs) (Duetz System) ................. 57
Results and Discussion ................................................................................................................................. 59
Visualisation of liquid hydrodynamics: Phase mixing and droplet size distribution ........................... 59
2L Fermenter ................................................................................................................................................ 59
Shake flask: 500mL and 1L .......................................................................................................................... 61
Microwell plates ............................................................................................................................................ 63
E.coli pGEc47 ∆J n-dodecane bioconversion .......................................................................................... 67
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E.coli pGEc47∆J n-dodecane bioconversion in Shake flasks ....................................................................... 67
E.coli pGEc47∆J n-dodecane bioconversion in 500mL and 1L SFs ............................................................. 68
E.coli pGEc47∆J n-dodecane bioconversion in 500mL and 1L SFs with PPG and Triton X-100 ................. 69
E.coli pGEc47∆J bioconversion of n-dodecane in 1L SFs. ........................................................................... 69
E.coli pGEc47∆J n-dodecane bioconversion in MTPs .................................................................................. 70
E.coli pGEc47∆J n-dodecane bioconversion in MTPs using the Duetz System ........................................... 71
E.coli pGEc47∆J n-dodecane bioconversion in MTPs using Duetz sytem with permeable membranes ...... 72
Application of Design Expert (DoE) .............................................................................................................. 74
Application of DoE on E.coli pGEc47∆J bioconversion of n-dodecane ......................................................... 74
Verification of the DoE design experience .................................................................................................... 78
Metrics from diverse E.coli pGEc47∆J bioconversion of n-dodecane experiences ............................. 79
Conclusion ...................................................................................................................................................... 82
Future Work ..................................................................................................................................................... 84
References....................................................................................................................................................... 85
Appendix ......................................................................................................................................................... 90
Appendix 1: Method of determination of dry cell weight (DCW) and concentration of 1-dodecanol and
dodecanoic acid ......................................................................................................................................... 90
Appendix 2: DoE Case Study: Fermentation Process Optimisation: Characterisation and
Optimisation of Soluble Recombinant Protein Expression in E.coli. .................................................... 92
Introduction to DoE ....................................................................................................................................... 92
Introduction to the Expression System ......................................................................................................... 92
DOE goal ...................................................................................................................................................... 93
Optimisation design to be used .................................................................................................................... 93
Data Analysis................................................................................................................................................ 94
Predicted Model Choice ............................................................................................................................... 94
ANOVA ......................................................................................................................................................... 94
Diagnostic plots ............................................................................................................................................ 96
Model Graphs and optimisation .................................................................................................................... 96
Final Optimised operating conditions ............................................................................................................ 97
Growth Period ............................................................................................................................................... 97
Growth agitation ........................................................................................................................................... 97
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Induction agitation ........................................................................................................................................ 98
Appendix 3: Raw Data from on E.colipGEc47∆J bioconversion of n-dodecane ................................... 99
Description of laboratorial experiences ......................................................................................................... 99
Experience 1: Effect of the SF Volume on E.colipGEc47 ∆J bioconversion of n-dodecane ......................... 99
Experience 2: Effect of SF volume on three different E.colipGEc47∆J bioconversion of n-dodecane cultures
conditions. .................................................................................................................................................... 99
Experience 3: E.colipGEc47∆J bioconversion of n-dodecane in 1L SFs. ..................................................... 99
Experience 4: E.coli pGEc47∆J bioconversion of n-dodecane in the thermomixer ...................................... 99
Experience 5: Effect of different Duetz Sandwich covers on the E.coli pGEc47∆J bioconversion of n-
dodecane on 24-DSW MTPs. ..................................................................................................................... 100
Experience 6: E.coli pGEc47∆J bioconversion of n-dodecane 24-DSW MTPs permeable membrabes..... 100
Experience 7: Design Experience (DoE) with E.coli pGEc47∆J bioconversion of n-dodecane in 24-SDW with
Duetz System. ............................................................................................................................................ 100
Experience 8: E.coli pGEc47∆J bioconversion of n-dodecane in 24-SDW with Duetz System and Triton X-
100. ............................................................................................................................................................ 108
GC Chromatograms ................................................................................................................................. 109
1-Dodecanol and Dodecanoic GC Standards ............................................................................................. 109
Samples ..................................................................................................................................................... 114
Cell Density and Volume of the aqueous phase, interface and organic phase. .................................. 126

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List of Figures

Figure 1: Classification of two-liquid phase biocatalytic processes on the solubility of both the substrate (S)
and product (P) molecules. The biocatalyst may be a whole-cell or an immobilized enzyme and is assumed to
be present in the aqueous phase. Dashed lines represent mass transfer processes. Image adapted from Lye
