by
AIMI AISHAH BINTI ARIFIN
Thesis submitted in fulfillment of the requirements for the degree
of Master of Science
FEBRUARY 2011
ii
made all things possible. I would like to acknowledge and extend my heartfelt gratitude to the following persons who have made the completion of this thesis.
Particularly, I owe my deepest gratitude to my supervisor, Dr. Mohamad Hekarl Uzir, for his vital encouragement and for giving me the opportunity to work with this project. I appreciate his understanding and patience guidance along the way. Funding from the USM Short Term Grants throughout the research period is also much appreciated. I also would like to make a special thank you to my co-supervisor, Assoc. Prof. Dr. Mashitah Mat Don for her dedication and valuable ideas to inspire me in achieving better performance regarding the project.
My gratitude also goes to the Dean of Chemical Engineering School, Professor Azlina Harun@Kamarudin for her support towards my postgraduate affairs. To our Deputy Dean (Postgraduate Studies), Assoc. Prof. Dr. Lee Keat Teong and Deputy Dean (Undergraduate Studies), Assoc. Prof. Dr. Zailani Abu Bakar, thank you very much. Sincere appreciation goes to all staffs and technicians in School of Chemical Engineering for their kindness and co-operation.
My sincere gratefulness goes to my adored family for their endless love and concern during the hard time of my research study. Last but not least, to all my lovely friends, Fauziah, Nadia, Fadzilah, Rabiatul, Umi Natrah, Ummi Kalsum and others for the valuable experiences, endless help and wonderful memories that we shared together.
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TABLE OF CONTENTS Page
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES viii
LIST OF FIGURES x LIST OF ABBREVIATIONS xiv LIST OF SYMBOLS xvii ABSTRAK xix ABSTRACT xxi CHAPTER ONE: INTRODUCTION 1 1.0 Overview 1
1.1 Natural flavor compounds 2 1.2 Biotransformation 3 1.3 Whole-cell biotransformation 5
1.3. 1 Growing and resting cells systems 6
1.3. 2 Cofactor regeneration in the whole-cell system 7
1.4 Yeast as a source of biocatalyst for biotransformation 8
1.5 Biotransformation in non-conventional systems 10
1.6 Problem statement 11
1.7 Objectives 12
1.8 Scope of study 13 1.9 Thesis structure 14
CHAPTER TWO: LITERATURE REVIEW 16 2.0 Overview 16 2.1 Production of flavors by microorganisms 16
iv
2.2 Stereospecific reduction mediated by Saccharomyces cerevisiae 23
2.2.1 Reduction of carbonyl compounds (C=O) 23
2.2.2 Reduction of carbon-carbon double bonds compounds (C=C) 31
2.2.3 Yeast oxidoreductases activities during baker’s yeast-mediated biotransformation 34 2.3 Saccharomyces cerevisiae: Growth and biotransformation conditions 36
2.4 Reduction of geraniol into citronellol 39
2.4.1 Geraniol and citronellol properties 39
2.4.2 Microbial versus chemical catalysis of citronellol 41
2.5 Non-conventional biotransformation systems 45 2.5.1 Modified whole-cell biocatalyst 46 2.5.2 Non-conventional media/bioreactor designs 48
2.5.2 (a) Two-phase system 48
2.5.2 (b) Ionic liquid system 49
2.5.2 (c) Membrane-based system 50
2.5.2 (d) Gas phase system 51
CHAPTER THREE: MATERIALS AND METHOD 54 3.0 Overview 54 3.1 Biocatalyst 54
3.2 Chemicals 54 3.3 Growth medium 55
3.4 Buffer solution 55
3.5 S. cerevisiae fermentation 55
3.6 Liquid-phase biotransformation in shake-flask culture 57
3.6.1 Biotransformation using growing cells 57
3.6.2 Biotransformation using resting cells 58
v
3.7 Biotransformation in the continuous closed-gas-loop bioreactor (CCGLB)
59
3.7.1 CCGLB configuration 59
3.7.2 CCGLB experimental setup 61
3.8 Analytical methods 63
3.8.1 Measurement of cell concentration 63
3.8.2 Glucose analysis 63
3.8.2 (a) Preparation of DNS reagent 63
3.8.2 (b) Measurement of glucose concentration 64
3.8.3 Gas chromatography (GC) analysis 64
3.9 Fermentation kinetic analysis 65
3.9.1 Determination of specific growth rate (𝜇) 65
3.9.2 Determination of glucose consumption rate (𝑄𝑠) 66 3.10 Enzyme kinetic analysis 66 3.10.1 Determination of initial rate of reaction and specific activity 67
3.10.2 Determination of Michaelis constant (𝐾𝑚) and maximum rate of reaction (𝑉𝑚𝑎𝑥) 67 3.11 Process methodology 68
CHAPTER FOUR: RESULTS AND DISCUSSION 69 4.1 Growth kinetics of S. cerevisiae 69
4.1.1 Effect of initial pH on growth 69
4.1.2 Effect of temperature on growth 72
4.1.3 Effect of initial glucose concentration on growth 75
4.1.4 General observations on growth profiles 79 4.2 Liquid-phase biotransformation 80 4.2.1 Biotransformation of geraniol using resting cells of S. cerevisiae 80 4.2.1 (a) Effect of initial pH 80
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4.2.1 (b) Effect of initial substrate concentration 85
4.2.2 Biotransformation of geraniol using growing cells of S. cerevisiae 88 4.2.2 (a) Effect of substrate for biotransformation on cell growth 88 4.3 Gas-phase biotransformation in a CCGLB system 90 4.3.1 Biotransformation of geraniol using resting cells of S. cerevisiae 92 4.3.1 (a) Effect of initial pH 92 4.3.1 (b) Effect of agitation rate 95
4.3.1 (c) Effect of substrate flow rate 98 4.3.1 (d) Effect of glucose concentration 102 4.3.2 Biotransformation of geraniol using growing cells of S. cerevisiae 106 4.3.2 (a) Effect of substrate (geraniol) on cell growth 107 4.3.2 (b) Effect of agitation rate 109 4.3.2 (c) Effect of substrate flow rate 113 CHAPTER FIVE: PROCESS MODELING OF BIOTRANSFORMATION IN CCGLB 116 5.1 Introduction 116
5.2 Modeling of a gas-liquid catalytic reactor 116
5.3 Model development of CCGLB 118
5.3.1 Mass balances 119
5.3.2 Parameters estimation 122
5.3.2 (a) Reaction kinetics 122
5.3.2 (b) Mass transfer kinetic 123
5.4 Model simulation 124
5.5 Discussion 125
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5.5.1 Model validation and application 125 5.5.2 Model sensitivity 127
CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS 129
6.1 Conclusions 129
6.2 Recommendations 132
REFERENCES 133
APPENDICES 151
viii
LIST OF TABLES Page
Table 1.1 Comparisons between whole-cell and enzymatic biotransformation (Ishige et al., 2005; Shin et al., 2007)
4 Table 1.2 Comparisons between biotransformation and chemical
catalysis
5 Table 2.1 Potential microorganisms for synthesis of flavors 20 Table 2.2 Baker’s yeast mediated reduction of four β-keto esters in
petroleum ether (Anthanasiou et al., 2001)
28 Table 2.3 List of enantioselective baker’s yeast-mediated
biotransformation of carbonyl compounds
31 Table 2.4 Operational parameters for bioreduction reactions and
fermentations in stirred-tank bioreactor
39 Table 2.5 Physical and chemical properties of geraniol and citronellol
(Perry’s Chemical Engineers’ Handbook, 1997; Chen and Viljoen, 2010)
41
Table 2.6 Details of immobilization procedures for ethanol fermentation
47
Table 3.1 List of chemicals 54
Table 3.2 A complex YPD medium for S. cerevisiae fermentation 55 Table 4.1 Effect of initial pH on S. cerevisiae growth in shake-flask
culture
71 Table 4.2 Effect of temperature on S. cerevisiae growth in shake-flask
culture
74 Table 4.3 Effect of glucose concentration on S. cerevisiae growth in
shake-flask culture
77 Table 4.4 Effect of initial pH on the biotransformation of geraniol into
citronellol using resting cells of S. cerevisiae in liquid phase system
83
Table 4.5 Effect of substrate concentration on the biotransformation of geraniol into citronellol using resting cells of S. cerevisiae in liquid phase system
87
Table 4.6 Effect of initial pH towards the biotransformation of geraniol into citronellol using resting cells of S. cerevisiae in the CCGLB system
94
ix
Table 4.7 Effect of agitation rate towards the biotransformation of geraniol into citronellol using resting cells of S. cerevisiae in the CCGLB system
98
Table 4.8 Effect of substrate flow rate towards the biotransformation of geraniol into citronellol using resting cells of S. cerevisiae in the CCGLB system
101
Table 4.9 Effect of initial glucose concentration towards the biotransformation of geraniol into citronellol using resting cells of S. cerevisiae in the CCGLB system
106
Table 4.10 Effect of agitation rate towards the biotransformation of geraniol into citronellol using growing cells of S. cerevisiae in the CCGLB system
111
Table 4.11 Effect of substrate flow rate towards the biotransformation of geraniol into citronellol using growing cells of S. cerevisiae in the CCGLB system
115
Table 5.1 Parameter constants 123
Table 5.2 Model parameters 124
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LIST OF FIGURES Page
Figure 1.1 Schematic representation of cofactor regeneration during the reduction of geraniol into citronellol by S. cerevisiae with glucose as co-substrate.
8
Figure 1.2 Electron micrograph showing Saccharomyces cerevisiae parent cell that has produced at least six daughter cells as shown by the circularly arranged bud scars (Hatzis and Porro, 2006).
9
Figure 2.1 Monoterpenoid derivatives structure; (1) geraniol (alcohol);
(2) citronellal (aldehyde); (3) pulegone (cyclic ketone).
17 Figure 2.2 Percentage of published papers on various biocatalysts used
in biotransformation of terpenes in the last ten years (de Carvalho and da Fonseca, 2006).
18
Figure 2.3 Chemical structure of (27) geraniol and nerol (28). 40
Figure 2.4 Chemical structure of citronellol. 41
Figure 2.5 Mechanism of geraniol hydrogenation in chemical catalysis (Tas et al., 1997).
43
Figure 2.6 Regioselective hydrogenation of geraniol (I) to citronellol (II) using a biocatalyst and Noyori’s reagent. Adopted from (Berger, 1995).
44
Figure 3.1 Schematic diagram of CCGLB system. 59
Figure 3.2 Overall experimental flow chart. 68
Figure 4.1 Fermentation profiles at different initial pH values. The fermentations were carried out in shake-flask culture for 84 hr (Conditions: 30°C, 150 rpm, 1 g/L baker’s yeast, 10 g/L glucose).
70
Figure 4.2 Plots of glucose consumptions at different initial pH values. 71 Figure 4.3 Fermentation profiles at different culture temperatures. The
fermentations were carried out in shake-flask culture for 84 hr (Conditions: pH 4, 150 rpm, 1 g/L baker’s yeast, 10 g/L glucose).
73
Figure 4.4 Plots of glucose consumptions at different temperatures. 74
xi
Figure 4.5 Fermentation profiles at different glucose concentrations.
The fermentations were carried out in shake-flask culture for 84 hr (Conditions: pH 4, 30°C, 150 rpm, 1 g/L baker’s yeast).
76
Figure 4.6 Plots of glucose consumptions at different glucose concentrations.
77 Figure 4.7 The product formation during biotransformation of geraniol
at different initial pH values. The reactions were carried out in shake-flask culture for 48 hr (Conditions: 30°C, 150 rpm , 10 g/L baker’s yeast, 3 g/L geraniol, 10 g/L glucose).
81
Figure 4.8 Initial rates of reaction of geraniol reduction by resting cells of S. cerevisiae at different initial pH values.
82 Figure 4.9 Plots of geraniol consumption and cell concentration during
the reduction reaction at pH 4.
83 Figure 4.10 The product formation during biotransformation of geraniol
at different substrate concentrations. The reactions were carried out in shake-flask culture for 48 hr (Conditions: pH 7, 30°C, 150 rpm, 10 g/L baker’s yeast, 10 g/L glucose).
86
Figure 4.11 Initial rates of reaction of geraniol reduction using resting cells of S. cerevisiae at different substrate concentrations.
87 Figure 4.12 Growth and biotransformation profiles using growing cells
of S. cerevisiae in shake-flask culture. The biotransformation was carried out at pH 4, 30°C, 150 rpm with 1 g/L geraniol and 10 g/L glucose. ‘ ’ represents growth without biotransformation (control); ‘ ’ represents growth with biotransformation; and ‘ ’ represents citronellol formation.
