MCL-PHA-Yong-Thesis

Fermentation Process Development for the Production

of Medium-chain-length Poly(3-hydroxyalkanoates) by

Pseudomonas putida KT2440

by

Zhiyong Sun

A thesis submitted to the Department of Chemical Engineering

in conformity with the requirements for the degree of Doctor of Philosophy

Queen’s University Kingston, Ontario, Canada

July, 2007

Copyright © Zhiyong Sun, 2007

i

Abstract

Fed-batch fermentation processes were developed for efficient and automated lab scale

production of saturated and unsaturated (functionalized) medium-chain-length

poly(3-hydroxyalkanoates) (MCL-PHA) by Pseudomonas putida KT2440. Four

automated glucose feeding strategies were first developed for the high-cell-density

cultivation of P. putida KT2440. An overall biomass productivity of 4.3 g X l-1 h-1

(highest reported for P. putida KT2440) was obtained by continuous feeding based on

cumulative glucose consumption estimation. An exponential feeding strategy proved to

be an effective way of applying carbon-limitation and controlling the specific growth rate

of the culture while also achieving a satisfactory cell concentration. Contrary to the

common literature belief, nitrogen limitation proved to be unnecessary for MCL-PHA

synthesis by P. putida KT2440. Instead, MCL-PHA production was achieved by

single-stage, carbon-limited, exponential nonanoic acid feeding fed-batch fermentations.

This approach resulted in an overall PHA productivity of 1.4 g PHA l-1 h-1 (highest

reported for PHA production using nonanoic acid as substrate) at a specific growth rate of

0.25 h-1 and a high final PHA content of 75% (highest reported final MCL-PHA content)

at a specific growth rate of 0.15 h-1. By co-feeding of nonanoic acid and glucose, the

nonanoic acid to PHA yield was increased up to 30% without significantly altering the

composition of the PHA or compromising the overall PHA productivity (1.4 g PHA l-1

h-1). This would lead to a substantial substrate cost reduction if MCL-PHA is produced at

a commercial scale. Unsaturated (functionalized) MCL-PHA was also produced at a

productivity of up to 1.1 g PHA l-1 h-1 (highest reported for unsaturated MCL-PHA

production) and a final content of up to 56% by using nonanoic acid with 10-undecenoic

acid as a co-substrate. The molar fraction of each monomeric component remained

relatively constant through such fermentations. A linear relationship between the degree

of unsaturation of the PHA product and the fraction of unsaturated carbon source in the

substrate mixture was demonstrated. Therefore, such a co-feeding fed-batch process can

be used to produce functionalized MCL-PHA with controlled compositions.

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Statement of Co-authorship

The author wishes to acknowledge the contribution of Dr. Bruce A Ramsay, Dr. Juliana A

Ramsay, and Dr. Martin Guay in the preparation of all manuscripts presented in this

thesis. All experiments were performed by the author.

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Acknowledgements

I would first like to thank my parents. I rarely performed as well as I should have in those

critical exams along my path of education, yet my parents have given me nothing but love

and encouragement, which made a boy became a useful man. When I decided to pursue

my graduate study abroad, they supported me without any hesitation. As their only child,

I have barely spent two months with them during the past five years, and probably this

will be even less in the near future. I know the only way I can make them happy is to

work hard and live a fruitful life. I miss them so much.

My thankfulness also goes to Xuan (Jade), my beloved wife, friend, and colleague. It

is a blessing to have the opportunity to work with her in the same lab. Her love, care, and

support made my life much brighter and my work much smoother. I cannot imagine what

a different study experience abroad would be like without her.

I also thank sincerely my supervisors and mentors, Dr. Bruce Ramsay, Dr. Juliana

Ramsay, and Dr. Martin Guay. Starting a graduate study is one thing. Starting a new life

by living in a different country and speaking a different language is another. Their

guidance, encouragement, and friendship made the transition smooth and joyfull. From

them, I learned advanced scientific knowledge, English, ways of being kind to people,

and even Canadian ways of humor. I consider myself quite lucky to have supervisors that

I can just walk in, sit down, and discuss my project with. I do not think there could be a

better graduate study experience than what they have provided to me. My gratefulness to

them is more than I can say.

Last but not least, thank you to all my labmates, Yinghao, Paul, Thiru, Bozhi, and

others. With all the equipment, I know I am working in an advanced lab and, with all you

guys, I feel like I am living with a warm family.

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Table of Contents

Abstract i Statement of Co-authorship ii Acknowledgements iii Table of Contents iv

List of Tables viii List of Figures ix

List of Abbreviations and Symbols xii CHAPTER 1 General Introduction 1

1.1 Background 1

1.2 Overall objective 4

1.3 Approaches 4

1.4 References 8

CHAPTER 2 Literature Review 10

Fermentation Process Development for the Production of Medium-chain-length Poly(3-hyroxyalkanoates) 10

2.1 Abstract 11

2.2 Introduction 11

2.3 Physiology and the kinetics of biosynthesis 13

2.3.1 General considerations 13

2.3.2 Carbon sources and their concentration 15

2.3.3 Growth rate and nutrient conditions 18

2.4 Process development 19

2.4.1 Continuous processes 20

2.4.2 Fed-batch processes 21

2.4.3 Production of MCL-PHAs containing functional groups 26

2.5 The effect of nutrient limitation on MCL-PHA accumulation 31

2.6 Economic considerations 32

2.7 Future commercial processes 35

2.8 References 36

CHAPTER 3 43

Automated Feeding Strategies for High-cell-density Fed-batch Cultivation of Pseudomonas putida KT2440 43

3.1 Abstract 44

3.2 Introduction 44

3.3 Materials and methods 47

3.3.1 Analytical procedures 47

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3.3.2 Microorganism and growth Medium 47

3.3.3 Fermentation conditions 48

3.3.4 Substrate Feeding Strategies 49

3.4 Results 51

3.4.1 Growth yield study 51

3.4.2 Exponential feeding with a predetermined μ 53

3.4.3 Exponential feeding based on initial μmax estimation 54

3.4.4 Semi-continuous feeding based on the detection of substrate limitation (CPR-based pulse feeding) 56

3.4.5 Continuous feeding with real-time glucose consumption estimation based on CCP analysis 57

3.5 Discussion 58

3.5.1 Yield values 58

3.5.2 Exponential feeding strategies 60

3.5.3 CPR-based pulse feeding 62

3.5.4 Continuous feeding based on CCP 63

3.5.5 Comparison of methodologies 64

3.6 References 66

CHAPTER 4 69

Increasing the Yield of MCL-PHA from Nonanoic Acid by Co-feeding Glucose During the PHA Accumulation Stage in Two-stage Fed-batch Fermentations of Pseudomonas-putida KT2440 69

4.1 Abstract 70

4.2 Introduction 70


4.4 Results and discussion 74

4.5 References 76

CHAPTER 5 78

Carbon-limited Fed-batch Production of Medium-chain-length Polyhydroxyalkanoates from Nonanoic Acid by Pseudomonas putida KT2440 78

5.1 Abstract 79

5.2 Introduction 79


5.3.1 Microorganism and growth medium 82


5.3.3 Substrate feeding and control methods 84


5.4 Results 86

5.4.1 Single-stage, exponential feeding of nonanoic acid (target µ= 0.15 h-1) 88

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5.4.2 Single-stage, exponential feeding of nonanoic acid (target µ= 0.25 h-1) 89

5.4.3 Single-stage, exponential feeding of nonanoic acid (target µ= 0.25 h-1) followed by N-limited culture on nonanoic acid 90

5.4.4 Biomass and PHA yield 92

5.5 Discussion 93

5.5.1 Process design implications 93

5.5.2 Yield 95

5.5.3 Kinetics of MCL-PHA synthesis 96

5.6 References 98

CHAPTER 6 103

Fed-batch Production of Medium-chain-length Polyhydroxyalkanoates by Pseudomonas putida KT2440 using Nonanoic Acid and Glucose Co-feeding 103

6.1 Abstract 104

6.2 Introduction 104






6.4 Results 110

6.4.1 Nonanoic acid and glucose co-feeding for MCL-PHA production 110

6.4.2 Monomeric compositions during co-substrate cultivation 113

6.4.3 Biomass and PHA Yield 114

6.5 Disucssion 118

6.5.1 High yield and PHA productivity by co-substrate carbon-limited fermentation 118

6.5.2 Yield potential of MCL-PHA synthesis 121

6.6 References 122

CHAPTER 7 125

Fed-batch Production of Unsaturated Medium-chain-length Polyhydroxyalkanoates with Controlled Composition by Pseudomonas putida KT2440 125

7.1 Abstract 126

7.2 Introduction 126






7.4 Results 132

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7.4.1 Production of unsaturated MCL-PHAs by exponential feeding of nonanoic acid and 10-undecenoic acid 132

7.4.2 Monomeric compositions 135

7.5 Discussion 137

7.5.1 Process design for unsaturated MCL-PHAs production 137

7.5.2 Effect of specific growth rate and varied substrate composition on PHA synthesis 138

7.5.3 Unsaturated MCL-PHAs with controllable compositions 139

7.6 References 141

CHAPTER 8 Conclusions 143

8.1 Summary and contributions 143

8.1.1 High-cell-density cultivation of P. putida KT2440 143

8.1.2 Carbon-limited, single-stage MCL-PHA production 144

8.1.3 Co-substrate feeding strategy for functionalized MCL-PHA production 145

8.1.4 Yield enhancement and substrate cost reduction 145

8.2 Recommendations for future work 146

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List of Tables

Table 2-1 Summary of MCL-PHA (regular and functionalized) production processes. 29

Table 3-1 Comparison of the four feeding strategies and recommendations 65

Table 6-1 Summary of fermentation results. 117

Table 6-2 Summary of literature MCL-PHA production and yield 117

Table 7-1 Fermentation results summary. 134

Table 7-2 Molar fraction of all 3-OH-alkanoates detected in the MCL-PHA product from different fermentations. 135

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List of Figures

Figure 2-1 Chemical structure of polyhydroxyalkanoates (PHAs), which are classified as short-chain-length PHAs (SCL-PHAs) when the repeating units contain 4 or 5 carbons and medium-chain-length PHAs (MCL-PHAs) when the repeating units contain 6 or more carbons; R pendant group may also contains functional groups resulting in functionalized PHAs. 13

Figure 2-2 PHA content of P. putida strains using alkanes or alkanoates of varying length. P. putida GPo1 (formerly known as P. oleovorans GPo1) grown on n-alkanoates (Gross et al. 1989), P. putida GPo1 on n-alkanes (Lageveen et al. 1988), and P. sp. DSY-82 on n-alkanoates (Kang et al. 2001). 16

Figure 3-1 Yield of dry biomass from the principal growth substrates. Different symbols indicate data from different fermentations. The slope of the line indicates the calculated yield value. Only data indicated by closed and open circles were used to calculate yield from PO4 in (c). 52

Figure 3-2 Fed-batch P. putida cultivation using simple exponential feeding designed to achieve μ= 0.25 h-1. 53

Figure 3-3 Online cumulative CO2 production (CCP) data regressions according to Eq. 2 for μmax estimation. (a) Regression at 10 h cultivation time. (b) Regression at 12 h cultivation time. 55

Figure 3-4 Fed-batch P. putida cultivation using exponential feeding with online μmax estimation. 55

Figure 3-5 Fed-batch P. putida cultivation using CO2 production rate (CPR) based pulse feeding. 57

Figure 3-6 Fed-batch P. putida cultivation using continuous glucose feeding based on glucose consumption as estimated from cumulative CO2 production data. 58

Figure 4-1 PHA subunits produced during the nitrogen-limited stage of each fermentation. Nitrogen-limitation began at about the same time as the first data point shown in each figure. (a) G-only PHA synthesis. (b) NA-only PHA synthesis. (c) NA+G co-feeding PHA synthesis. The PHA subunits are 3-HHp, 3-hydroxyheptanoate, 3-HO, 3-hydroxyoctanoate, 3-HN, 3-hydroxynonanoate, 3-HD, 3-hydroxydecanoate. 75

Figure 4-2 Relationship between total carbon of nonanoic acid (NA) or glucose (G) consumed and total carbon accumulated into the corresponding PHA subunits. Closed squares are from G-only PHA synthesis, open squares

x

from NA-only PHA synthesis, and half-closed squares from NA+G co-feeding. 75

Figure 5-1 Growth of P. putida KT2440 on glucose followed by N-limited growth on nonanoic acid (NA). The division between growth phase and N-limited phase is indicated by the dashed line. The nonanoic acid feeding rate (FNA) was increased from 0.47 g NA l-1 h-1 to 1.01 g NA l-1 h-1 at 27 h during the N-limited phase, as indicated by the dash-dot line. 87

Figure 5-2 Carbon-limited growth of P. putida KT2440 on nonanoic acid (NA) at µ= 0.15 h-1. 89

Figure 5-3 Effect of growth rate on PHA synthesis in P. putida KT2440 under nonanoic acid limitation. Closed and open symbols represent data from the µ= 0.15 h-1 and the µ= 0.25 h-1 fermentations, respectively. qPHA(Xr) is the specific PHA synthesis rate (g PHA g Xr

-1 h-1). 91

Figure 5-4 Two-stage, fed-batch using nonanoic acid (NA) as the sole carbon source. Exponential feeding (µ= 0.25 h-1) was used during the growth phase with all other nutrients in excess. The division between the growth and N-limited phases is indicated by the dashed line. The nonanoic acid feed rate was increased from 1.31 g NA l-1 h-1 to 1.82 g NA l-1 h-1 at 41.5 h during the N-limited phase as indicated by the dash-dot line. 92

