Non-Saccharomyces wine yeast production in aerobic fed-batch culture

102 Pages·2015·2.26 MB·English

by  J Barkhuizen

Non-Saccharomyces wine
yeast production in aerobic
fed-batch culture
by
Jan-Harm Labuschagne Barkhuizen
Thesis presented in partial fulfilment
of the requirements for the degree
of
MASTER OF ENGINEERING
(CHEMICAL ENGINEERING)
in the Faculty of Engineering
at Stellenbosch University
Supervisor
Prof Johann F Görgens
December 2015
Stellenbosch University https://scholar.sun.ac.za
Declaration
By submitting this thesis electronically, I declare that the entirety of the work contained therein is my
own original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that
reproduction and publication thereof by Stellenbosch University will not infringe any third party rights
and that I have not previously in its entirety or in part submitted it for obtaining any qualification.
This dissertation includes 0 original papers published in peer reviewed journals or books and
1 unpublished publications. The development and writing of the papers (published and
unpublished) were the principal responsibility of myself and, for each of the cases where this
is not the case, a declaration is included in the dissertation indicating the nature and extent of
the contributions of co-authors.
Date: 25 November 2015
Copyright © 2015 Stellenbosch University
All rights reserved
Stellenbosch University https://scholar.sun.ac.za
Abstract
The production of the non-Saccharomyces wine yeasts Metschnikowia pulcherrima,
Issatchenkia orientalis and Lachancea thermotolerans was optimised in aerobic fed-batch
cultures for commercial application. These non-Saccharomyces have been used in sequential
wine fermentations where they are employed to enhance the organoleptic characteristics of
wine. The primary goal was to produce these organisms at a maximum biomass yield (Y )
x/s
through aerobic fed-batch cultivations where a dynamic feed regime was used to ensure
accurate control over the specific growth rate (µ ) of each culture.
spec
By controlling the µ at preferred points during cultivation at 9 L scale, Y maxima of 0.83
spec x/s
g g-1, 0.68 g g-1 and 0.76 g g-1could be achieved for I. orientalis, M. pulcherrima and L.
thermotolerans, respectively. This was higher than the 0.51 g g-1 achieved in Saccharomyces
cerevisiae cultures, due to the Crabtree-positive behaviour of the latter. When producing L.
thermotolerans at 90 L pilot scale a maximum Y of 0.54 g g-1 was achieved, which was
x/s
significantly lower than the 0.76 g g-1 achieved at 9 L bench scale.
A secondary goal was to determine what effect different production growth rates has on the
culture’s subsequent fermentative performance or yeast quality. The fermentative performance
of the yeasts produced under various culture conditions were evaluated by measuring the
acidification power of the yeast and evaluating the yeast in synthetic wine fermentations. The
tests indicated that the yeast can be produced at a growth rate where the Y is at a maximum
x/s
value without compromising the quality of the yeast culture. This allowed the selection of
conditions where a maximum Y is produced for industrial yeast production.
x/s
The non-Saccharomyces yeasts M. pulcherrima, I. orientalis and L. thermotolerans should be
produced at 0.10 h-1, 0.11 h-1 and 0.12 h-1, respectively. These growth rates will ensure the
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highest possible biomass yield on sugar with any compromise to the fermentative performance
of the yeast product.
Opsomming
Die produksie van die nie-Saccharomyces wyngiste Metschnikowia pulcherrima, Issatchenkia
orientalis en Lachancea thermotolerans is geoptimaliseer in aerobiese gevoerde-lotkulture vir
kommersiële toepassings. Hierdie nie-Saccharomyces wyngiste toon potensiaal wanneer hulle
in kombinasie met Saccharomyces cerevisiae gebruik word om die organoleptiese eienskappe
van die wyn te bevorder. Die hoofdoel van die projek was om die giste by ‘n maksimale
biomassa-opbrengs (Y ) te produseer deur middel van aerobiese gevoerde-lotkulture waar ‘n
x/s
dinamsie voermodel gebruik is om die kultuur by ‘n konstante spesifieke groeisnelheid (µ )
spec
te handhaaf.
‘n Maksimum Y van 0.83 g g-1, 0.68 g g-1 en 0.76 g g-1 kon bereik word vir onderskeidelik I.
x/s
orientalis, M. pulcherrima en L. thermotolerans deurdat die µ by sleutelpunte gehandhaaf
spec
is tydens kulture by 9 L kon. Dit is hoër as die 0.51 g g-1 wat in S. cerevisiae kulture bereik is.
Wanneer L. thermotolerans op proefskaal (90 L) geproduseer is, is ‘n maksimum Y van 0.54
x/s
g g-1 bereik, wat noemenswaardig laer was as die 0.76 g g-1 wat op klein skaal (9 L) bereik is.
‘n Sekondêre doelwit was om die gistingsprestasie van die gis te toets na afloop van die
afsonderlike kulture. Die gistingsprestasie is getoets deur die aansuringsvermoë van die
afsonderlike kulture te toets. Die gis se gistingsprestasie is verder getoets in sintetiese
wyngisting. Die toetse het gewys dat die gis by ‘n groeisnelheid gekweek kan word waar ‘n
maksimum biomassa-opbrengs bereik kan word sonder dat die werkverrigting tydens
wyngisting benadeel word. Hierdie bevinding bevestig dat die giste by lae groeisnelhede
gekweek kan word op kommersiële vlak.
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Die nie-Saccharomyces gis M. pulcherrima, I. orientalis en L. thermotolerans moet teen
afsonderlike groeisnelhede van 0.10 h-1, 0.11 h-1 and 0.12 h-1 produseer word om ‘n maksimale
biomass opbrengs op suiker te lewer. By hierdie kondisies word die gistingsprestasie van die
finale gis produk nie nadelig beïnvloed nie.