89
Figure 4.13 The product formation during biotransformation of geraniol using resting cells of S. cerevisiae at different initial pH values in the CCGLB system. The reactions were carried out for 48 hr (Conditions: 30°C, agitation rate of 200 rpm, substrate flow rate of 8 L/min, 10 g/L baker’s yeast, 10 g/L glucose).
93
Figure 4.14 Variation in the initial rates of reaction relative to the initial pH.
94 Figure 4.15 The product formation during biotransformation of geraniol
using resting cells of S. cerevisiae at different agitation rates in the CCGLB system. The reactions were carried out for 48 hr (Conditions: pH 7, 30°C, substrate flow rate of 8 L/min, 10 g/L baker’s yeast, 10 g/L glucose).
96
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Figure 4.16 Variation in the initial rates of reaction relative to the agitation rate.
97 Figure 4.17 The product formation during biotransformation of geraniol
using resting cells of S. cerevisiae at different substrate flow rates in the CCGLB system. The reactions were carried out for 48 hr (Conditions: pH 7, 30°C, agitation rate of 350 rpm, 10 g/L baker’s yeast, 10 g/L glucose).
100
Figure 4.18 Variation in the initial rates of reaction relative to the substrate flow rate.
100 Figure 4.19 The product formation during biotransformation of geraniol
using resting cells of S. cerevisiae at different glucose concentrations in the CCGLB system. The reactions were carried out for 48 hr (Conditions: pH 7, 30°C, agitation rate of 350 rpm, substrate flow rate of 8 L/min, 10 g/L baker’s yeast).
104
Figure 4.20 Variation in the initial rates of reaction relative to the initial glucose concentration.
104 Figure 4.21 Plots of glucose consumptions at different initial glucose
concentrations.
105 Figure 4.22 Growth and biotransformation profiles during the
biotransformation of geraniol using growing cells of S.
cerevisiae. The reaction was carried out for 48 hr (Condition: pH 4, 30°C, agitation rate of 350 rpm, substrate flow rate of 8 L/min). ‘ ’ represents growth with free substrate (control) ‘ ’ represents growth with biotransformation, ‘ ’ represents citronellol formation.
108
Figure 4.23 The product formation during biotransformation of geraniol using growing cells of S. cerevisiae at different agitation rates in the CCGLB system. The reactions were carried out for 33 hr (Conditions: pH 4, 30°C, substrate flow rate of 8 L/min, 10 g/L glucose).
110
Figure 4.24 The growth of S. cerevisiae during the biotransformation of geraniol into citronellol at different agitation rates.
110 Figure 4.25 Variation in the initial rates of reaction at different agitation
rates.
111 Figure 4.26 The product formation during biotransformation of geraniol
using growing cells of S. cerevisiae at different substrate flow rates in the CCGLB system. The reactions were carried out for 33 hr (Conditions: pH 4, 30°C, agitation rate of 500 rpm, 10 g/L glucose).
114
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Figure 4.27 The growth of S. cerevisiae during the biotransformation of geraniol into citronellol at different substrate flow rates.
114 Figure 4.28 Variation in the initial rates of reaction at different substrate
flow rates.
115 Figure 5.1 The boundary of CCGLB system used in the modeling
work.
118 Figure 5.2 Reciprocal initial rate (1/𝑟) versus reciprocal initial
concentration of geraniol (1/𝑆) for biotransformation of geraniol.
123
Figure 5.3 Comparison of experimental and simulated data at different initial substrate flow rates. Symbols and lines represent experimental and simulated data, respectively. ‘ ’ represents flow rate of 8 L/min; ‘ ’ represents flow rate of 6 L/min; ‘ ’ represents flow rate of 4 L/min.
126
Figure 5.4 Concentration of geraniol in the CCGLB predicted using the proposed model at different substrate flow rates.
127 Figure 5.5 Concentration of citronellol in the CCGLB predicted using
the proposed model at different 𝑘𝐿𝑎 values.
128 Figure 5.6 Concentration of geraniol in the CCGLB predicted using
the proposed model at different 𝑘𝐿𝑎 values.
128
xiv
LIST OF ABBREVIATIONS
Abbreviation Description
DNA Deoxyribonucleic acid
ATP Adenosine triphosphate
NADH Nicotinamide adenine dinucleotide
NAD(P)H Nicotinamide adenine dinucleotide phosphate
NAD+ Oxidized form of NADH
NADP+ Oxidized form of NADPH
GRAS Generally recognized as safe
CCGLB Continuous-closed-gas-loop bioreactor
e.e. % Percentage enantiomeric excess
YADH Yeast alcohol dehydrogenase
rpm Rotation per minute
hr Hour
PTFE Polytetrafluoroethylene
DO Dissolved oxygen
OD680 Optical density at wavelength of 680 nm
ODE Ordinary differential equation
DNS Dinitrosalicyclic acid
U Units of productivity
VOC Volatile organic compound
MBS Membrane-based system
YPD Medium for fermentation of yeast
ISPR In-situ product removal
HCl Hydrochloric acid
KOH Potassium hydroxide
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KH2PO4 Potassium dihydrogen phosphate K2HPO4 Potassium hydrogen phosphate
s Second
min Minute
T Temperature
R Ideal gas constant
vvm Volume liquid per volume of reactor per minute
Eqn. Equation
N Nitrogen
S Sulphur
P Potassium
NaOH Sodium hydroxide
FID Flame ionized detector
GC Gas chromatography
M Molar
mM Milimolar
CO2 Carbon dioxide
atm Atmospheric
g Gram
L Liter
mL Mililiter
µm Micrometer
µg Microgram
nmol Nanomol
S. cerevisiae Saccharomyces cerevisiae
sp. Species
v/v Ratio volume per volume
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EMP Embden Mayerhof Pathway
M-M Michaelis-Menten
N.A Not available
TCA Tricarboxylic acid
MPa Megapascal
OSN Organic solvent nanofiltration
ILs Ionic liquids
i.d Internal diameter
ATCC American Type Culture Collection
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LIST OF SYMBOLS
Symbol Description Unit