Figure 5-5 Yield of total biomass, PHA, and residual biomass (Xr) from nonanoic acid (NA). Closed and open symbols represent data from μ= 0.15 h-1 and the μ= 0.25 h-1 fermentations, respectively. The slopes of the curves indicate yields from nonanoic acid. 93

Figure 5-6 The specific rate of PHA synthesis (per gram of PHA) approaches a constant value as the concentration of PHA in the biomass increases. 98

Figure 6-1 Single-stage MCL-PHA production by P. putida KT2440 using nonanoic acid (NA) and glucose (G) co-feeding strategy with NA:G= 1:1 (w/w). Exponential feeding stopped and linear feeding (constant feed rate of 17 g l-1 h-1) began at 24.9 h. Only DO data around the occurance of oxygen limitation (25 h) are shown, before which DO was always maintained around 40% air saturation. 112

Figure 6-2 Single-stage MCL-PHA production by P. putida KT2440 using nonanoic acid (NA) and glucose (G) co-feeding strategy with NA:G= 1:1.5 (w/w). Exponential feeding was applied throughout the fermentation. 113

Figure 6-3 (a) 3-OH-nonanoate (C9) to 3-OH-heptanoate (C7) ratio from NA:G= 1:1.5 co-feeding fermentation and NA sole feeding fermentation. (b) PHA composition throughout the NA:G= 1:1.5 fermentation. C5, 3-OH-valerate;

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C7, 3-OH-heptanoate; C9, 3-OH-nonanoate; C-even, total of mainly 3-OH-hexanoate, 3-OH-octanoate and 3-OH-decanoate. m is the slope of the corresponding regression line. 114

Figure 6-4 (a) Substrate to biomass yield. (b) nonanoic acid (NA) to PHA yield plots. m is the slope of the corresponding linear regression line. All linear regression curves were forced through origin. 116

Figure 6-5 The relationship between PHA content and the cumulative nonanoic acid to PHA yield ( /

overallPHA NAY ). /

overallPHA NAY is the cumulative PHA yield from nonanoic

acid at a given sampling point. 122

Figure 7-1 Fermentation a, exponential feeding of nonanoic acid (NA) and 10-undecenoic acid (UDA=) (molar ratio 4.98:1) with desired μ= 0.15 h-1. The dissolved oxygen data are shown after 30 h, before when it was kept around 40% air saturation. Curve fitting (dashed line) was done by nonlinear regression function of Sigmaplot 9.01. 133

Figure 7-2 Biomass, PHA concentration and PHA content of fermentations b and c, with the same μ (0.25 h-1) and different NA to UDA= ratio (5.07:1 and 2.47:1, respectively). 134

Figure 7-3 (a) Linear relationship between total amount of saturated components and total amount of unsaturated components from three fermentations. (b) Relative monomeric composition of the MCL-PHA synthesized during fermentation b. (c) Relative monomeric composition of the MCL-PHA synthesized during fermentation c. The dash-dot line in (b) and (c) divides saturated and unsaturated components. C5, 3-OH-valerate, C7, 3-OH-heptanoate, C7:1, 3-OH-heptenoate, C9, 3-OH-nonanoate, C9:1, 3-OH-nonenoate, C11, 3-OH-undecanoate, C11:1, 3-OH-undecenoate. The small amount of even carbon number 3-OH-alkanoates is not included these plots. 136

Figure 7-4 Relationship between unsaturation of the MCL-PHA product and the substrate for fermentations at desired µ= 0.25h-1 (fermentations b and c). The r2 is 0.99 for the regression curve. 140

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List of Abbreviations and Symbols 3HB or C4 3-hydroxybutyrate 3HV or C5 3-hydroxyvalerate 3HHx or C6 3-hydroxyhexanoate 3HHp or C7 3-hydroxyheptanoate 3HO or C8 3-hydroxyoctanoate 3HN or C9 3-hydroxynonanoate 3HD or C10 3-hydroxydecanoate 3HDD or C12 3-hydroxydodecanoate C14 3-hydroxy-cis-5-tetradecanoate C7:1 3-hydroxyheptenoate C9:1 3-hydroxy-8-nonenoate C11:1 3-hydroxy-10-undecenate C12:1 3-hydroxy-cis-6-dodecenoate C14:1 3-hydroxy-cis-5-tetradecenoate C10O 3-hydroxy-7-oxooctanoate HCDC High-cell-density cultivation or culture pH-stat A substrate feeding strategy to maintain the pH of the culture at a

certain value or within a certain range DO-stat A substrate feeding strategy to maintain the dissolved oxygen of

the culture at a certain value or within a certain range PHA Polyhydroxyalkanoates or PHA concentration, g l-1 PHB Poly(3-hydroxybutyrate) PHB/V Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHB/HHx Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) PHO Poly(3-hydroxyoctanoate) SCL Short-chain-length MCL Medium-chain-length G Glucose NA Nonanoic acid UDA= 10-undecenoic acid DO Dissolved oxygen expressed as percentage of air saturation, % D Dilution rate in continuous fermentation, h-1 µ Specific growth rate, h-1 µmax Maximum specific growth rate, h-1 X0 Initial biomass at the time of inoculation, g l-1 X Biomass, g l-1

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Xr Residual biomass (X minus PHA concentration), g l-1 ∆St Substrate amount required at time t, g qPHA(Xr) Specific PHA synthesis rate based on Xr, dP/dt/Xr, g PHA g Xr

-1 h-1

qPHA(PHA) Specific PHA synthesis rate based on PHA, dP/dt/PHA, g PHA g PHA-1 h-1

F Substrate (nonanoic acid or glucose) feed rate, g l-1 h-1 PHA productivity g PHA l-1 h-1 X productivity g X l-1 h-1 C Carbon N Nitrogen P Phosphorus Mg Meganesium Fe Iron C/N Carbon to nitrogen ratio % PHA PHA content expressed as percent of PHA among total biomass, % YX/G Yield of biomass from glucose, g g-1 YX/NA Yield of biomass from nonanoic acid, g g-1 YX/NH4 Yield of biomass from ammonium, g g-1 YX/PO4 Yield of biomass from phosphate, g g-1 YX/Mg Yield of biomass from magnesium, g g-1 YX/S Yield of biomass from substrate, g g-1 YCO2/G Yield of CO2 from glucose, g g-1 YCO2/X Yield of CO2 from biomass, g g-1 YCO2/S Yield of CO2 from substrate, g g-1 YP/S Yield of PHA from substrate, g g-1 YPHA/NA Yield of PHA from nonanoic acid, g g-1 Y(C7+C9)/NA Yield of C7 and C9 PHA components from nonanoic acid, g g-1 or

mol mol-1 Y(C8+C10)/G Yield of C8 and C10 PHA components from glucose, g g-1 or mol

mol-1 CPR CO2 production rate, g CO2 h-1 CCP Cumulative CO2 production, g CGC Cumulative glucose consumption, g t Cultivation time, h rNA Proportion of the mass of nonanoic acid among the total NA+G

feed, % rG Proportion of the mass of glucose among the total NA+G feed, %

1

CHAPTER 1 General Introduction

1.1 Background

Plastics have been produced by the chemical industry since the 1930s. Their formability

and durability make them among the most versatile materials ever developed and they

have found their way into nearly every line of business. Despite their versatility and wide

applicability, their drawbacks are becoming increasingly obvious and cannot be ignored.

Most plastics are produced from petroleum, a non-renewable resource. Furthermore, after

being disposed, most synthetic materials are very persistent in the environment. This

causes serious problems in waste management and has a negative impact on the

environment. Therefore, people have been trying for decades to develop new plastic

materials that are biodegradable and derived from renewable resources. Biological

polyesters such as poly(hydroxyalkanoates) (PHAs) and poly(lactic acid) may fulfill such

requirements.

PHAs have drawn great attention as a group of biopolyesters with properties similar

to petroleum-based thermoplastics and thermoelastomers. They are mainly synthesized by

microorganisms as intracellular carbon and energy storage materials. They are commonly

classified as short-chain-length PHA (SCL-PHA), which contains 4 or 5 carbons in their

repeating units; and medium-chain-length PHA (MCL-PHA), which contains 6 or more

2

carbons in the repeating units. At present, approximately 150 different constituents

occurring in PHAs as homopolyesters or copolyesters have been identified (Steinbüchel

and Lütke-Eversloh 2003). Generally speaking, the longer the chain length of PHA, the

lower its crystallinity and melting temperature (van der Walle et al. 2001). Biosynthesized

PHAs are commonly known as biodegradable and biocompatible. They are not only

promising as environmental friendly materials, but also as biomaterials for medical

applications (Zinn et al. 2001).

Poly(3-hydroxybutyrate) (PHB) was the first PHA discovered by Lemoigne in 1926.

Studies on the physiology, biosynthesis, properties and applications of SCL-PHAs have

been carried out intensively since the 1960s. Although PHB and poly(3-hydroxybutyrate-

co-3-hydroxyvalerate) (PHB/V) had been commercialized under the trademark Biopol™

in the 1980s, their initial mission to provide a biodegradable replacement for

petrochemical plastics was hindered by the high production cost. With the further

development of better production strains and fermentation techniques to lower the

production cost, the increasing price of petroleum, and the general increasing desire for

sustainable and environmentally benign plastics, interest in PHA has grown in recent

years. Metabolix Inc. and Archer Daniels Midland Company (ADM) recently announced

a joint venture to build their first commercial scale plant in Clinton, Iowa, USA. They are

3

expecting to produce MirelTM Natural Plastics, which are a series of SCL-PHAs (Peoples

2006), at an annual rate of 110 million pounds beginning in 2008.

In contrast to SCL-PHA, studies on MCL-PHA have taken place only within the last

two decades since the discovery of poly(3-hydroxyoctanoate) (PHO) (de Smet et al.

1983). Production techniques are not as well established as for SCL-PHA. One challenge

is the availability of suitable microorganisms. Although many genera of bacteria have the

ability to synthesize significant amount of SCL-PHAs, only certain species of

Pseudomonas are capable of accumulating MCL-PHAs. Among those, the physiology of

MCL-PHA production seems to differ considerably from one strain to another, rendering

the fermentation process to be strain-specific. The major process challenge lies in control

of the carbon source concentration, as well as process automation. Most MCL-PHA

fermentation processes reported so far involve a substantial level of instrumental

complexity and of manual operation. Hence, they are not suitable for industrial

development. The current high cost of suitable carbon substrates (mainly aliphatics) for

MCL-PHAs synthesis present another challenge for industrial scale production. Certain

substrates, such as oleic acid, albeit supporting substantial MCL-PHA synthesis, result in

PHAs with a fixed but complex composition, and relatively undesirable mechanical

properties.

4

It is well known that MCL-PHAs have wide diversity and are good candidates for

chemical modification. Therefore, they are very promising in a wide range of potential

applications, such as adhesives, paints, coatings (van der Walle et al. 2001), drug delivery

(Pouton and Akhtar 1996), tissue engineering (Chen and Wu 2005) and other medical

uses. It is now essential to develop highly efficient, repeatable and reliable MCL-PHA

production processes in order to increase the availability of these materials for

application-related research and commercial production.

1.2 Overall objective

The overall objective of this research is to develop automated, reliable, well-controlled,

productive lab-scale fermentation processes for the production of MCL-PHAs from

Pseudomonas putida KT2440 (ATCC 47054).

1.3 Approaches

The overall objective was achieved in five phases of study.

It was widely accepted in the literature that MCL-PHAs synthesis is stimulated by the

limitation of certain major growth nutrients (such as nitrogen or phosphorus) and an

excess supply of carbon source. Therefore, a two-stage fed-batch fermentation scheme is

usually employed. During the first stage (growth stage), the microorganism is allowed to

grow to a high cell concentration, which provides the basis for potential high intracellular

5

PHA synthesis. Then a major nutrient is limited but carbon supply is continued, such that

the culture stops growth and begins PHA synthesis. Such a two-stage fed-batch scheme

was initially adopted for our process development.

To make such a two-stage process more economically favorable, an inexpensive

carbon source (glucose) was used for the growth stage, while a more expensive but also

more efficient MCL-PHA-synthesis substrate (nonanoic acid) was used for most of the

PHA-synthesis stage. Key aspects of a successful fed-batch fermentation process involve

the selection of a suitable microorganism, the formulation of a balanced cultivation

medium, and medium feeding strategies. Pseudomonas putida KT2440 was chosen and a

balanced medium was formulated. Four substrate feeding strategies were developed to

achieve high-cell-density culture. Each of these feeding strategies can be fully automated,

and resulted in biomass productivities that were similar or higher than previous literature

work with related strains of Pseudomonas. This phase of research is covered in Chapter 3,

“Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas

putida KT2440 ”, published in Applied Microbiology and Biotechnology 71 (4): 423-431,

2006.