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Dedication
I dedicate this thesis to the loved ones in my life, especially my wife Truidi-lee, who supported
me throughout my studies.
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Acknowledgments
I would like to express my gratitude to my supervisor, Prof Johann F Gӧrgens, for his guidance
throughout my master’s studies.
I am also grateful to Dr Eugéne van Rensburg for critically reading the manuscripts, and for
his academic and moral support throughout my entire study. I am truly grateful for his never-
ending patience and guidance.
I special thanks to Samantha Fairbairn from the Institute of Wine Biotechnology for her
technical assistance with synthetic wine fermentations.
Finally, I would like to thank Emmanuel Anane for his mentorship. Without him this study
would not have been concluded.
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Table of Contents
Abstract ....................................................................................................................................... i
Dedication ................................................................................................................................. iv
Acknowledgments...................................................................................................................... v
List of Figures ............................................................................................................................ x
List of Tables ........................................................................................................................... xii
Nomenclature ......................................................................................................................... xiii
Chapter 1 Introduction .......................................................................................................... 1
Chapter 2 Theoretical Considerations .................................................................................. 4
2.1 Yeast metabolism ........................................................................................................ 4
Substrate transport into yeast cell ........................................................................ 4
Glycolysis ............................................................................................................ 5
Aerobic Respiration ............................................................................................. 5
Anaerobic Fermentation....................................................................................... 6
2.2 The Crabtree effect ...................................................................................................... 7
Rationale behind the Crabtree effect .................................................................... 8
The short-term Crabtree effect ............................................................................. 8
The long-term Crabtree effect ............................................................................ 11
2.3 Process kinetics for industrial yeast production ........................................................ 12
Inocula production ............................................................................................. 13
Batch medium considirations ............................................................................. 13
Batch phase ........................................................................................................ 14
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Feed medium considerations.............................................................................. 17
Nitrogen in the feed medium ............................................................................. 18
Fed-batch phase ................................................................................................. 19
Fermentative performance ................................................................................. 26
2.4 Process conditions ..................................................................................................... 28
Oxygen supply in aerobic cultivations ............................................................... 28
Effects of Temperature ...................................................................................... 31
Effects of pH ...................................................................................................... 31
Mixing Considerations ....................................................................................... 32
Vessel pressure................................................................................................... 32
Process conditions required for non-Saccharomyces yeast production ............. 32
2.5 Scale-up procedures .................................................................................................. 33
2.6 Experimental plan and objectives ............................................................................. 34
Chapter 3 Non-Saccharomyces wine yeast production in aerobic fed-batch culture ......... 37
3.1 Abstract ..................................................................................................................... 38
3.2 Introduction ............................................................................................................... 39
3.3 Materials and methods .............................................................................................. 42
Yeast strain and inoculum preparation .............................................................. 42
Medium preparation ........................................................................................... 42
Cultivation control ............................................................................................. 43
Exponential feed profile ..................................................................................... 44
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Scale-up cultivations .......................................................................................... 46
Specific growth rate calculations ....................................................................... 48
Yield calculations............................................................................................... 48
Acidification Power test ..................................................................................... 49
Analytical methods ............................................................................................ 50
Synthetic wine fermentation sample preparation ............................................... 51
Synthetic wine fermentation .............................................................................. 51
Synthetic fermentation: kinetics and analytic methods ..................................... 51
3.4 Results ....................................................................................................................... 52
Batch cultures..................................................................................................... 52
Exponential feed validation ............................................................................... 53
Fed-batch culture at 9 L scale ............................................................................ 55
Acidification Power (AP) .................................................................................. 59
Synthetic wine fermentations ............................................................................. 60
Fed-batch cultivations at pilot scale ................................................................... 62
3.5 Discussion ................................................................................................................. 65
The Crabtree effect ............................................................................................ 66
Dynamic fed-batch control ................................................................................ 66
Biomass yield ..................................................................................................... 67
Sugar utilisation ................................................................................................. 69
Transient effects and overfeeding in fed-batch cultures .................................... 70
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