𝜇 Specific growth rate hr-1
𝑄𝑠 Glucose consumption rate g/L.hr
𝑋 Cell concentration g/L
𝑡 Time hr
𝑋𝑜 Initial concentration of cell g/L
𝑆 Substrate concentration g/L
𝑟𝑖 Mass transfer rate of component i g/L.hr
𝑘𝐿𝑎 Overall mass transfer coefficient hr-1
𝐶∗ Equilibrium concentration at interface g/L
𝑖 Component i -
𝐶𝑖 Concentration of component i g/L
𝑃𝑖 Partial pressure of component i atm
𝐻 Henry’s coefficient L.atm/mol
𝐶𝐺,𝑔 𝑖𝑛 Concentration of geraniol in gas phase at bioreactor inlet
g/L 𝐶𝐺,𝑔 𝑜𝑢𝑡 Concentration of geraniol in gas phase at bioreactor
outlet
g/L 𝐶𝐶,𝑔 𝑜𝑢𝑡 Concentration of citronellol in gas phase at bioreactor
outlet
g/L
𝐶𝐺,𝑙 Concentration of geraniol in bulk liquid g/L
𝐶𝐶,𝑙 Concentration of citronellol in bulk liquid g/L
𝑄 Substrate flow rate L/hr
𝑉𝑔 Volume of gas L
𝑉𝑇 Volume of bioreactor L
𝑉𝑙 Volume of liquid L
xviii
𝐶𝐺,𝑙∗ Equilibrium concentration of geraniol at interface g/L 𝐶𝐶,𝑙∗ Equilibrium concentration of citronellol at interface g/L
𝑅 Ideal gas constant L.atm/mol.K
𝑇 Temperature K
𝑟 Rate of reaction g/hr.gcell
𝐾𝑚 Michaelis constant g/L
𝑉𝑚𝑎𝑥 Maximum rate of reaction g/hr.gcell
U Units of production µmol/min
xix ABSTRAK
Kajian ini dijalankan bagi mengkaji keupayaan keseluruhan sel Saccharomyces cerevisiae mentransformasikan monoterpen yang bersifat hidrofobik, iaitu geraniol kepada sitronellol di dalam reaktor gelung gas tertutup berterusan (CCGLB). Geraniol yang mempunyai tekanan wap dan kemeruapan yang tinggi serta kebolehlarutan yang rendah di dalam medium akuas amat sesuai digunakan dalam sistem ini. Kajian kinetik terhadap proses biotransformasi menggunakan dua jenis keadaan sel; iaitu sel tumbuh dan sel rehat telah dijalankan. Kajian awal menunjukkan sistem biotransformasi fasa cecair hanya sesuai dijalankan dengan penggunaan sel rehat di mana perencatan substrat terhadap sel telah diperhatikan apabila sel tumbuh digunakan. Walaubagaimanapun, kedua-dua jenis sel telah berjaya mentransformasikan geraniol di dalam CCGLB. Sistem gelung gas tertutup tersebut telah menghasilkan kepekatan sitronellol yang maksimum iaitu sebanyak 2.38 g/L dengan aktiviti spesifik 7.9 U/gcell. Ia telah dihasilkan dengan menggunakan sel rehat sebagai pemangkin bio pada pH 7, 350 rpm, kadar aliran substrat pada 8 L/min dan kepekatan glukosa sebanyak 50 g/L. Proses tersebut berjaya meningkatkan produktiviti sitronellol sebanyak 5 kali ganda berbanding biotransformasi di dalam mod kelompok (biotransformasi fasa cecair). Bagi proses biotransformasi menggunakan sel tumbuh pula, didapati kehadiran substrat biotransformasi telah mempengaruhi penumbuhan sel di mana penurunan kepekatan sel sebanyak 60% telah direkodkan. Proses tersebut telah menghasilkan sitronellol yang maksimum sebanyak 1.18 g/L pada keadaan kendalian pH 7, 500 rpm dan kadar aliran substrat pada 8 L/min. Sistem CCGLB juga berkebolehan menyingkirkan produk secara in-situ di mana produk yang terhasil adalah jernih tanpa memerlukan sebarang proses hilir. Pemodelan sistem biotransformasi
xx
menggunakan sel rehat di dalam CCGLB telah dibina dengan menggabungkan model Michaelis-Menten serta model dua filem. Pemalar-pemalar kinetik yang diperoleh daripada eksperimen mod kelompok adalah 0.015 g/hr.gcell bagi 𝑉𝑚𝑎𝑥 dan 17.9 g/L bagi 𝐾𝑚. Model penyelakuan telah dipadankan dengan keputusan eksperimen pada kadar aliran substrat yang berbeza. Dari kerja pemodelan tersebut, pekali pemindahan jisim keseluruhan (𝑘𝐿a) bagi geraniol boleh ditentukan. Nilai yang diperoleh adalah 4, 7 dan 10.8 hr-1 bagi kadar aliran substrat 4, 6 dan 8 L/min.
xxi ABSTRACT
The present study describes the biotransformation of hydrophobic monoterpene, geraniol into citronellol by whole cells of Saccharomyces cerevisiae in a continuous closed-gas loop bioreactor (CCGLB). Geraniol which has high vapor pressure, high volatility and low solubility in aqueous medium is highly suited to be used in this system. The kinetics of the biotransformation using both states of cells;
growing and resting cells were investigated. The preliminary results showed that the liquid-phase biotransformation system was only practical with resting cells where the growing cells were incapable to transform geraniol. However, both growing and resting cells have successfully performed the biotransformation of geraniol in CCGLB. The gas loop led to a maximum citronellol concentration of 2.38 g/L and specific activity of 7.9 U/gcell. This was obtained by the biotransformation using resting cells at pH 7, 350 rpm, substrate flow rate of 8 L/min and glucose concentration of 50 g/L. Process improvements achieved a 5-fold increase in the citronellol production over the shake-flask performance (liquid-phase biotransformation). For the biotransformation using growing cells, it was found that the cell growth was affected by the presence of substrate for biotransformation with about 60% reduction of final cell concentration was observed. The process gives a maximum citronellol formation of 1.18 g/L at conditions of pH 7, 500 rpm and 8 L/min of substrate flow rate. The CCGLB system also brings advantage of in-situ product removal where a clear product was obtained without a need of downstream processes. A modeling work of the biotransformation using resting cells in the CCGLB was further developed with a combination of Michaelis-Menten and two- film models. The kinetic constants of 𝑉𝑚𝑎𝑥 and 𝐾𝑚 were determined from the shake- flask experiments to be 0.015 g/hr.gcell and 17.9 g/L, respectively. The simulations
xxii
results were validated to the experimental results at different substrate flow rates.
From the modeling work, overall mass transfer coefficient (𝑘𝐿𝑎) of geraniol could be predicted and were found to be 4, 7 and 10.8 hr-1 at substrate flow rates of 4, 6 and 8 L/min, respectively.