Following the high-cell-density growth phase of P. putida KT2440, the second-stage

(PHA-synthesis stage) was investigated using nonanoic acid as the major carbon substrate,

and also by imposing nitrogen limitation. According to the literature, nonanoic acid is the

6

most efficient carbon substrate for MCL-PHA synthesis in P. putida, and can be

considered a renewable carbon source since it is produced from oleic or other carboxylic

acids derived from temperate plants such as canola. However, no published fermentation

process had used nonanoic acid as the carbon substrate, partly due to its toxicity to the

culture. In my study of two-stage fed-batch fermentation, a nonanoic acid linear feeding

strategy was developed based on the estimated substrate consumption rate, and resulted in

a final PHA content of nearly 30%. A nonanoic acid and glucose co-feeding strategy was

also established such that the yield of MCL-PHA from nonanoic acid was almost doubled

by the co-feeding of glucose. This phase of research is covered in Chapter 4, “Increasing

the yield of MCL-PHA from nonanoic acid by co-feeding glucose during the PHA

accumulation stage in two-stage fed-batch fermentations of Pseudomonas putida

KT2440”, published in the Journal of Biotechnology, March, 2007 online.

Although it was potentially possible to achieve a high final PHA content using the

two-stage fed-batch process, the overall PHA productivity was low, due to prolonged

fermentation time. A chemostat study on the optimal PHA synthesis conditions

demonstrated that substantial PHA synthesis was possible in P. putida KT2440 when

carbon-limited, while other growth nutrients (nitrogen or phosphorus) were in excess.

Based on this finding, a single-stage exponential nonanoic acid feeding fed-batch process

was developed. Substantial MCL-PHA synthesis occurred during the exponential growth

7

of P. putida KT2440. This process is by far the easiest and most automated approach for

the production of MCL-PHAs, and resulted in the highest final MCL-PHA content (75%)

reported in the literature. This phase of research is covered in Chapter 5, “Carbon-limited

fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid

by Pseudomonas putida KT2440”, published in Applied Microbiology and Biotechnology,

74 (1): 69-77, 2007.

Since the cost of carbon substrate(s) usually accounts for more than 30% of the total

production cost for either SCL-PHA production (Choi and Lee 1997) or MCL-PHA

(Hazenberg and Witholt 1997), it is highly desirable to reduce the cost of substrate and/or

increase the PHA yield as much as possible. Therefore, a carbon-limited single-stage

MCL-PHA fermentation process using nonanoic acid with glucose as co-substrate was

developed. Compared to the nonanoic acid as sole substrate process, the co-substrate

process resulted in a similar final PHA content and overall volumetric productivity, a

similar PHA composition, and a nonanoic acid to PHA yield enhancement of 30%.

Consequently, the total carbon substrate cost may be reduced by about 15%. This phase

of research is covered in Chapter 6, “Fed-batch production of medium-chain-length

poly(3-hydroxyalkanoates) by Pseudomonas putida KT2440 using nonanoic acid and

glucose co-feeding”, to be submitted to Journal of Biotechnology in June, 2007.

8

The great potential of MCL-PHAs in many applications is largely due to the wide

diversity of the possible structures. Besides saturated MCL-PHAs, functionalized

MCL-PHAs (with functional groups in the side chains) can also be synthesized. Among

possible functionalized MCL-PHAs, those with olefin groups (unsaturated MCL-PHAs)

are considered the most useful functionalized MCL-PHAs, as many chemical

modifications can be made to these polymers (Hazer and Steinbüchel 2007). Based on the

single-stage nonanoic acid exponential feeding process, nonanoic acid and 10-undecenoic

acid co-feeding processes were developed and unsaturated MCL-PHA productivity as

high as 1.1 g PHA l-1 h-1 (highest reported for unsaturated MCL-PHA production) was

achieved. A linear relationship between the co-substrate mixture composition and the

resulted MCL-PHA composition were confirmed, demonstrating that the production of

functionalized MCL-PHA with controllable composition by fed-batch fermentation is

possible. This phase of research is covered in Chapter 7, “Fed-batch production of

unsaturated medium-chain-length poly(3-hydroxyalkanoates) with controlled composition

by Pseudomonas putida KT2440”, to be submitted to Applied Microbiology and

Biotechnology in June, 2007.

1.4 References

Chen GQ, Wu Q (2005) The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26:6565-6578

9

Choi JI and Lee SY (1997) Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng 17:335-342

de Smet MJ, Eggink G, Witholt B, Kingma J, Wynberg H (1983) Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane. J Bacteriol 154:870-878

Hazenberg W and Witholt B (1997) Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: Economic considerations. Appl Microbiol Biotechnol 48:588-596

Hazer B, Steinbüchel A (2007) Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications. Appl Microbiol Biotechnol 74:1-12

Peoples OP (2006) PHA natural plastics: a disruptive technology for a sustainable future. International Symposium on Biological Polyesters 2006, Minneapolis/St. Paul, Minnesota, USA

Pouton CW, Akhtar S (1996) Biosynthetic polyhydroxyalkanoates and their potential in drug delivery. Adv Drug Deliv Rev 18:133-162

Steinbüchel A, Lütke-Eversloh T (2003) Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem Eng J 16:81-96

van der Walle GAM, de Koning GJM, Weusthuis RA, Eggink G (2001) Properties, modifications and applications of biopolyesters. In Babel W and Steinbüchel A edit, Adv Biochem Eng Biotechnol 71:263-291

Zinn M, Witholt B, Egli T (2001) Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53:5-21

10

CHAPTER 2 Literature Review

Fermentation Process Development for the Production

of Medium-chain-length Poly(3-hydroxyalkanoates)

Zhiyong Sun, Juliana A. Ramsay, Martin Guay, Bruce A. Ramsay

Originally published June, 2007 in Applied Microbiology and Biotechnology, 75 (3): 475-485

11

2.1 Abstract

This paper presents a review of existing fermentation processes for the production of

medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHAs). These biodegradable

polymers are usually produced most efficiently from structurally related carbon sources

such as alkanes and alkanoic acids. Unlike alkanoic acids, alkanes exhibit little toxicity

but their low aqueous solubility limits their use in high density culture. Alkanoic acids

pose little mass transfer difficulty, but their toxicity requires that their concentration be

well controlled. Using presently available technology, large-scale production of

MCL-PHA from octane has been reported to cost from 5 to 10 US $ kg-1 with

expenditures almost evenly divided between carbon source, fermentation process and the

separation process. However, MCL-PHAs, even some with functional groups in their

subunits, can also be produced from cheaper unrelated carbon sources, such as glucose.

Metabolic engineering and other approaches should also allow increased PHA cellular

content to be achieved. These approaches, as well as a better understanding of

fermentation kinetics, will likely result in increased productivity and lower production

costs.

2.2 Introduction

Poly(3-hydroxyalkanoates) (PHAs) have attracted extensive interest due to their

biocompatibility and biodegradability. Since approximately 150 different PHA subunits

12

have been identified (Steinbüchel and Lütke-Eversloh 2003), PHAs exhibit a wide variety

of properties and thus may have many different applications. Short-chain-length PHAs

(SCL-PHAs) such as poly(3-hydroxybutyrate) (PHB) have been studied in depth and

have been produced on a commercial scale. Due to structural differences (Figure 2-1), the

physical properties of medium-chain-length PHAs (MCL-PHAs), are generally quite

different from PHB and other SCL-PHAs (Gagnon et al. 1992). PHAs are more expensive

to produce than conventional plastics so they must find high value applications to be

commercially viable. MCL-PHAs have shown promise as thermoelastomers for

biomedical applications, such as drug delivery (Pouton and Akhtar 1996) and tissue

engineering (Williams et al. 1999; Chen and Wu 2005). However, to date, the process

development for the production of MCL-PHAs has been much less extensive than for

SCL-PHAs. The consequent lack of availability has hampered application development.

Efficient MCL-PHA synthesis occurs when using structural-related carbon sources, such

as alkanes and aliphatic acids (Lageveen et al. 1988). These carbon sources are either

poorly miscible with water and/or toxic to bacteria at relatively low concentrations. The

major process challenge lies in automation of the control of these carbon source(s)

concentration which generally govern the rates of growth and production. The highly

reduced nature of these aliphatic carbon sources results in higher oxygen demand than

carbohydrates and they are generally much more expensive than conventional

13

fermentation substrates such as carbohydrates. Increasing the efficiency of their use in

PHA synthesis is critical to the overall economics.

Figure 2-1 Chemical structure of polyhydroxyalkanoates (PHAs), which are classified as short-chain-length PHAs (SCL-PHAs) when the repeating units contain 4 or 5 carbons and medium-chain-length PHAs (MCL-PHAs) when the repeating units contain 6 or more carbons; R pendant group may also contains functional groups resulting in functionalized PHAs.

In this paper, the effects of physiological conditions on biosynthesis kinetics of

MCL-PHA-synthesizing bacteria and fermentation strategies for MCL-PHA production

will be reviewed. Economic considerations will be briefly discussed.

2.3 Physiology and the kinetics of biosynthesis

2.3.1 General considerations

The occurrence of MCL-PHAs was first observed in Pseudomonas oleovorans GPo1

(ATCC 29347) (de Smet et al. 1983). Early screening studies showed that only certain

species of fluorescent pseudomonads could synthesize MCL-PHAs (Huisman et al. 1989;

Timm and Steinbüchel 1990). More recently, 48 Pseudomonas species belonging to the

rRNA homology group I were examined for their ability to synthesize PHA from

octanoate (Diard et al. 2002). These organisms have been grouped into intrageneric

clusters I and II (Yamamoto et al. 2000). The species in cluster II, as well as certain

O O

R O

OH

R O R

OH

O

n

14

strains of P. aeruginosa in cluster I synthesized MCL-PHAs with 3HO as the major

component. Other cluster I organisms, including P. oleovorans strains, synthesized only

PHB. It is important to note that one of the mostly frequently studied strains, P.

oleovorans GPo1 (ATCC 29347) for the MCL-PHA synthesis studies, has been

reclassified as P. putida GPo1 (ATCC 29347) (van Beilen et al. 2001; Diard et al. 2002).

This strain is unique among MCL-PHA-synthesizing Pseudomonas, in that it can utilize

alkanes such as octane for MCL-PHA synthesis due to its OCT plasmid (Huisman et al.

1989). Alkanoates such as octanoate are common MCL-PHA carbon sources for all

MCL-PHA-synthesizing Pseudomonas.

Since the metabolism of MCL-PHA synthesis is quite different from that of

SCL-PHA synthesis, early studies attempted to understand the physiological and kinetic

fundamentals of MCL-PHA production. The most interesting aspects for process

development are the effects of carbon sources and their concentration, specific growth

rate (µ), and the ratio of carbon to other major nutrients on cell growth and MCL-PHA

synthesis. Physiological conditions directly affect subunit composition, cellular PHA

content (%), specific PHA synthesis rate based on residual biomass (qPHA(Xr), g PHA g

Xr-1 h-1), and overall PHA productivity (g PHA l-1 h-1). Throughout this review, “biomass”

(X) is used to indicate dry biomass while “residual biomass” (Xr) is defined as dry

biomass minus PHA.

15

2.3.2 Carbon sources and their concentration

It has been clearly demonstrated that fatty acid β-oxidation is involved in supplying the

precursors for MCL-PHA synthesis from structurally related carbon sources (Eggink et al.

1992). MCL-PHA synthesis results in copolyesters containing C6 to C14 units but carbon

sources, such as octane, octanoate, and nonanoate, are considered to be the most efficient

substrates for MCL-PHA synthesis (Madison and Huisman 1999), also shown in Figure

2-2. When P. putida GPo1 was grown on octanoate and nonanoate under nitrogen

limitation in a chemostat study (µ= 0.2 h-1), comparable PHA content, 42% and 31% were

respectively obtained (Durner et al. 2001), but such results are not likely the maximum

obtainable from these substrates since PHA content often depends on µ (Preusting et al.

1991; Ramsay et al. 1991). During the PHA accumulation phase of the batch cultivation

of P. putida GPo1, the qPHA(Xr) using nonanoate was considerably higher than using

octanoate (0.082 g PHA g Xr-1 h-1 vs. 0.036 g PHA g Xr

-1 h-1) (Durner et al. 2001),

suggesting that nonanoate is a more efficient carbon source for PHA synthesis. To

produce MCL-PHAs from cheaper and more widely available resources, vegetable oils

and animal fats are of interest as many Pseudomonas are able to utilize such carbon

sources. Oleic acid, a common carboxylic acid in vegetable oils, has been used in the

efficient production of MCL-PHAs by P. putida KT2442 (Huijberts and Eggink 1996;

Lee et al. 2000). Other complex substrates, such as coconut oil and tallow (Solaiman et al.

1999; Thakor et al. 2005), co-products of soy-based biodiesel production (Ashby et al.

16

2004), and soy molasses (Solaiman et al. 2006) have been reported to support MCL-PHA

synthesis by various Pseudomonas strains. Although some strains may accumulate as

much as 40% PHA using such carbon sources, the yields are usually much lower than

pure carboxylic acids. And since most of these studies were done in shake-flask culture or

lab-scale batch fermentations, further work is required to evaluate their economic

potential.

Figure 2-2 PHA content of P. putida strains using alkanes or alkanoates of varying length. P. putida GPo1 (formerly known as P. oleovorans GPo1) grown on n-alkanoates (Gross et al. 1989), P. putida GPo1 on n-alkanes (Lageveen et al. 1988), and P. sp. DSY-82 on n-alkanoates (Kang et al. 2001).