G. L., et. al., 2001.
Figure 2: Integrated design strategy for two-liquid phase biocatalytic processes: (a) may need to compromise
on solvent selection to satisfy solubility constraints; (b) opportunities for protein/genetic engineering; (c) may
need to compromise on reactor operation to satisfy downstream processing constraints. Image adapted from
Lye G. L., et. al., 2001.
Figure 3: Classification of the two-liquid biocatalytic systems. A) Two-liquid phase mixture: emulsion of the
aqueous phase in the organic; B) Two-liquid mixture: emulsion of the organic phase in the aqueous; C) and D)
Immobilised biocatalyst in a porous support on a biphasic system; E) Microencapsulated Biocatalyst in a
inverse micelle; F) Modified biocatalyst with Polyethylene glycol (PEG) and dissolved on organic phase; H)
Lyophilized biocatalyst and in crystal from, suspended on the organic phase. Image adapted from Aires-Barros
M.R. et. al., 2003.
Figure 4: Process flowsheets for (a) conversion of a poorly water-soluble substrate into a poor water-soluble
product, and (b) conversion of a poorly water-soluble substrate into a water-soluble product..Dashed lines,
represent options for organic phase recycle while dotted lines represent options for biocatalyst recycle. Image
adapted from Lye G. L., et. al., 2001.
Figure 5: Schematic diagram of substrate, product, and heat fluxes throughout a multiphase bioreactor
medium. Gaseous, apolar, and solid substrates must cross phase boundaries to be taken up by cells.
Likewise, products excreted by cells, if they do not accumulate in the aqueous phase, are extracted into the
organic solvent phase, stripped by the continuous flow of gas through the reactor, or adsorbed to a solid
absorbent. Heat produced by the culture is transferred to the cooling system of the reactor. In a mass-heat
transfer-limited culture one of these fluxes indicates by the shaded arrows, is rate limiting. Image adapted from
Schmid A. et.al., 1998.
Figure 6: Theoretical hydrophobic substrate concentration profiles to several biocatalytic systems. Image
adapted from Aires-Barros M.R. et. al. (2003).
Figure 7: Illustration of the trade off in information output versus HT capability that currently exists for various
cell cultivation devices at different scales. This figures shows that as bioreactors increase in scale, typically
more process information is available due to improved monitoring a control systems. Image adapted from
Jonathan I. B., et. at., 2003.
Figure 8: A) Schematic diagram of individual microwell formats: (a) 96-deep square well format; (b) 24-round
well format; (c) 96-round well format. V represents the total well volu me, SA represents the static surface
W
area available for gas–liquid mass transfer. Image adapted from Lye G.L. et. al.,2003. B). Duetz and coworkers
(2000) described a closure system consisting of a soft silicone layer with a small hole above the center of each
well.
Figure 9: Prototyps from MSBR and MBCR developed in UCL. A) Technical illustration of an 18 ml working
volume miniature stirred bioreactor (MSBR) prototype. B) Diagram of the miniature bubble column reactor
(MBCR) prototype. Image adapted from Jonathan I. B. and et. al. 2003.
Figure 10: Overview of the substrate range of alkane hydroxylase with respect to alkanes. sMMO Soluble
methane monooxygenase; pMMO particulate methane monooxygenase; PMO propane monooxygenase; BMO
butane monooxygenase; CYP153; pAH1 medium-chain-length integral membrane alkane hydroxylase ; pAH2
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