1 1.0 Overview
In recent years, biocatalytic process has become a popular method in organic compound synthesis. A wide range of compounds can be produced in a highly chemo-, regio- and enantioselective manner using such a method through a process called biotransformation. Monoterpenoids are one of the most important starting materials in biotransformation and are widely used as flavors in the fine chemicals sector such as pharmaceuticals and food industries (Bull et al., 1999; Miller and Nagarajan, 2000). In the biocatalytic branch, microbial cells are of the preferential biocatalysts for reduction and oxidation (redox) reactions as they provide an in-situ cofactor regeneration system (Carballeira et al., 2009). The addition of external cofactors is necessary when dealing with an isolated enzyme system (Liu and Wang, 2007).
Conventionally, whole-cell biotransformation is carried out in an aqueous medium where the cells are most active. However, the substrates involved, which are usually organic, have low solubility in the aqueous phase and the substrates and/or products may cause inhibition to the cells at high concentrations (Leon et al., 1998;
Marques et al., 2010). In order to overcome such problems, a prominent development has been made approaching several ways such as metabolic engineering, reaction medium engineering and also through the introduction of new concepts in bioreactor design. The combined effect of advances in these areas is found to contribute to the success in many biotransformation processes. Such
2
technologies could promote the biological-based process for use in important sectors which have long been dominated by the ordinary chemical-based process.
1.1 Natural flavor compounds
Flavor compounds are considered ‘natural’ if they were synthesized through fermentation, enzymatic process or extraction from plants (Serra et al., 2005).
According to the regulations of US Food and Drug Administration Guidelines, the natural product must be identical in physical and chemical aspects to the natural substance that is already known to exist in nature (de Carvalho and da Fonseca, 2006). Product from chemical catalysis is considered synthetic because its chemical property differs to that of the natural substance even though it gives similar taste and odor (Brenna et al., 2003). A rising demand for natural products in food processing and drug manufacturing over the last decade has triggered off significant research activities in flavors production using biocatalysis (Berger, 1995; Demyttenaere, 2001). Two main factors owing to such a high demand are the specific isomer (chiral) property of product and the strong preference towards ‘natural’ instead of synthetic product among customers. Chirality is an important feature in flavors as well as in fragrances since two different isomers of the same compound can have quite different odors and tastes. For example, R-(+)-limonene gives orange scent while S-(-)-limonene gives turpentine scent (Berger, 1995). There are various types of flavors that have been successfully produced through biocatalytic process. A comprehensive overview of the specific biocatalytic reaction involved in the synthesis of flavors has been well presented in the literature (Cheetham, 1993;
Berger, 1995; Demyttenaere, 2001; de Carvalho and da Fonseca, 2006).
3 1.2 Biotransformation
Biotransformation is described as a selective modification of a compound into other specific compound with the use of biological catalysts such as plant cells, microorganisms or isolated enzymes (Kieslich, 1984). The process involves a vast array of enzymatic reactions, which is always referred to as a biocatalytic reaction.
Historically, the first biotransformation was discovered by Pasteur in 1862 for acetic acid production using a pure culture of Bacterium xylinum (Csuk and Glanzer, 1991).
Both whole cells and isolated enzymes have great potentials for biotransformation purposes. The selection is mainly based upon reaction type. Hydrolases-type enzyme such as lipase and esterase are the most predominantly used in catalyzing hydrolysis reaction while the whole cells are of choice in reactions involving electron transfer such as oxidation and reduction as they provide in-situ cofactor recycling as the reaction progresses. Industrial application of enzymes in redox reaction has not yet been recognized due to a high cost of enzymes as well as the external cofactors pool present (Turner, 1995; Straathof et al., 2002). Often, the cofactors are more expensive compared to that of the desired products (Liu and Wang, 2007), which makes the choice of isolated enzymes not practical. Nevertheless, several cofactors can now be effectively regenerated using enzymatic method, but they are still scarcely used in industry (Shin et al., 2007).
In comparison to the enzymatic system, whole-cell biotransformation generally perform slower rate of reaction due to the fact that there exist a permeability barrier of cell membrane to substrate and/or product. Fortunately, the drawback has been addressed using molecular engineering approach of recombinant DNA to resolve the problem (Shin et al., 2007). Apart from that, multiple enzymes in the whole cells always lead to a number of side reactions. This phenomenon has also
4
been controlled which resulted in the increase of optical purity (Chartrain et al., 2001). The pros and cons between these two methodologies are presented as in Table 1.1. The whole-cell biotransformation system is discussed at length in Section 1.3 as this study is particularly focused on the similar type of system.
Table 1.1: Comparisons between whole-cell and enzymatic biotransformation (Ishige et al., 2005; Shin et al., 2007)
Whole-cell biotransformation Enzymatic biotransformation Favorable in redox reaction Requires addition of expensive
cofactor(s) for redox reaction Readily available Required protein purification steps Cheaper biocatalyst Expensive biocatalyst due to extraction
and recycling costs Side reactions may occur due to multi-
enzyme system
Clean process Protected by cell envelope
(stable)
Exposure to external environment (rather unstable)
Biotransformation offers a product with outstanding properties of chemo-, regio- and stereospecificity. The chemospecificity refers to a restricted single chemical reaction when several functional groups present at the compound’s structure; and thus, avoids side reactions. Regiospecificity gives an indication of substrate molecules to react at the same site of enzymes while stereospecificity indicates the enzyme preference to attack with one of enantiomers from two entities of R- or S- configurations resulted in a single enantiomer compound, thereby avoiding difficulties of racemic mixtures (Leuenberger, 1990). Such a problem is a key issue recognized in the chemical catalysis which makes it unfeasible choice for the production of compound that requires high selectivity.
Generally, the conditions used in carrying out biotransformation are comparatively mild; with temperature as low as 30 to 40°C and at atmospheric
5
pressure (Bommarius and Riebel, 2004). In contrary, a chemical catalysis normally requires extreme conditions which results in high energy consumption.
Biotransformation is environmentally friendly, in contrast to chemical catalysis due to the usage of heavy metal catalysts (Gotor, 2002). The advantages offered by biotransformation over chemical catalysis are summarized in Table 1.2.
Table 1.2: Comparisons between biotransformation and chemical catalysis
Biotransformation Chemical catalysis
Operated at mild operating conditions Operated at high temperature and pressure
Product specificity Product with racemic mixtures Environmentally friendly Used heavy metal catalysts
Acknowledged as ‘natural’ Acknowledged as artificial/synthetic
The introduction of biotransformation technique in chemical synthesis is unlikely to replace the conventional chemical method completely. In most industries, a close co-operation between these two methodologies is essential for their success.