Number of carbons

5 6 7 8 9 10 11 12 13

% P

HA

0

20

40

60

80P. putida GPo1 on n-alkanoateP. putida GPo1 on n-alkaneP. sp. DSY-82 on n-alkanoate

17

Structurally unrelated carbon sources, such as gluconate, can also be used for PHA

synthesis by most MCL-PHA-synthesizing Pseudomonas (Huijberts et al. 1992; Timm

and Steinbüchel 1990), but this is generally less efficient than structurally related

substrates (Madison and Huisman 1999). However, P. putida IPT046 (Table 2-1) has

demonstrated very high MCL-PHA-synthesizing ability from carbohydrates (Sanchez et

al. 2003) and has been evaluated in lab scale fermentations (Diniz et al. 2004).

The toxic nature of some carbon sources impedes process development.

Hydrocarbons such as octane was often used as an organic second phase in the cultivation

of P. putida GPo1 and exhibited little apparent toxicity (Preusting et al. 1993b), but

growth at high cell concentration may be mass transfer limited (Hazenberg and Witholt

1997). Octane is also flammable. In contrast, alkanoate salts are miscible in aqueous

media but exhibit toxicity. Growth of P. putida GPo1 was inhibited by 4.65 g l-1

octanoate (Ramsay et al. 1991) while P. putida KT2440 (ATCC 47054) was inhibited by

3-4 g l-1 nonanoic acid (Sun et al. 2007/Chapter 5). Vegetable oils are generally less

inhibitory as are longer chain fatty acids. Oleic acid was good for growth of P. putida

KT2442 if maintained below 15 g l-1 (Lee et al. 2000). Glucose did not inhibit the growth

of P. putida if kept below 40 g l-1 (Hofer et al. 2002; Kim et al. 1996). The inhibitory

effects of these carbon sources commonly used in MCL-PHA synthesis pose considerable

difficulty in the development of high-cell-density production processes.

18

2.3.3 Growth rate and nutrient conditions

Chemostat studies of P. putida GPo1 on octane (Preusting et al. 1991) and octanoate

(Durner et al. 2000; Ramsay et al. 1991), and P. putida KT2442 on oleic acid (Huijberts

and Eggink 1996) have shown that cellular PHA content decreases with increasing

dilution rate (equals to µ at steady state) with the highest content obtained at the lowest µ.

The effects of nutrient conditions on the synthesis of MCL-PHAs by various strains

differed considerably. Although MCL-PHA synthesis was significant during nutrient (e.g.

N or P) limitation with excess carbon, as shown by many batch and chemostat

experiments (Hazenberg and Witholt 1997; Kim et al. 1997; Lageveen et al. 1988), this is

not a necessity to obtain MCL-PHA for some strains using certain carbon sources. P.

putida GPo1 synthesized PHA during batch exponential growth at a rate even higher than

biomass formation; and synthesized 19% PHA under carbon limited chemostat conditions

when grown on nonanoate but not octanoate (Durner et al. 2001). P. putida KT2440

synthesized up to 75% PHA during high-cell-density carbon-limited exponential growth

(Sun et al. 2007/Chapter 5). Its rifampicin-resistant derivative P. putida KT2442 also

demonstrated significant PHA-synthesizing ability during batch exponential phase on

octanoate (Huisman et al. 1992) and has been cited as not requiring nutrient limitation for

MCL-PHA synthesis (Kessler et al, 2001).

19

In a chemostat study of P. putida GPo1 growing on octanoate at µ= 0.2 h-1, the PHA

content increased linearly from 2% to 42% as the carbon to nitrogen (C/N) ratio increased

while both C and N were limited, but was constant at 40% as C/N ratio was further

increased (the N-limited zone) (Durner et al. 2001). In another study with the same strain,

constant PHA content (15%) was also observed at an increasing C/N ratio under N

limitation (Ramsay et al. 1991). In contrast, chemostat study of P. resinovorans ATCC

14235 on octanoate (Ramsay et al. 1992) and P. putida KT2442 on oleic acid (Huijberts

and Eggink 1996), showed that the highest PHA content was obtained via nitrogen

limitation (17 mol C mol N -1 at 0.25 h-1 and 20 mol C mol N -1 at 0.1 h-1, respectively).

Therefore, it cannot be generally stated that MCL-PHA synthesis is stimulated by

non-carbon nutrient limitation. Based on the results reported, it is necessary to assess the

optimal PHA-synthesis conditions when any new Pseudomonas strain, carbon source, or

nutrient composition is to be employed.

2.4 Process development

To date, P. putida GPo1, P. putida KT2440 and KT2442, P. putida BM01 and P. putida

IPT046 have been employed for fermentation process development using octane,

octanoate, oleic acid, or carbohydrates (Table 2-1). Although efforts have been made to

express MCL-PHA in recombinant Escherichia coli (Langenbach et al. 1997; Qi et al.

1998; Ren et al. 2000) or to improve PHA synthesis with recombinant Pseudomonas

20

(Kraak et al. 1997), few fermentation processes have been based on recombinant strains

(Prieto et al. 1999). Due to the common belief that PHA synthesis is stimulated by

nutrient limitation, almost all MCL-PHA production processes have incorporated an N or

P limitation.

2.4.1 Continuous processes

Continuous fermentation is not only an appropriate method to study microbial

physiology, but also a good way to achieve high productivity. Studies have been carried

out to find suitable strategies for PHA production using this mode of operation.

High-cell-density, continuous P. putida GPo1 fermentation processes using octane have

been developed to high productivity levels by Witholt and coworkers. In their early study,

P. putida GPo1 was cultivated at a cell concentration of 11.6 g l-1 containing 25% PHA

with 12.5% (v/v) octane as the organic phase and ammonium limited conditions in a

chemostat operated at D= 0.2 h-1 (Preusting et al. 1993a). The yields of biomass from

major nutrients (P, Mg, Fe, N) were determined to allow medium composition

optimization. This led to PHA productivity of 0.56 g PHA l-1 h-1. This process was

operated continuously for 30 days with no significant degeneration of the culture. By

further improving the medium composition and mixing capability of the bioreactor,

enhanced PHA productivity (0.76 g PHA l-1 h-1) was achieved (Hazenberg and Witholt

1997). As previously mentioned, PHA content decreased with increased µ (or D in steady

21

state of continuous fermentations). Thus, a compromise between PHA content,

concentration and productivity is required in a single-stage continuous process if a

reasonable percent of PHA is to be attained in the biomass. To overcome this limitation, a

two-stage continuous process was developed (Jung et al. 2001). Dilution rates to achieve

maximum overall volumetric PHA productivity and content were determined to be 0.21

h-1 for cell growth (1st stage) and 0.16 h-1 for N-limited PHA-accumulation (2nd stage),

using transient experiments. Under these conditions, a high-cell-density continuous

fermentation was carried out, resulting in 18 g l-1 cell concentration containing 63% PHA

in the second stage, and an overall PHA productivity of 1.06 g PHA l-1 h-1.

High-cell-density continuous processes of P. putida KT2442 using oleic acid have

also been developed (Huijberts and Eggink 1996). Fed-batch operation was used to obtain

a high cell concentration before switching to continuous mode. A dilution rate of 0.1 h-1

and a C/N ratio of 20 mol mol-1 was used to achieve maximum PHA productivity.

Although the productivity was 0.67 g PHA l-1 h-1, comparable to that of P. putida GPo1

process (Hazenberg and Witholt 1997; Jung et al. 2001), the PHA content was only 23%.

2.4.2 Fed-batch processes

Fed-batch cultivation is often employed to achieve high product concentrations. Many

different feeding control techniques have been reported (Lee et al, 1999). Fed-batch

strategies have been applied to high-cell-density cultivation of P. putida GPo1 in a

22

two-phase system, using octane. Since octane does not appear to inhibit culture growth,

ammonium limited feeding strategies were applied. In one study (Preusting et al. 1993b),

ammonium was fed at a constant rate of 0.23 g h-1 for 38 h which led to a final biomass of

37.1 g l-1 containing 33% PHA (0.25 g PHA l-1 h-1). A high oxygen transfer rate was

generally needed to obtain high cell density. Using a reactor with better oxygen-transfer

capacity and optimized medium composition, P. putida GPo1 was grown up to 90 g l-1

using linear ammonium feeding and 112 g l-1 using exponential ammonium feeding

(Kellerhals et al. 1999a). However, a maximum PHA content of only 25% was obtained

in the middle of the process and decreased significantly until the end of the fermentation.

Based on information from previous chemostat work (Hazenberg and Witholt 1997), it

was possible that an octane mass transfer limitation hindered PHA accumulation.

Therefore such two-phase systems might not allow efficient MCL-PHA production in

high cell density fed-batch mode.

Alkanoates have also been used in MCL-PHA processes; but controlling their

concentrations at a non-toxic level is a major challenge in achieving high-cell-density.

Octanoate has been used as an alternative to octane in fed-batch processes for P. putida

GPo1. In one such study, ammonium octanoate was first fed at an exponential rate

resulting in a µ of 0.19 h-1, then adjusted to a constant rate to prevent oxygen limitation

(Dufresne and Samain 1998). The pH was regulated by addition of octanoic acid which

23

also served as an additional carbon source. A final biomass of 47 g l-1 was obtained

containing 55% PHA. Similarly, pH-stat feeding of a mixture of octanoic acid and

ammonium nitrate was applied by Kim (2002). The C/N ratio of 20 g octanoic acid g-1

ammonium nitrate resulted in 75% PHA but a lower overall productivity (0.63 g PHA l-1

h-1) compared to C/N of 10 g g-1 that result in 1 g PHA l-1 h-1, mainly due to a prolonged

fermentation time.

Other P. putida strains have been employed in fed-batch production of MCL-PHA.

The P. putida BM01, isolated by Kim’s group, can grow on glucose up to 100 g l-1 cell

concentration, but synthesized little PHA (Kim et al. 1996). Therefore a two-stage

fed-batch process using glucose as growth substrate and octanoic acid as PHA-synthesis

substrate was developed so that the overall yield of PHA from octanoic acid, which is

much more expensive than glucose, could be improved (Kim et al. 1997). It was also

found that a slight supplement of octanoic acid while using glucose as the main carbon

source during the growth stage could shorten the adaptation period of the culture for the

PHA-accumulating stage. Using such a two-stage process, biomass (55 g l-1) containing

65.5% of PHA was obtained and the overall yield of PHA from octanoate was 0.4 g g-1. It

was not clear, however, how the octanoate concentration was maintained at an

appropriate level during the feeding process.

24

Growth of P. putida KT2442 on oleic acid was also investigated (Weusthuis et al.

1997). One approach employed a DO-stat strategy in an oleic acid fed-batch process.

Pulses of oleic acid below the inhibitory concentration were fed whenever an increase in

dissolved oxygen indicated a depletion of carbon source. This feeding strategy produced

92 g l-1 of biomass containing 47% PHA (1.6 g PHA l-1 h-1). In another study, using the

same strain and carbon source, phosphorus limitation was applied instead of nitrogen

limitation (Lee et al. 2000). By extending the process using pH-stat feeding strategy

following DO-stat feeding, the final biomass, PHA concentration and productivity was

improved to 141 g l-1, 51% PHA and 1.9 g PHA l-1 h-1, respectively. Similar processes

have been reported using a 30 l pilot-scale bioreactor (Kellerhals et al. 2000), but it

seemed that nitrogen limitation hampered the increase in PHA content in the biomass and

hence the overall productivity.

An alternative to the pH-stat or DO-stat feeding strategy is to control the feed directly

by determining the actual carbon source concentration using rapid off-line measurement.

Such off-line measurement equipment is now commonly used in glucose fermentations

(Kim et al. 1996) but rare when alkanoates are the carbon source. A closed-loop off-line

gas chromatography measurement system was developed to directly measure and control

the medium concentration of octanoate within a selected range for the cultivation of P.

putida KT2442 (Kellerhals et al. 1999b). The octanoate concentration was very well

25

maintained below 25 mM, but the PHA content declined from 50% during the middle of

the fermentation to 34% at the end of the fermentation, and resulted in only 17.4 g l-1 of

PHA. Despite the fact that such a system is excellent for the direct monitoring and control

of toxic substrates, its complexity renders it less likely to be used commercially if,

simpler, albeit indirect, feeding strategies are available.

In our recent study (Sun et al. 2007/Chapter 5), simple yet efficient exponential

feeding strategies were applied to cultivate P. putida KT2440 using nonanoic acid. This

approach was possible because MCL-PHAs synthesis occurs significantly along with cell

growth, and no nutrient-limitation conditions were required. PHA content of 75% was

obtained using a lower controlled specific growth rate (0.15 h-1) while higher overall

productivity was achieved at higher controlled specific growth rate (0.25 h-1). This

process was subsequently repeated in a 150 l bioreactor with similar success (unpublished

data). From the reported results it seems that oxygen limitation was the only reason

hampering the further increase of cell concentration and PHA content. Although most

MCL-PHA-synthesizing strains can accumulate PHA from unrelated carbon sources

(Huijberts et al. 1992; Timm and Steinbüchel 1990), the achievable PHA content is

usually unacceptably low. Many such strains also accumulate large amounts of

undesirable polysaccharides if grown on carbohydrates. The exception is P. putida

IPT046 (Sanchez et al. 2003) for which fermentation processes have been developed

26

(Diniz et al. 2004). Glucose and fructose pulse feeding, constant rate feeding and

exponential feeding strategies with no nutrient limitation were all tested, but resulted in

little difference in the final achievable cell concentration (about 30 g l-1). Following an

exponentially-fed growth phase, ammonium limited or phosphate limited

PHA-accumulation phases were investigated and resulted in a final biomass 40 g l-1

containing 21% PHA or 50 g l-1 containing 63% PHA, respectively. Such a process has

industrial appeal as the control strategies are relatively simple, and more importantly, the

carbon sources are cheaper and more widely available than alkanes and alkanoates.

2.4.3 Production of MCL-PHAs containing functional groups

In addition to MCL-PHAs containing solely 3-hydroxyalkanoates subunits, MCL-PHAs

containing various functional groups, such as olefins (Fritzsche et al. 1990), branched

alkyls (Hazer et al. 1994), halogens (Kim et al. 1996), phenyl (Hazer et al. 1996), and

esters (Scholz et al. 1994) can often be incorporated when grown on suitable carbon

substrates with such groups. However, the ability to utilize these carbon sources may

differ considerably between strains (Kim et al. 2000). Few of these polymers have been

produced at even gram quantities due to the toxicity of the carbon substrates, inefficient

accumulation by the cultures employed, and unavailability of such substrates as many of

them require dedicated chemical synthesis.

27

Among such functionalized PHAs, the synthesis of PHAs containing unsaturated

subunits draws the most interest. Medium chain length alkenes and alkenoic acids, which

are more easily available than many other functional substrates, have been used in such

studies. When using octene or nonene as sole carbon source for P. oleovorans (Lageveen

et al. 1988), the resulting PHA contained up to 55% of unsaturated subunits, probably due

to the β-oxidation at either end of the alkene. The biomass and PHA yields were lower

from alkenes than from alkanes (Kim et al. 1995; Lageveen et al. 1988). To overcome

such inefficiency but still obtain an unsaturated fraction in synthesized PHA,

10-undecenoic acid was mixed with octanoic or nonanoic acid as co-substrate (Kim et al.

1995), and the fraction of unsaturated units in the resulting polymers increased in

proportion to the fraction of undecenoic acid in the mixture. This kind of co-substrate

cultivation not only leads to better growth, but also allows control of the fraction of

unsaturated subunits. Using the closed-loop gas chromatography system,

sodium-octanoate and sodium-10-undecenoate were co-fed via a PID feedback loop in a

fed-batch fermentation (Kellerhals et al. 1999b). Although this system was effective in

maintaining the carbon compounds below their toxic level, the final cell concentration

and MCL-PHA content were only 30 g l-1 and 33%, respectively, probably due to a

limited nitrogen supply and insufficient fermentation time. In Hartmann’s study, the

variation in polymer compositions from batch and chemostat fermentations co-fed with

octanoic and 10-undecenoic acids were compared (Hartmann et al. 2006). It was

28

concluded that chemostat cultivation is the most suitable method of obtaining

functionalized MCL-PHAs with defined and consistent monomer compositions. Besides

unsaturated MCL-PHAs, up to 2 g l-1 PHAs containing oxo side chains from octane and

2-octanone mixtures, and PHAs containing acetoxy side chain from octane and

n-octylacetate mixture, have been produced using a two-phase continuous culturing

technique described previously (Jung et al. 2000). As demonstrated in these studies,

processes for producing functionalized PHAs can be very similar to that of regular

MCL-PHAs. Such processes normally involve at least two carbon sources, one being a

standard medium-chain-length carbon source and the other containing the functional

group desired. It should be mentioned, however, that unsaturated subunits of MCL-PHAs

can also be produced from unrelated carbon sources, such as glucose (Sanchez et al.

2003). This approach could potentially avoid the necessity of using expensive and

inhibitory co-substrates for functionalized MCL-PHA production.

29

Table 2-1 Summary of MCL-PHA (regular and functionalized) production processes.

Fermentation mode

Microorganism Carbon source (s)

Cell concentration (g l-1)

PHA concentration (g l-1)

PHA content (%)

PHA productivity (g PHA l-1 h-1)

PHA subunits (high to low)

Reference

Continuous (D= 0.2 h-1)

P. putida GPo1 (ATCC 29347)

Octane 11.6 2.9 25 0.56 C8, C6 (Preusting et al. 1993a)


P. putida GPo1 Octane 12.4 3.7 30 0.74 C8, C6 (Hazenberg and Witholt 1997)

Two-stage continuous (D= 0.21 h-1/ 0.16 h-1)

P. putida GPo1 Octane 10.50; 18 4.04; 11.34 38.5; 63 1.06 C8, C6 (Jung et al. 2001)


P. putida KT2442 Oleic acid 30 6.9 23 0.69 C8, C10, C12, C14:1, C6, C12:1

(Huijberts and Eggink 1996)

Fed-batch P. putida GPo1 Octane 37.1 12.1 33 0.25 C8, C6 (Preusting et al. 1993b)

Fed-batch P. putida GPo1 Octane 112 11.2 10 0.19 C8, C6 (Kellerhals et al. 1999a)

Fed-batch P. putida GPo1 Ammonium octanoate/ octanoic acid

47 25.9 55 0.53 C8, C6 (Dufresne and Samain 1998)

Fed-batch P. putida GPo1 Octanoic acid

63 39 62 1 C8, C6 (Kim 2002) 55 41 75 0.63

Two-stage fed-batch

P. putida BM01 Octanoic acid/ glucose

55 36 65.5 0.90 C8, C6, C10 (Kim et al. 1997)

Fed-batch P. putida KT2442 Oleic acid 92 43 47 1.60 C8, C10, C12, C14:1, C6

(Weusthuis et al. 1997)

Fed-batch P. putida KT2442 Oleic acid 141 72 51 1.90 C8, C10, C6, C12, C14,

(Lee et al. 2000)

Fed-batch P. putida KT2442 Octanoic acid

51.5 17.4 34 0.40 C8, C6, C10 (Kellerhals et al. 1999b)

Fed-batch P. putida KT2442 Oleic acid 89.8 18.0 20 0.56 C8, C10, C12, C14:1, C6

(Kellerhals et al. 2000)

Octanoic acid

51.5 18.0 35 0.41 C8, C6, C10

Vegetable fatty acid

73 25 34 0.56 C8, C10, C6, C12:1, C14:1

Fed-batch P. putida KT2440 (ATCC 47054)

Nonanoic acid

70 52.5 75 1.11 C9, C7 (Sun et al. 2007) 56 37.4 67 1.44 C9, C7

30

Fed-batch P. putida IPT046 Glucose/fructose

50 31.5 63 0.75 C10, C8 (Diniz et al. 2004; Sanchez et al. 2003)


P. oleovorans (putida) POMC1 (recombinant)

Octanoic acid/ citric acid

1.50 0.65 43 0.016 C8, C6 (Prieto et al. 1999)

Fed-batch E. coli 193MC1 (recombinant)

Octanoic acid/ glycerol

3.55 0.43 12 0.011 C8, C10, C6 (Prieto et al. 1999)

Continuous (D= 0.1-0.3 h-1)

P. putida GPo1 Octanoate (50%)/ 10-undecenoate (50%)

1.11-1.44 0.13-0.47 12-37 0.04-0.07 C8, C9:1, C11:1, C6, C7:1

(Hartmann et al. 2006)

Fed-batch P. putida GPo1 2-octanone/octane

9.5 2.52 26.5 0.063 C8, C10O, C6 (Jung et al. 2000)

C6, 3-hydroxyhexanoate; C7, 3-hydroxyheptanoate; C7:1, 3-hydroxy-6-heptenoate; C8, 3-hydroxyoctanoate; C9, 3-hydroxynonanoate; C9:1, 3-hydroxy-8-nonenoate; C10, 3-hydroxydecanoate; C10O, 3-hydroxy-7-oxooctanoate; C11:1, 3-hydroxy-10-undecenoate; C12, 3-hydroxydodecanoate; C12:1, 3-hydroxy-cis-6-dodecenoate; C14, 3-hydroxy-cis-5-tetradecanoate; C14:1, 3-hydroxy-cis-5-tetradecenoate; P. putida GPo1 was formerly known as P. oleovorans GPo1.

31

2.5 The effect of nutrient limitation on MCL-PHA

accumulation

When comparing the MCL-PHA production processes described in this report and

summarized in Table 2-1, it is necessary to consider the effects of nutrient limiting and

nutrient depleted conditions. Processes involving complete nutrient depletion are usually

two-stage fed-batch processes with PHA accumulation occurring mainly during the

nutrient-depleted stage (Kim et al. 1997; Lee et al. 2000; Diniz et al. 2004). In other

studies, all nutrients are provided continuously, while ensuring that a key nutrient (most

often N) is the growth-limiting factor. This is achieved either by limiting nitrogen feeding

(Dufresne and Samain 1998; Kellerhals et al. 1999a) or by adjusting the C to N ratio in

the feed (Kim 2002). Therefore, although not directly pointed out in these reports, it can

be assumed that PHA synthesis occurred in conjunction with growth. One explanation for

the increased PHA content that often results from nutrient depletion or limitation is that

the growth of biomass other than PHA (i.e. residual biomass) is limited while PHA

synthesis continues unabated (Ramsay et al. 1991). This is a very different phenomenon

than that which occurs in many SCL-PHA accumulating bacteria, such as Ralstonia

eutropha, where it has been clearly demonstrated that the rate of PHA synthesis is greatly

increased when nutrients such as N are in short supply (Oeding and Schlegel 1973).

Simultaneous rapid growth and PHA accumulation has been recently demonstrated in our

32

studies (Sun et al. 2007/Chapter 5). The continuously increasing qPHA(Xr) (g PHA g Xr-1

h-1) in our results indicated that PHA was synthesized at a rate higher than the residual

biomass synthesis rate, suggesting that such PHA synthesis is not strictly associated with

cell growth. The biochemical and genetic mechanisms controlling such kinetics needs to

be understood if simple yet highly efficient MCL-PHA production processes similar to

those available for SCL-PHA are to be developed.

2.6 Economic considerations

Little has been published concerning the cost of production of MCL-PHA. However, the

technology is very similar to SCL-PHA production whose economics have been well

studied (Choi and Lee 1999). Before transfer of their technology to Monsanto, Zeneca

sold Biopol (their family of P(HB-HV) copolymers) for about 10 US $ kg-1. It has been

estimated that large-scale production of MCL-PHA from octane would cost from 5 (de

Koning et al. 1997) to 10 (Hazenberg and Witholt 1997) US $ kg-1 with costs almost

evenly split between the carbon source, the fermentation process and the separation

process. MCL-PHA production costs considerations differ from those for SCL-PHA in

several ways. The substrates used for MCL-PHA production are generally different and

more expensive. The fermentation processes developed to date have much lower PHA

productivity comparing to industrial SCL-PHA processes and would lead to higher capital

and labour costs. The solubility of the polymers in common solvents such as acetone and

33

the granule density are also different from SCL-PHAs, which affect separation costs. For

example, PHA is recovered from the biomass either by solvent extraction or by

dissolution of the residual biomass. If the latter approach is used, a latex suspension of

PHA granules results. SCL-PHA suspensions are much easier to centrifuge since their

density is significantly greater than that of MCL-PHA granules. As was previously

discussed, fats, oils or carboxylic acids are usually used as substrates since β-oxidation

generally results in much higher yields than β-addition pathways. Use of fats or oils

requires lipase production by the cells or addition of lipase to the fermentation medium.

This renders process control difficult for two reasons. Unless there is an excess of lipase,

it is difficult to predict how much carboxylic acid is released into the growth medium, and

these acids are toxic at relatively low concentrations. In addition, the release of glycerol

provides an additional substrate which further complicates the process. Alkanes are less

toxic than carboxylic acids but require the appropriate oxidase lacking in MCL-PHA

producers unless they possess the OCT plasmid. They are also flammable and their mass

transfer into the aqueous phase may limit the growth rate. Thus carboxylic acids,

especially those in the C6 to C10 range, although inhibitory to growth above a certain

concentration, are preferred substrates for MCL-PHA production. Unfortunately, they are

expensive and often not readily available on a large scale. Commercial octanoic acid is

usually derived from coconut oil while nonanoic acid can be easily synthesized from oils

such as canola, which are rich in linoleic acid. These feedstocks are renewable, which is

34

an important environmental consideration. They are also becoming increasingly in

demand for other “green” products, especially biodiesel.

Although this review deals with fermentation processes, separation accounts for up to

30% of the overall production cost. The fermentation process also greatly influences PHA

separation cost since this cost is inversely related to the PHA content in the biomass. As

with SCL-PHA, MCL-PHA may be separated from cells either by solvent extraction,

dissolution of biomass other than PHA, or a combination of both of these approaches.

Solvent extraction of MCL-PHA would be less expensive than for SCL-PHA since it is

soluble in a wider range of solvents including ones such as acetone, which can be

produced via fermentation of renewable materials. In fact, it is often a co-product of

butanol which can be used as a replacement for gasoline in automobiles. Dissolution of

non-PHA biomass could be more challenging than in SCL-PHA processes. The released

PHA granules are typically separated by centrifugation but capital and energy costs

increase greatly if the granule density approaches that of the aqueous phase. The density

of SCL-PHA granules are nearly 1.2 times the density of water, while those of MCL-PHA

are very similar to that of water and may even float, depending on the subunit

composition. A variety of methodologies such as froth flotation (van Hee et al. 2006)

have been investigated to avoid centrifugation. A cost analysis on a non-PHA biomass

dissolution approach employing filtration concluded that the separation cost could be as

low as 1 US $ kg-1 if the PHA content in the biomass from the fermentor exceeded 80%

35

(de Koning et al. 1997). In summary, production of MCL-PHA is likely to be somewhat

more expensive than SCL-PHA production due to additional substrate and separation

costs. However, it is thought that MCL-PHAs may be more suitable for high value

applications such as in the biomedical industry (Pouton and Akhtar 1996; Williams et al.