The possible routes for each target compound are influenced by a number of considerations. The availability of starting materials, product specificity, the number of steps involved, environmental considerations, scalability, downstream processing and development time are initially evaluated (Shaw et al., 2003). Therefore, biotransformation in particular, can be a useful tool in cases where product is difficult to obtain via conventional chemical catalysis.
1.3 Whole-cell biotransformation
Nowadays, rapid advances in the life sciences especially in microbiology have greatly increased the usage of whole cells in biotransformation processes.
Whole cells can either come from microorganisms such as fungi, yeast, bacteria, or
6
may be from plant cells culture. In order to understand the catalytic reaction using the whole cells of microorganisms, one must explore their cell physiology as well as its metabolism. An explanation of these topics is detailed-out in the following sub- sections.
1.3.1 Growing and resting cell systems
The whole-cell biotransformation is generally classified according to the culture state as growing or resting phenomenon (Kieslich, 1984). The difference between both systems is of the different ways of media preparation. A simple media preparation of buffer solution or even water with an addition of carbon source is enough to build the resting cell biotransformation system while the growing cells require other nutrient sources, such as nitrogen and amino acid (Chin-Joe et al., 2002). Selection of the best system normally relies on the availability and also the cost of microorganisms. Normally, the former was used due to restricted availability of the microorganisms in market. Nevertheless, the best example of commercially applied resting cells is the Saccharomyces cerevisiae (baker’s yeast). Baker’s yeast represents the largest bulk production of any single-cell microorganism throughout the world with several million tons of fresh baker’s yeast cells are produced annually for human consumption (Di Serio et al., 2001).
In growing cell biotransformation, substrate can be induced during the inoculation time or in the later phases of the microbial growth. As the biotransformation progresses, the cells will grow and increasing in their numbers.
When dealing with such a biotransformation system, substrate concentration is always crucial. High concentration of substrates could inhibit the cell activity. Due to this reason, the biotransformation processes are normally carried out at late
7
exponential or during stationary phase where the cells and their enzymes are at their maximum density (Kieslich, 1984). On the other hand, cells in resting state are viable, but they are unable to grow as the biotransformation is in progress due to limited amount of nutrient. Generally, the resting cell biotransformation is performed in order to investigate in particular, the biocatalytic reaction without the interference of cell growth which could affect the production of the responsible enzyme (Wang et al., 2005).
1.3.2 Cofactor regeneration in the whole-cell system
Microorganisms are capable to catalyze almost all chemical reactions such as isomerization, racemization, oxidation, reduction and hydrolysis. Among these reactions, oxidation and reduction reactions are relatively complex as they require cofactor-dependent enzymes, or known as oxidoreductases (Presečki and Vasić- Rački, 2005). The cofactor pool exists naturally in the whole cells as a result of cellular metabolism, and can be recycled by the addition of co-substrates such as glucose (Buque et al., 2002). In order to understand how the microorganism generates these cofactors, one should explore the main centre of the cellular metabolism which is known as the glycolysis process.
Glycolysis involves a degradation of carbon source, which in this work is glucose, into smaller molecules of pyruvate with concomitant production of an amount of energy in the form of ATP, which is the form needed by the cells. Most of pyruvates are further degraded to produce ethanol with the release of carbon dioxide.
Simultaneously, the reduced form of cofactor, NAD(P)H is oxidized into NADP+ (Shuler and Kargi, 1992). When a new sub-system is employed, for instance the addition of substrate of biotransformation, the cofactor will interact with the new
8
sub-system and performs a side reaction. The substrate is reduced into the corresponding product while at the same time, the NAD(P)H is continuously oxidized into NADP+ (Chin-Joe et al., 2002). The process continues as long as there is enough glucose to carry out the reaction. A schematic diagram which represents the cofactor recycling in yeast cell with geraniol as the substrate is shown in Figure 1.1.
H3C
H3C
CH3 HO
Geraniol
H3C
H3C
CH3 HO
Citronellol
Figure 1.1: Schematic representation of cofactor regeneration during the reduction of geraniol into citronellol by S. cerevisiae with glucose as co-substrate.
1.4 Yeast as a source of biocatalyst for biotransformation
Yeast is a well-known microorganism and probably the only group of microorganisms, that has a cultural history dating back to prehistoric time.
Saccharomyces, Candida, Kluyveromyces and Pichia are among the genus of yeast that have been manipulated as biocatalysts (Li et al., 2010). The yeast can be found in various sugar-rich fruits, in water or in soils. The characteristics of yeast have been extensively discussed in a number of textbooks (Wang et al., 1979; Shuler and Kargi, 1992; Wainwright, 1992). In short, the yeast cells are eukaryotes, having cellular organelles such as mitochondria, a nucleus, etc. They are all mostly unicellular and reproduce asexually by budding off daughter cells from a single mother cell. Figure 1.2 shows a parent cell of S. cerevisiae that produced new cells
CO2 + Ethanol
NADPH NADP+
Yeast cell
Glucose
9
from budding process. The size of yeast is normally larger than bacteria with about 5-12 µm in diameter. A prominent feature of all yeast cells is that the cell wall is mainly made up of polysaccharides which give strength and rigidity to the cell. In the yeast family, S. cerevisiae or commercially known as baker’s yeast is the most popular and most studied species of yeast. The species was the first eukaryote to have its entire genome sequenced (Vassarotti et al., 1995). Saccharomyces means
‘sugar fungi’, and indeed they grow well in various types of sugar such as glucose, glycerol and acetate (Zhang et al., 2003). S. cerevisiae has been used for fermentative food processing since the ancient times; and thus, are considered to be quite safe for industrial use (Servi, 1998).
Figure 1.2: Electron micrograph showing Saccharomyces cerevisiae parent cell that has produced at least six daughter cells as shown by the circularly arranged bud scars (Hatzis and Porro, 2006).
The uniqueness of baker’s yeast is that, it can be easily grown without a sterile condition. As reported by several researchers, baker’s yeast is genetically stable and very robust organism, non-toxic, easy to handle and not a strict anaerobiosis (Servi, 1998; Perles et al., 2008). In the field of biotransformation, the
10
ability of baker’s yeast to perform an asymmetric reduction has been first presented by Dumas in 1874, and nowadays, there are a huge number of studies being published which employ the microorganism especially for the reduction of carbonyl and carbon-carbon double bonds compounds. A brief description on the biotransformation using baker’s yeast as biocatalyst is presented in the next chapter.