1999; Chen and Wu 2005).

2.7 Future commercial processes

Many of the studies presented in this review were process studies as opposed to process

optimizations. The amount of PHA accumulated as a % of dry biomass is much less than

in optimized SCL-PHA processes but there is no reason why similar productivity cannot

be achieved in MCL-PHA processes. Advances in metabolic and biochemical engineering

are likely to produce significant improvements in the near future. Although MCL-PHA

production would be much simpler and cheaper using highly productive processes based

on glucose fermentation or recombinant plants, use of related carbon sources such as

octanoate allows tailoring of the final product to fit commercial demands. Thus, it is

likely that such demand will be initially fulfilled by fermentation processes based on

mixtures of glucose and carbon sources such as octanoate, especially those in which most

or all of the glucose is used to supply energy and produce residual biomass.

36

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Diniz SC, Taciro MK, Gomez JG, da Cruz Pradella JG (2004) High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates. Appl Biochem Biotechnol 119:51-70

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Durner R, Zinn M, Witholt B, Egli T (2001) Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources. Biotechnol Bioeng 72:278-288

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Eggink G, Dewaard P, Huijberts GNM (1992) The role of fatty-acid biosynthesis and degradation in the supply of substrates for poly(3-hydroxyalkanoate) formation in Pseudomonas putida. FEMS Microbiol Rev 103:159-163

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Gagnon KD, Lenz RW, Farris RJ, Fuller RC (1992) The mechanical-properties of a thermoplastic elastomer produced by the bacterium Pseudomonas oleovorans. Rubber Chemistry and Technology 65:761-777

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Hazenberg W, Witholt B (1997) Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic considerations. Appl Microbiol Biotechnol 48:588-596

Hazer B, Lenz RW, Fuller RC (1996) Bacterial production of poly-3-hydroxyalkanoates containing arylalkyl substituent groups. Polymer 37:5951-5957

Hazer B, Lenz RW, Fuller RC (1994) Biosynthesis of methyl-branched poly(beta-hydroxyalkanoate)s by Pseudomonas oleovorans. Macromolecules 27:45-49

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Huijberts GNM, Eggink G (1996) Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures. Appl Microbiol Biotechnol 46:233-239

Huijberts GNM, Eggink G, Dewaard P, Huisman GW, Witholt B (1992) Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-Hydroxyalkanoates) consisting of saturated and unsaturated monomers. Appl Environ Microbiol 58:536-544

38

Huisman GW, de Leeuw O, Eggink G, Witholt B (1989) Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl Environ Microbiol 55:1949-1954

Huisman GW, Wonink E, de Koning G, Preusting H, Witholt B (1992) Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains. Appl Microbiol Biotechnol 38:1-5

Jung K, Hany R, Rentsch D, Storni T, Egli T, Witholt B (2000) Characterization of new bacterial copolyesters containing 3-hydroxyoxoalkanoates and acetoxy-3-hydroxyalkanoates. Macromolecules 33:8571-8575

Jung K, Hazenberg W, Prieto M, Witholt B (2001) Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates). Biotechnol Bioeng 72:19-24

Kang HO, Chung CW, Kim HW, Kim YB, Rhee YH (2001) Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanaotes by Pseudomonas sp. DSY-82. Antonie Van Leeuvenhoek 80:185-191

Kellerhals MB, Hazenberg W, Witholt B (1999a) High cell density fermentations of Pseudomonas oleovorans for the production of mcl-PHAs in two-liquid phase media. Enzyme Microb Technol 24:111-116

Kellerhals MB, Kessler B, Witholt B (1999b) Closed-loop control of bacterial high-cell-density fed-batch cultures: Production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrate and cofeeding conditions. Biotechnol Bioeng 65:306-315

Kellerhals MB, Kessler B, Witholt B, Tchouboukov A, Brandl H (2000) Renewable long-chain fatty acids for production of biodegradable medium-chain-length polyhydroxyalkanoates (mcl-PHAs) at laboratory and pilot plant scales. Macromolecules 33:4690-4698

Kessler B, Weusthuis R, Witholt B, Eggink G (2001) Production of microbial polyesters: fermentation and downstream processes. Adv Biochem Eng Biotechnol 71: 159-182

Kim BS (2002) Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans. Biotechnol Lett 24:125-130

39

Kim DY, Kim YB, Rhee YH (2000) Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseudomonas putida. Int J Biol Macromol 28:23-29

Kim GJ, Lee IY, Choi DK, Yoon SC, Park YH (1996) High cell density cultivation of Pseudomonas putida BM01 using glucose. Journal of Microbiology and Biotechnology 6:221-224

Kim GJ, Lee IY, Yoon SC, Shin YC, Park YH (1997) Enhanced yield and a high production of medium-chain-length poly(3-hydroxyalkanoates) in a two-step fed-batch cultivation of Pseudomonas putida by combined use of glucose and octanoate. Enzyme Microb Technol 20:500-505

Kim O, Gross RA, Hammar WJ, Newmark RA (1996) Microbial synthesis of poly(beta-hydroxyalkanoates) containing fluorinated side-chain substituents. Macromolecules 29:4572-4581

Kim YB, Lenz RW, Fuller RC (1995) Poly-3-hydroxyalkanoates containing unsaturated repeating units produced by Pseudomonas oleovorans. Journal of Polymer Science Part A-Polymer Chemistry 33:1367-1374

Kraak MN, Smits THM, Kessler B, Witholt B (1997) Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains. J Bacteriol 179:4985-4991

Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans - effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924-2932

Langenbach S, Rehm BH, Steinbüchel A (1997) Functional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in poly(3-hydroxyalkanoate) synthesis. FEMS Microbiol Lett 150:303-309

Lee J, Lee SY, Park S, Middelberg APJ (1999) Control of fed-batch fermentations. Biotechnol Adv 17: 29-48

Lee SY, Wong HH, Choi JI, Lee SH, Lee SC, Han CS (2000) Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation. Biotechnol Bioeng 68:466-470

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Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): From DNA to plastic. Microbiology and Molecular Biology Reviews 63:21-53

Oeding V and Schlegel HG (1973) β-ketothiolase from Hydrogenomons eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism. Biochem J 134: 239-248


Preusting H, Hazenberg W, Witholt B (1993a) Continuous production of poly(3-hydroxyalkanoates) by Pseudomonas oleovorans in a high-cell-density, 2-liquid-phase chemostat. Enzyme Microb Technol 15:311-316

Preusting H, Kingma J, Witholt B (1991) Physiology and polyester formation of Pseudomonas oleovorans in continuous 2-liquid-phase cultures. Enzyme Microb Technol 13:770-780

Preusting H, Vanhouten R, Hoefs A, Vanlangenberghe EK, Favrebulle O, Witholt B (1993b) High-cell-density cultivation of Pseudomonas oleovorans - growth and production of poly (3-hydroxyalkanoates) in 2-liquid phase batch and fed-batch systems. Biotechnol Bioeng 41:550-556

Prieto MA, Kellerhals MB, Bozzato GB, Radnovic D, Witholt B, Kessler B (1999) Engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates. Appl Environ Microbiol 65:3265-3271

Qi Q, Steinbüchel A, Rehm BH (1998) Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid. FEMS Microbiol Lett 167:89-94

Ramsay BA, Saracovan I, Ramsay JA, Marchessault RH (1992) Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans. Appl Environ Microbiol 58:744-746

Ramsay BA, Saracovan I, Ramsay JA, Marchessault RH (1991) Continuous production of long-side-chain poly-beta-hydroxyalkanoates by Pseudomonas oleovorans. Appl Environ Microbiol 57:625-629

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Ren Q, Sierro N, Witholt B, Kessler B (2000) FabG, an NADPH-dependent 3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors for medium-chain-length poly-3-hydroxyalkanoate biosynthesis in Escherichia coli. J Bacteriol 182:2978-2981

Sanchez RJ, Schripsema J, da Silva LF, Taciro MK, Pradella JGC, Gomez JGC (2003) Medium-chain-length polyhydroxyalkanoic acids (PHA(mcl)) produced by Pseudomonas putida IPT 046 from renewable sources. European Polymer Journal 39:1385-1394

Scholz C, Fuller RC, Lenz RW (1994) Growth and Polymer Incorporation of Pseudomonas oleovorans on Alkyl Esters of Heptanoic Acid. Macromolecules 27:2886-2889

Solaiman DKY, Ashby RD, Foglia TA (1999) Medium-chain-length poly(beta-hydroxyalkanoate) synthesis from triacylglycerols by Pseudomonas saccharophila. Curr Microbiol 38:151-154

Solaiman DKY, Ashby RD, Hotchkiss AT, Foglia TA (2006) Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy molasses. Biotechnol Lett 28:157-162


Sun Z, Ramsay J, Guay M, Ramsay B (2007) Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440. Appl Microbiol Biotechnol 74:69-77

Thakor N, Trivedi U, Patel KC (2005) Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils. Bioresour Technol 96:1843-1850

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van Hee P, Elumbaring ACMR, van der Lans RGJM, van der Wielen LAM (2006) Selective recovery of polyhydroxyalkanoate inclusion bodies from fermentation broth by dissolved-air flotation. J Colloid Interface Sci 297:595-606

Weusthuis RA, Huijberts GNM, Eggink G (1997) Production of mcl-poly(hydroxyalkanoates) (review). In: Eggink G, Steinbüchel A, Poirer Y, Witholt B (eds) 1996 International Symposium on Bacterial Polyhydroxyalkanoates. NRC Research Press, Ottawa

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CHAPTER 3

Automated Feeding Strategies for High-cell-density

Fed-batch Cultivation of Pseudomonas putida KT2440


Originally published July, 2006 in Applied Microbiology and Biotechnology, 71 (4): 423-431

44

3.1 Abstract

Four automatic substrate feeding strategies were developed and investigated in this study

to obtain rapid, repeatable and reliable high cell density cultivation of Pseudomonas

putida KT2440 from glucose. Growth yield data of the key nutrients, YX/Glucose, YX/NH4,

YX/PO4, YX/Mg, and YCO2/Glucose, were determined to be 0.41, 5.44, 13.70, 236, and 0.65 g

g-1, respectively. Although standard exponential feeding strategy worked well when the

predetermined μ was set at 0.25 h-1, an exponential glucose feeding strategy with online

μmax estimation resulted in a higher average biomass productivity (3.4 vs. 2.8 g X l-1 h-1).

A CO2 production rate based pulse glucose feeding strategy also resulted in good overall

productivity (3.0 g X l-1 h-1) and can be used as an alternative to pH-stat or DO-stat

feeding. A cumulative CO2 production based continuous feed with real-time cumulative

glucose consumption estimation resulted in much higher biomass productivity (4.3 g X l-1

h-1) and appears be an excellent and reliable approach to fully automating high cell

density fed-batch cultivation of P. putida.

3.2 Introduction

Many industrial fermentation processes require rapid growth to a high cell density.

Fed-batch processes are considered to be the most efficient way of achieving high cell

density cultures (HCDC) (Lee et al. 1999; Riesenberg and Guthke 1999) but depend on

45

suitable substrate feeding strategies to control key nutrient concentrations (typically the

carbon source). Ideally feeding should be based on direct measurement of the substrate

whose concentration requires control. Unfortunately automatic analysis of key substrates

usually involves a substantial time delay, expensive, dedicated analytical equipment and

moderately difficult computer interfacing, but there are alternatives. For example,

substrate feeding may be based on knowledge of a microorganism's predicted growth rate.

In this approach a predetermined exponential feeding rate designed to obtain a growth

rate close to μmax is typically used for the growth phase, while a linear or decaying linear

feed rate is often used in a nongrowth-associated production phase (Riesenberg et al.

1991). Feeding may also be based on physiology as in pH-stat and DO-stat techniques

(Lee et al. 2000), where feeding depends on acid production or oxygen utilization. Lee et

al (1999) have written an extensive review on fed-batch fermentation control technology.

Pseudomonas putida KT2440 and its rifampicin-resistant derivative, strain KT2442,

are metabolically versatile and well-characterized (Timmis 2002). The KT2440 genome

has been sequenced and the putative gene products have been identified (Nelson et al.

2002). These strains are being exploited in a wide variety of applications, such as

catabolic pathway designs for pollutant biodegradation (Rojo et al. 1987), biological

desulphurization of petroleum feedstocks and products (Galan et al. 2000), production of

recombinant proteins (Thuesen et al. 2003), and synthesis of medium-chain-length

46

poly(3-hydroxyalkanoates) (MCL-PHA) (Huijberts et al. 1992). Some of these

applications, such as recombinant protein and MCL-PHA production, are most

economically performed in fed-batch mode. While standard methods are available for the

fed-batch cultivation of Escherichia coli (Riesenberg 1991; Kim et al. 2004), these are

only now being developed for P. putida.

Kim et al. (1996) obtained 100 g l-1 of P. putida by maintaining glucose

concentration within the range of 5-20 g l-1 using a glucose analyzer. Cell concentrations

greater than 100 g l-1 have been obtained using a combination of pH-stat and DO-stat

feeding strategies (Lee et al. 2000), while 38 g l-1 of a recombinant P. putida strain was

obtained using an exponential glucose feeding (Thuesen et al. 2003). However, the

substrate feeding and control strategies employed in those studies either required very

complex online analysis, significant manual manipulation or have not been optimized to

achieve maximum biomass productivity.

Although product yields depend on the type of carbon and energy source utilized,

glucose, often obtained by starch hydrolysis, is by far the most commonly used source of

carbon and energy in the fermentation industry. Glucose does not inhibit growth of P.

putida below a concentration of at least 40 g l-1 (Hofer et al. 2002; Kim et al. 1996),

provides reasonable yield efficiency, and has excellent aqueous solubility. Glucose is also

cheap and widely available. Therefore, in this study, we investigated four automated

47

glucose feeding strategies designed to obtain rapid, repeatable, and reliable high cell

densities of P. putida KT2440. Detailed growth yield information was also obtained. It is

hoped that this information can be used for the development of specific fermentation

processes using P. putida KT2440 or strains and species with similar physiology.

3.3 Materials and methods

3.3.1 Analytical procedures

During fermentations, an Accu-Chek Advantage blood glucose meter (Roche

Diagnostics, Canada) was sometimes used to monitor real time glucose concentration

according to an established standard curve. The cell dry weight was measured after

centrifugation of 5 ml of culture broth at 17000 x g for 15 min, followed by

lyophilization. A colorimetric assay of glucose using 4-hydroxybenzoic hydrazide and

NaOH as reagents was used to determine the glucose concentration in all samples (Lever

1972). Phosphate concentration was measured via reduction of phosphomolybdate to

molybdene blue (Clesceri et al. 1999). Ammonium concentration was determined by the

phenol-hypochlorite reaction (Weatherburn MW 1967).

3.3.2 Microorganism and growth Medium

P. putida KT2440 (ATCC 47054) was maintained in lyophiles and on nutrient agar plates

at 4 oC. The shake flask mineral salts medium contained the following components per

liter: 4.70 g (NH4)2SO4, 0.80 g MgSO4·7H2O, 12.00 g Na2HPO4·7H2O, 2.70 g KH2PO4,

48

9.00 g glucose and 1.00 g nutrient broth. The initial fermentation culture medium

contained the following components per liter: 4.70 g (NH4)2SO4, 0.80 g MgSO4·7H2O,

18.00 g Na2HPO4·7H2O, 4.05 g KH2PO4, 1 to 15.00 g of glucose (according to the

feeding strategy) and 8 ml of trace element solution. Each liter of trace element solution

contained 10 g FeSO4·7H2O, 3 g CaCl2·2H2O, 2.2 g ZnSO4·7H2O, 0.5 g MnSO4·4H2O,

0.3 g of H3BO3, 0.2 g CoCl2·6H2O, 0.15 g Na2MoO4·2H2O, 0.02 g NiCl2·6H2O, 1.00 g

CuSO4·5H2O. Nitrogen source was supplied by using 14% (w/v) NH4OH to control the

pH. Each liter of feeding medium contained 600 g glucose and 10 g MgSO4·7H2O.

3.3.3 Fermentation conditions

Seed culture was incubated in 500-ml shake flasks with 100 ml mineral salts medium at

30±1 oC and 200 rpm for 13 h before inoculation into the bioreactor. Fed-batch

fermentations were all carried out at 31±1 oC with 0.8 l of initial working volume in a

Multigen F-2000 2 l stirred-tank bioreactor (New Brunswick Scientific, Edison, N.J.).

Data acquisition (dissolved oxygen, CO2 outlet gas concentration, NH4 and feed medium

addition, and pH) and control was conducted with an IBM-compatible PC using

LabVIEW 6.1 (National Instruments) software. pH was controlled between 6.80 and 6.90

by automatic addition of 14% (w/v) NH4OH with an Ingold sterilizable probe and a

Bioengineering AG (Wald, Switzerland) controller. The dissolved oxygen was measured

with an Ingold polarographic probe (Westech Industrial Ltd, Calgary, AB, Canada) and

49

maintained at or above 40% saturation (except where indicated), first, by adjusting the

agitation speed up to 900 rpm and, thereafter, by controlling air and pure oxygen flow via

mass flow controllers at a total gas flow rate of 1.5 l min-1. Exit gas (CO2) content was

measured with an infrared monitor (Guardian Plus, Topac Inc. Hingham, MA, USA).

Feed medium was continuously weighed on an Ohaus (Pine Brook, NJ, USA) GT 8000

balance. Feeding was conducted with a Cole-Parmer Ltd. (Vernon Hills, Il, USA)

peristaltic pump automatically controlled with LabVIEW based on the weight of the feed.

3.3.4 Substrate Feeding Strategies

3.3.4.1 Exponential feeding with a predetermined μ

With 1 g l-1 of initial glucose, substrate feeding was started at the beginning of the

fermentation according to feed Eq.1 such that the culture grew at a predetermined specific

growth rate.

0

/ /

tt

X S X S

tX XS eY Y

µ⋅∆ = = ⋅ (1)

where ∆St is the total amount of substrate required to produce a certain amount of

biomass, Xt, at cultivation time t; YX/S is the yield of biomass from glucose, obtained

previously and assumed to be constant; X0 is the initial biomass, obtained by measuring

the optical density650 of the shake-flask inoculum culture before inoculation; and μ is the

desired specific growth rate (0.25 h-1 was used in this study).

50

3.3.4.2 Exponential feeding based on initial maximum specific growth rate (μmax) estimation

Fermentations were started in batch mode with 10 g l-1 of initial glucose. CO2 content in

the exit gas flow was automatically measured once per minute, converted to CO2

production rate (CPR; g h-1), then integrated into cumulative CO2 production (CCP; g)

with respect to time. Assuming the CO2 production yield from biomass (YCO2/X) is

constant during the growth phase, then:

2 2/ / 0t t

t CO X t CO XCCP Y X Y X e a eµ µ⋅ ⋅= ⋅ = ⋅ ⋅ = ⋅ (2)

where a is the product of YCO2/X and X0. After 10 hours of cultivation time, the CCP data

were regressed nonlinearly using Sigmaplot 9.0.1 (Systat Software Inc. Point Richmond,

CA, USA) to obtain the parameters a and μ for the above equation. The μ value obtained

was considered to be the μmax that this culture could reach under these growth conditions.

Once the model parameters were obtained, the substrate was fed according a modified

form of equation (1).

max max

2 2

0

/ / / / /

tt

X S X S CO X X S CO S

t ttX X a aS e e eY Y Y Y Y

µ µµ ⋅ ⋅⋅= = ⋅ = ⋅ = ⋅⋅

(3)

51

3.3.4.3 Semi-continuous feeding based on the detection of substrate limitation

Fermentations began with 15 g l-1 of glucose. When CPR dropped by 10% of its previous

maximum (indicating a substrate limitation), sufficient glucose to raise the concentration

by 10 g l-1 was fed automatically.

3.3.4.4 Continuous feeding based on cumulative glucose consumption (CGC) estimation

Fermentations began with 10 g l-1 of glucose. Glucose was fed continuously as a function

of cumulative CO2 production (CCP) and YCO2/G (Eq. 4). The cumulative substrate

addition at time t (∆St) was kept equal to the estimated CGCt, hence, maintaining the

estimated glucose concentration in the reactor at 10 g l-1.

2 /

tt t

CO S

CCPS CGCY

∆ = = (4)

3.4 Results

3.4.1 Growth yield study

To obtain the information necessary to compose balanced growth media, data for the

consumption of key nutrients from several nutrient-unlimited fed-batch fermentations

were collected (Figure 3-1). Growth yields of biomass from glucose (YX/G), ammonia

(YX/NH4), phosphate (YX/PO4), and the production of CO2 from glucose (YCO2/G) were

determined as 0.41 g g-1 (r2= 0.98), 5.44 g g-1 (r2= 0.99), 13.7 g g-1 (r2= 0.95) (open and

closed circle data only), and 0.65 g g-1 (r2= 0.99), respectively, using linear regression. In

52

addition, YX/Mg was determined as 236 g g-1 by imposing periodic magnesium limitation

followed by pulse additions of magnesium sulfate to verify that magnesium was the

growth-limiting nutrient.

Figure 3-1 Yield of dry biomass from the principal growth substrates. Different symbols indicate data from different fermentations. The slope of the line indicates the calculated yield value. Only data indicated by closed and open circles were used to calculate yield from PO4 in (c).

(a) Biomass vs. glucose consumption

Glucose consumption (g)

0 20 40 60 80 100 120 140 160

Bio

mas

s (g

)

0

10

20

30

40

50

60

70

(b) Biomass vs. NH4 consumption

NH4 consumption (g)

0 2 4 6 8 10 12 14 16

Bio

mas

s (g

)0

20

40

60

80

100

(c) Biomass vs. PO4 consumption

PO4 consumption (g)

0 1 2 3 4 5 6 7

Bio

mas

s (g

)

0

20

40

60

80

100

(d) CO2 vs. glucose consumption

Glucose consumption (g)

0 20 40 60 80 100 120 140

Cum

ulat

ive

CO

2 pr

oduc

tion

(g)

0

20

40

60

80

100

53

3.4.2 Exponential feeding with a predetermined μ

Exponential feeding with a predetermined μ was examined first, since it is the simplest

and most commonly employed method of HCDC (Riesenberg and Guthke 1999). To

avoid over-feeding, the μ value in the feeding Eq. 1 was set at the conservative value of

0.25 h-1. CPR (g h-1), an indicator of culture growth rate, exhibited an unlimited

exponential increase during first seven hours (μ= 0.56 h-1), and then a steep decline

(Figure 3-2). After this point, the CPR trajectory followed the glucose feeding curve

(Figure 3-2), indicating a substrate-limited growth. By 22 h, the dissolved oxygen could

no longer be maintained at 40% of saturation as oxygen demand surpassed the

bioreactor's mass transfer capabilities. During the portion of the growth phase when

growth was substrate-limited, glucose concentration was below 0.5 g l-1 and the total

amount of dry biomass produced was 76.4 g (61.6 g l-1), with an average productivity of

2.8 g X l-1 h-1.

Figure 3-2 Fed-batch P. putida cultivation using simple exponential feeding designed to achieve μ= 0.25 h-1.

Culture time (h)

0 5 10 15 20 25

Bio

mas

s (g

)

0

20

40

60

80

Glu

cose

con

cent

ratio

n (g

l-1)

0

1

2

3

4

5C

PR

(g h

-1)

0

10

20

30

40

50

Glu

cose

add

ed

0

50

100

150

200

250

300

350

400

BiomassGlucose concentrationCPRGlucose added

54

3.4.3 Exponential feeding based on initial μmax estimation

The previous exponential feeding strategy was based on a conservative choice of μ for the

feeding equation. An online estimation of the actual μ would allow for more rapid feeding

and thus a more productive fermentation. This fermentation again began in batch mode.

Data analysis 10 h after inoculation determined parameters a and μmax of Eq. 2 to be 0.15

g and 0.38 h-1, respectively (Figure 3-3a). These values, along with the YCO2/S for glucose

of 0.65 g g-1, were then used in exponential feeding Eq. 3 to begin feeding. Since glucose

feeding was based on ∆St in Eq. 3, additional glucose was immediately automatically

added in a step-wise fashion to compensate for the estimated use of glucose up to that

point. This was followed by exponential addition to maintain the estimated ∆St. At 12 h,

the overall a and μmax values were redetermined as 0.09 g and 0.45 h-1, respectively

(Figure 3-3b). The feeding equation was updated accordingly, resulting in more glucose

being automatically added in a stepwise fashion to reach the updated ∆St value. Again

this was followed by exponential addition to maintain ∆St as it increased with time.

Biomass accumulated exponentially up to 64.6 g (54.4 g l-1) at 16 h, at which time the

dissolved oxygen concentration could no longer be maintained at or above 40% saturation

due to excessive O2 demand (Figure 3-4). This average biomass productivity throughout

this fermentation was 3.40 g X l-1 h-1.

55

Figure 3-3 Online cumulative CO2 production (CCP) data regressions according to Eq. 2 for μmax estimation. (a) Regression at 10 h cultivation time. (b) Regression at 12 h cultivation time.

Figure 3-4 Fed-batch P. putida cultivation using exponential feeding with online μmax estimation.

Culture time (h)

0 2 4 6 8 10 12

CC

P (g

)

0

2

4

6

8

CCPRegression curve

Culture time (h)

0 2 4 6 8 10 12 14

CC

P (g

)

0

5

10

15

20

CCPRegression curve

CCP=0.15*EXP(0.38*t) CCP=0.09*EXP(0.45*t)

Culture time (h)

0 2 4 6 8 10 12 14 16 18

Bio

mas

s (g

) and

cum

ulat

ive

CO

2 pro

duct

ion

(g)

0

20

40

60

80

100

120

140

160

180

200

Glu

cose

add

ed (g

) and

glu

cose

con

sum

ed (g

)

0

20

40

60

80

100

120

140

160

180

200

0

20

40

60

80

100

120

140

160

180

200

Glu

cose

con

cent

ratio

n (g

l-1)

0

20

40

60

80

100

120

140

160

180

200

BiomassGlucose addedGlucose consumedCCPGlucose concentration

(a) (b)

56

3.4.4 Semi-continuous feeding based on the detection of substrate limitation (CPR-based pulse feeding)

The preceeding feeding method is dependent on an accurate YCO2/G value. A method

allowing the actual level of glucose to be detected should be more reliable. Limitation of

growth by falling glucose levels will cause the CPR to decline. Addition of a quantity of

glucose insufficient to inhibit growth as soon as glucose limitation is detected in the CPR

data should allow the glucose concentration to be maintained near its optimal value

throughout the majority of the fermentation. In this approach, 10 g l-1 of glucose was fed

automatically each time the CPR declined by 10% of its previous maximum value. Liquid

samples were taken during the period of CPR decline. As expected, the glucose

concentration during each CPR decline was very close to 0, which indicated that overall,

the glucose concentration was always controlled at or below 10 g l-1 (Figure 3-5). Oxygen

transfer became problematic at the 10 h point since increasingly large oxygen demands

occurred after each addition of glucose. 45.9 g (30.0 g l-1) of biomass had been produced

by that time giving an overall biomass productivity of 3.0 g X l-1 h-1.