1.5 Biotransformation in non-conventional systems
Biotransformation of organic compounds in non-conventional systems have been extensively explored due to several weaknesses associated in conventional system such as low substrate solubility and substrate and/or product inhibition (Leon et al., 1998). This causes only a small amount of substrate to come into contact with the cells, which could lead to a decrease in the volumetric productivity. Apart from that, a mixture of biocatalyst and product in the reaction medium requires several downstream processes for product separation. A number of non-conventional systems have been proposed to tackle the solubility problem including; water- immiscible organic solvent system (Nikolova and Ward, 1993; Doig et al., 1998b;
Leon et al., 1998; Kansal and Benerjee, 2009); ionic liquid system (Itoh and Tomoko, 2007; Sureshkumar and Lee, 2009), supercritical fluid system (Al-Duri et al., 2001) and compressed solvent system (Oliveira et al., 2006). An integrated design of bioreactor with in-situ product removal (ISPR) technique has also been introduced to eliminate the downstream processes such as the membrane-based system (Collins and Daugulis, 1997; Doig et al., 1998a; Onken and Berger, 1999;
Krieg et al., 2000; Carvalho et al., 2001; Valadez-Blanco et al., 2008); solid-gas system (Lamare and Legoy, 1993; Maugard et al., 2001; Yeom and Daugulis, 2001;
Létisse et al., 2003; Marchand et al., 2008); and the gas loop system (Steinig et al., 2000; Pescheck et al., 2009).
11 1.6 Problem statement
Flavor and fragrance compounds can be obtained through three major techniques which are; the physical extraction from plants, chemical catalysis and biocatalysis. A traditional method of physical extraction from plants has been abandoned since such a technique suffers from seasonal variation, low yield of the final product and high production cost due to the application of many downstream processes. For a long time, these compounds are synthesized using ordinary chemical catalysis. However, this technique is lacking of producing an enantiomerically pure compound where the undesirable racemic mixtures have often formed. Enantiomeric purity is a key issue in synthetic chemistry especially for use in food and consumer products. The quest for enantiomerically pure compounds is motivated by the increasing health concerns among consumers since non-purity compounds lead to adverse side effects to the human body. Due to this reason, an application of biocatalysis has started to spread because of its extraordinary capability to produce compounds with highly enantiomeric purity. In the area of biotransformation, non- conventional systems are gaining importance with the aim to achieve high productivity as well as to provide ease of handling process. A continuous-closed-gas- loop bioreactor (CCGLB) is a new design approach of bioreactors and has been applied in this work. The CCGLB possesses a continuous gas phase reaction which can overcome some major problems associated with the conventional system and these include; 1) eliminating mass transfer limitation which resulted in an increase in the biotransformation rate; 2) providing in-situ product removal thus, reduce a number of downstream processes and; 3) reducing substrate and/or product inhibition.
12
A model of reaction proposed in this work is an asymmetric reduction of geraniol into citronellol using the whole cells of Saccharomyces cerevisiae (baker’s yeast type-II). Citronellol which has a pleasant rose scent has been recognized as safe (GRAS) and commercially used in food, fragrance and pharmaceutical sectors.
Baker’s yeast type-II was chosen because it has shown a great performance in reduction reaction, its availability in the market is in bulk and also generally inexpensive. Those aspects make it favorable for this particular investigation. An effort in optimizing parameters that might give a strong impact to the biotransformation rate was closely investigated in the experimental work.
1.7 Objectives
The main goal of this research is to investigate the efficiency of CCGLB system in conducting the biotransformation of geraniol into citronellol mediated by baker’s yeast. Therefore, in order to achieve this goal, a few integrated objectives have been addressed as follows:
1) To investigate the compatibility and performance of CCGLB for biotransformation.
2) To compare the performance of baker’s yeast in catalyzing gas-phase biotransformation to that of the liquid-phase biotransformation.
3) To determine the optimum parameters such as biomedium pH, agitation rate, substrate flow rate and glucose concentration for which highest product formation can be obtained in the CCGLB.
4) To explore the baker’s yeast efficiency in catalyzing biotransformation during its growth.
13
5) To determine the kinetic parameters and simulate the biotransformation process in CCGLB.
1.8 Scope of study
The biotransformation of geraniol into citronellol using the whole cells of S.
cerevisiae was conducted in liquid and gas phase systems. The difference between these two systems is in the form of geraniol being consumed during the reaction. For the liquid-phase biotransformation, experiments were conducted in shake-flask culture, while the gas-phase biotransformation was conducted in the CCGLB. Taking into account the highest production of citronellol obtained, the reaction was optimized at several parameters such as initial pH and substrate concentration. While in the CCGLB system, the effects of agitation rate, substrate flow rate and amount of glucose required were also investigated.
The study begins with a preliminary work where the kinetics of yeast growth were observed at several fermentation conditions such as initial pH, temperature and glucose concentration. Such work is important in order to maximize the productivity of biomass during S. cerevisiae cultivation. In addition, the optimum temperature obtained from this work was further used in later biotransformation experiments.
Specific growth rate was determined in order to evaluate the cell growth behavior.
Liquid-phase biotransformation of geraniol was then conducted in shake- flask culture. The potential of growing and resting cells of baker’s yeast in catalyzing the reaction were investigated. The purpose of this work is mainly to determine the efficiency of S. cerevisiae to transform geraniol using both states of cells. From this work, one might explore the behavior of yeast growth with and without the presence of substrate for biotransformation (geraniol). It also provides information of the
14
affinity of yeast to grow and simultaneously to perform the biotransformation. In contrast, the biotransformation using resting cells strictly focused on the rate of reaction at several specified parameters.
In the CCGLB system, the same states of cells were applied as in the aforementioned preliminary work. Parameters that give major effects to the rate of reaction such as biomedium pH, agitation rate, substrate flow rate and glucose concentration were closely investigated. These experiments were carried out with the aim to observe the performance of CCGLB and later to compare with the results of the liquid-phase biotransformation.
The last part of the work is on process modeling of the biotransformation with regard to the CCGLB system. This was carried out by preparing material balances of substrate and product in gas and liquid phases. A complete solution of the model is then numerically solved using codes implemented in MATLAB® and later graphically simulated for further understanding of the working system.
Validation of the model was undertaken by comparing the simulated results to the experimental data at different substrate flow rates. The model was also used to estimate the overall mass transfer coefficient of geraniol within the system.
1.9 Thesis structure
This thesis contains six chapters. Chapter 1 introduces the background and the objectives in this study. Chapter 2 gives a survey of the literature relevant to this study, which includes the baker’s yeast mediated biotransformation, biological and non-biological catalysis of geraniol, and describes the previous work and development in the area of biotransformation. Chapter 3 illustrates the experimental procedures and materials applied in this project. The experimental results are
15
presented and discussed in Chapter 4, which includes the experimental results from yeast growth study, liquid-phase biotransformation, and the results from the study in the CCGLB. Chapter 5 presents and discusses the modeling of biotransformation of geraniol into citronellol in the CCGLB, and the results predicted by the model.
Chapter 6 draws the conclusions from this work and suggests some recommendations for future studies.
16
CHAPTER TWO
LITERATURE REVIEW
2.0 Overview
In this chapter, the published knowledge of flavors production by microorganisms is reported. Further review on stereoselective biotransformation mediated by baker’s yeast is presented in order to explore the versatility of this microorganism in catalyzing variety of useful compounds. Factors that influence the yeast growth as well as the biotransformation was summarized, which can be helpful for future investigation. Apart from that, a compilation of citronellol production using whole-cell biocatalysis as well as chemical catalysis is also presented. The last part of this chapter discusses on the improvements in the field of biotransformation processes including whole-cell engineering, medium engineering and bioreactor design.
2.1 Production of flavors by microorganisms
Fruits and flowers have their own perception of flavors and odors due to the occurrence of sensory substances in the form of essential oils. Traditionally, man has extracted these essential oils directly from plants for commercial use as food additives and also as components in consumer products such as perfumes and cosmetics. However, the demand of flavor compounds can no longer be met by the supply of plant sources. This is due to the fact that, this method usually produces low concentration of the desired compound. Apart from that, the extraction process is rather complex which implies to high production cost (Naik et al., 1989). Nowadays, majority of artificial flavors were prepared in bulk quantity by chemical catalysis.
Alongside with this means, the employment of biological catalysis especially in food
17
processing and pharmaceutical manufacturing has increased considerably in the past decades because the process is regarded as safe (Straathof et al., 2002).
Terpenes are the major class of natural products found in nature and widely used as starting materials in flavor synthesis. Classification of terpene family is based on the number of isoprene units (C5) incorporated in the molecular skeleton of the compound. For example, two isoprene units represent monoterpene (C10H16) while three isoprene units represent sesquiterpene (C15H24). Modified terpenes with the addition of an oxygen atom in their structures are known as terpenoids and being the largest constituents in essential oils. About 23,000 single terpenoids were found in nature and most of them are in the form of monoterpenoids (Demyttenaere, 2001).
Almost all terpenoids are acknowledged by US Food and Drug Administration (FDA) as GRAS (generally recognized as safe) for their intended use as flavoring substances (The Terpene Consortium, 2002).
Monoterpenoids can exist as aldehydes, alcohols, ketones or ethers, in acyclic and cyclic structures. Figure 2.1 gives the structure of some representative monoterpenoids.
CH3
H3C CH3 O
(3)
CH3
H3C CH3 O
(2)
CH3
H3C CH3 OH
(1)
Figure 2.1: Monoterpenoid derivatives structure; (1) geraniol (alcohol); (2) citronellal (aldehyde); (3) pulegone (cyclic ketone).
18
Prior to the biocatalysis, microorganisms, plant cells and isolated enzymes have shown their capability in catalyzing various types of terpene compounds (Demyttenaere, 2001). According to a survey conducted by de Carvalho and da Fonseca (2006), approximately two-thirds of the published works on the terpenes biotransformation in the last decade had employed microorganisms as the biocatalyst and indeed, bacteria and fungi are the most dominant as indicated in Figure 2.2. Plant cells contributed about 11% while only 7% had used isolated enzymes.
Figure 2.2: Percentage of published papers on various biocatalysts used in biotransformation of terpenes in the last ten years (de Carvalho and da Fonseca, 2006).
The reason why the microorganisms are of choice is eventually because they are capable to catalyze a broad range of chemical reactions and also much cheaper than that of isolated enzymes. In comparison to the plant cells, the growth of microorganisms is much faster, which makes it feasible for use in biotransformation.
Almost all classes of microorganisms have been utilized as biocatalysts in synthesis of flavors which are from bacteria, fungi, yeast and higher fungi families
Bacteria 41%
Yeasts Enzymes 2%
7%
Microalgae 4%
Plants 11%
Fungi 33%
Cyanobacteria 2%
19
(Berger, 1995). Bacillus, Pseudomonas and Acetobacter species are among bacteria employed (Hua et al., 2007), while Aspergillus, Basidiomycetes and Penicillium are of favorable strains from higher fungi (Lomascolo et al., 1999; Pescheck et al., 2009). In the yeast family, Candida, Saccharomyces and Rhodutorula are among the most popular strains used in the synthesis (Miyazawa et al., 1995; Muller et al., 2006). The strain of S. cerevisiae has successfully transformed a number of monoterpenes such as monoterpene ketones of menthone, carvone, isopiperitenone and phenylalanine to the corresponding alcohols (Xu et al., 2007; Yadav et al., 2007), allylic alcohols such as geraniol to citronellol (Gramatica et al., 1982), linalool to α-terpineol (King and Dickinson, 2000) and monoterpene aldehyde of citral to citronellal (Muller et al., 2006). Other potential microorganisms in the synthesis of flavors are presented in Table 2.1.
The development in terpene synthesis has led to an improvement of the corresponding reaction systems such as the increase in their yield and productivity.
Such a work may involve utilizing different types of microorganisms, changing reaction conditions and optimizing the duration of biotransformation (Aniol and Huszcza, 2005). The improvement in this system with an integration of other methods such as genetic engineering, cell immobilization and medium engineering could increase the potential of whole cells in organic synthesis purposes.
20 Table 2.1: Potential microorganisms for synthesis of flavors
Biocatalyst Substrate Product Flavoring
assessment
Reference
Serratia
Amycolatopsis
Pseudomonas sp.HR 199
O HO
O OH
Ferulic acid
O OH
Eugenol
OH
O
Isoeugenol
HO
O O
Vanillin
Vanilla (Xu et al., 2007)
Ischnoderma
H2N
O
OH
L-Phenylalanine
O
Benzaldehyde
Cherry (Xu et al., 2007)