57

Figure 3-5 Fed-batch P. putida cultivation using CO2 production rate (CPR) based pulse feeding.

3.4.5 Continuous feeding with real-time glucose consumption estimation based on CCP analysis

The three previous glucose feeding strategies all allow glucose to fall to growth-limiting

levels for at least part of the fermentation. Besides being at least moderately sub-optimal

in terms of biomass productivity, allowing glucose to limit growth may trigger

undesirable physiological responses. If glucose limitation is to be avoided and automatic

measurement of glucose concentration is impractical, accurate knowledge of YCO2/S

values may allow substrates to be controlled based on CPR data. To assess whether this

could be achieved, glucose was fed in a continuous manner according to feeding Eq. 4.

Using this approach, the glucose concentration was maintained between 5 and 19 g l-1

(Figure 3-6) which is commonly considered to be neither growth-limiting nor

Culture time (h)

0 2 4 6 8 10 12

Bio

mas

s (g

)

0

10

20

30

40

50

Glu

cose

con

cent

ratio

n (g

l-1)

0

2

4

6

8

10

12

14

CO

2 pro

duct

ion

rate

(CP

R, g

h-1

)

0

5

10

15

20

25

30

35

40

Glu

cose

add

ed (g

)

0

20

40

60

80

100

120

BiomassGlucose concentrationCPRGlucose added

58

growth-inhibiting. By 12.5 h of fermentation the dissolved oxygen concentration had

dropped to near 0 % so the fermentation was abandoned. By that time 63.5 g (54.2 g l-1)

of biomass had been produced, giving an overall biomass productivity of 4.3 g X l-1 h-1.

Figure 3-6 Fed-batch P. putida cultivation using continuous glucose feeding based on glucose consumption as estimated from cumulative CO2 production data.

3.5 Discussion

3.5.1 Yield values

The yield values obtained are similar to literature data for other P. putida strains. P.

putida IPT 046 was found to have a dry biomass yield from a mixture of glucose and

fructose of 0.40 g g-1 (Diniz et al. 2004), while we found a dry biomass yield from

glucose of 0.41 g g-1 for KT 2440. The YX/NH4 for P. putida KT 2440 (5.44 g g-1) was also

Culture time (h)

0 2 4 6 8 10 12 14

Bio

mas

s (g

)

0

10

20

30

40

50

60

70

Glu

cose

con

cent

ratio

n (g

l-1)

0

10

20

30

40

50

Cum

ulat

ive

CO

2 pro

duct

ion

(g) a

nd G

luco

se a

dded

(g)

0

20

40

60

80

100

120

140

160

180

200

BiomassGlucose concentrationCCPGlucose added

59

very similar to that of IPT 046 (5.50 g g-1). These differences appear to be within the

range of experimental error and are thus essentially identical.

An analysis of data for P. putida KT2442 grown on oleic acid showed a YX/PO4 of

less than or equal to 16 g g-1 (Lee et al. 2000) compared to 13.7 g g-1 for KT2440 on

glucose. As stated previously, some phosphate data was excluded from our calculations.

It is believed that significant precipitation of phosphate occurred in fermentations from

which the excluded data was collected. The formation of insoluble phosphate complexes

with other minerals should be considered during medium formulation and preparation,

especially since phosphate limitation is believed to lead to the accumulation of

poly(3-hydroxyalknoates) in P. putida (Lee et al. 2000).

Further analysis of data for P. putida KT2442 grown on oleic acid indicated a YX/Mg

of less than or equal to 265 g g-1 (Lee et al. 2000), compared to 236 g g-1 for KT2440 on

glucose. These values are lower than those typically found in bacterial cultures (Pirt,

1975). MgSO4 is generally added in small batches during high density fermentations or

fed into the reactor with glucose as it too has a tendency to precipitate, possibly in

complexes with phosphate.

CO2 production was found to be directly associated with glucose consumption in this

strict aerobe (Figure 3-1d). Since the exit gas flowrate and the CO2 content can be easily

60

and accurately measured online, the CPR and consequently, the cumulative CCP, was

used in various manners during the development of automatic substrate feeding strategies.

3.5.2 Exponential feeding strategies

Exponential feeding is a simple yet efficient automatic substrate feeding strategy that has

been applied extensively in fed-batch high cell density cultivation of E. coli and other

microorganisms to achieve desired specific growth rate in order to prevent accumulation

of inhibitory products or substrates (Riesenberg et al. 1991; Tada et al. 2000; Yoon et al.

1994). It has been applied to HCDC of P. putida for intercellular protein (Thuesen et al.

2003) and MCL-PHA (Diniz et al. 2004) production. In our study, we began to evaluate

this feeding strategy using a μ of 0.25 h-1 in Eq. 1. There was little risk of glucose

overfeeding as the μmax of this species is about 0.67 h-1 when grown on glucose in a

mineral salts medium (Diniz et al. 2004; Givskov et al. 1994). The glucose concentration

limited growth rate to the desired value throughout the fermentation. Nonlinear regression

of the CCP data with respect to time during the bulk of the growth phase (7.5 h to 21 h)

according to a two parameter exponential growth model, y= a·exp(bt) showed the

damping parameter b to be 0.248 h-1, which was essentially the target value of μ (0.25

h-1). This demonstrated that CCP data can be used to fit the growth model. In addition,

since CCP is the integral of CPR with respect to time, it is naturally filtered of noise.

61

Therefore CCP data are a good choice for off-line μ determination when biomass data are

poor or, more importantly and practically, for online μ estimation.

Simple exponential feeding requires only basic equipment, interfacing and

programming, and is commonly practiced. However, conservative feeding rates are

usually chosen since μ may vary from batch to batch or during a particular fermentation

due to the variations in the inoculum and physiological conditions. This results in

sub-optimal biomass productivity. Online adjustment of feeding to the actual μmax would

be preferable. As had been shown in our exponential feeding study (Figure 3-2) and also

observed in other exponential feeding studies (Riesenberg et al. 1991), the culture usually

exhibits a period of rapid growth early in the fermentation since all substrates are in

excess. This may be considered as the μmax for the conditions in that portion of that

particular fermentation. Sub-optimal feeding results in a rapid drop in CPR as excess

carbon source is exhausted after the period of rapid growth. Using the CCP data collected

online, we can determine the μmax before carbon source limitation, allowing us to set an

appropriate glucose feeding rate and thus avoid limitation. This process was used to

produce the data in Figure 3-3. As expected, "μmax" did vary, but the updated feeding

model resulted in a very good match between the glucose addition and the actual glucose

consumption during cultivation time 13 to 15 h (Figure 3-4). After that, subsequent

glucose analysis showed a deviation of the actual glucose consumption from the glucose

62

feeding trajectory, which indicated that further actual μmax updating was required.

However, using exponential feeding with online μmax estimation two estimations), the

biomass productivity (3.40 g X l-1 h-1) and specific growth rate were enhanced and were

comparable or higher than calculated from or reported in the literature data for other P.

putida strains (Diniz et al. 2004; Thuesen et al. 2003). In order to accurately calculate the

variation of μ and adjust the feeding rate accordingly, we are trying to develop a recursive

least square algorithm to estimate μ in a real time. More frequent updating of μ estimation

using this algorithm and consequently more frequent adjustment in the feeding rate

should allow for even greater productivity.

3.5.3 CPR-based pulse feeding

DO-stat and pH-stat feeding methodologies have been developed to prevent the

accumulation of substrate to inhibitory levels. These are based on the fact that DO

increases rapidly when a key substrate is depleted (Cutayar and Poillon 1989) and that pH

may also increase due to the excretion of ammonium ions (Suzuki et al. 1990). Both of

these feeding strategies share the similar indirect feedback control scheme of keeping the

substrate concentration between 0 and a noninhibitory level by pulse feeding, and are

often used in fed-batch cultivation, including fermentations of Pseudomonas (Kim 2002;

Lee et al. 2000). CPR, which can be obtained in real time almost as easily and rapidly as

DO or pH, can also be used as the indication of substrate limitation. Although it was

63

previously claimed that such pulse feeding requires a full-time operator (Diniz et al.

2004), a control algorithm was written to add glucose automatically at every significant

drop in CPR. Using this methodology, we were able to control the glucose concentration

in the medium between 0 and 10 g l-1, leading to a biomass productivity of 3.0 g X l-1 h-1.

This CPR-based feeding strategy can be an alternative method to DO-stat and pH-stat

feeding strategies during the fed-batch cultivation of microorganisms, such as P. putida,

especially when DO-stat or pH-stat feeding is not feasible, such as when pH is not

significantly affected by substrate depletion (Kim et al. 1996).

3.5.4 Continuous feeding based on CCP

A simple continuous feeding strategy was developed based on real-time glucose

consumption estimation. Without a complicated control algorithm and programming

requirements, only real-time CCP data and an accurate YCO2/S are needed to control the

substrate feeding according to Eq. 4. YCO2/S was already found to be highly stable during

both glucose limitation and excess (Figure 3-1d). The regression of the CCP data with

respect to time indicated that the μmax of the culture during the growth phase was 0.48 h-1,

which was slightly higher than the μmax value obtained using the exponential feeding with

updated μ strategy, and resulted in a high overall biomass productivity at 4.3 g X l-1 h-1.

As can be seen from the increase of glucose concentration from 10 to 12 h of this

fermentation, glucose consumption was overestimated, indicating either error in a

64

measurement or that the YCO2/S during that growth period must have varied to below the

assumed value (0.65 g g-1). However, glucose was always maintained within a

concentration range of 5-20 g l-1 in four fermentations using the same YCO2/S setting (data

not shown). Thus the reliability and performance using this simple feeding strategy is

satisfactory. Perfectly accurate CCP determinations could result in even better control.

3.5.5 Comparison of methodologies

In this study, we evaluated traditional exponential feeding strategy, and developed and

investigated three other substrate feeding strategies for high cell density fed-batch

cultivation of P. putida KT2440. In each case productivity was limited by the glucose

feeding methodology and, at the higher oxygen demands found late in the fermentations,

by the oxygen transfer capacity of the bioreactor. Although continuous feeding based on

real-time CGC estimation produced the best results in terms of overall biomass

productivity (Table 3-1), each of these approaches might be suitable for a particular set of

conditions. Choice of feeding strategy would depend on availability of equipment and

expertise as well as the microorganism. For example, some strategies require more

sophisticated programming expertise (Table 3-1, automation programming). CPR based

pulse feeding demands a tracking and comparison algorithm whose design is fairly

challenging. Exponential feeding with μmax estimation requires automation of regression

analysis which may be even more difficult to achieve. Continuous feeding with real-time

65

glucose consumption estimation is probably the most reliable methodology in terms of

achieving a high biomass density since it can compensate for disturbances in the growth

conditions (temperature, pH etc) that may affect μ (Table 3-1, reliability). However, this

adaptability may make it less repeatable than simple exponential feeding at a conservative

μ (Table 3-1, repeatability). In addition, most P. putida strains carry plasmids and copy

number may be of importance depending on the application. Unless selective medium is

used, none of the aforementioned growth strategies may be optimal, since plasmid

replication is often independent of μ. The feeding strategies relying on YCO2/S would

obviously not be suitable for organisms whose YCO2/S varies depending on physiological

conditions (such as the facultative anaerobe E. coli). Nevertheless, all of these strategies

offer the possibility of complete automation, allowing productive high density fed-batch

cultivation of bacteria with physiologies similar to that of P. putida KT 2440.

Table 3-1 Comparison of the four feeding strategies and recommendations

Feeding strategies investigated

Evaluation criteria Overall biomass productivity (g X l-1 h-1)

Automation programming Reliability Repeatability Recommendations &

further improvements

Exponential with predetermined μ Fair Easy Good Good Good for strictly

controlled μ application

Exponential with μmax estimation Good Difficult Fair Fair

More frequent online μ estimation required for reliable results

CPR based pulse feed Fair Fair Fair Fair

Good alternative to pH-stat or DO-stat feeding strategies

Continuous feed based on real-time CGC estimation

Best Easy Best Fair Good for maximizing biomass productivity and production

66

Acknowledgement This project was supported by the Natural Science and Engineering

Research Council of Canada (NSERC).

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Timmis KN (2002) Pseudomonas putida: a cosmopolitan opportunist par excellence. Environ Microbiol 4:779-781

Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971-974

Yoon SK, Kang WK, Park TH (1994) Fed-batch operation of recombinant Escherichia coli containing Trp promoter with controlled specific growth rate. Biotechnol Bioeng 43:995-999

69

CHAPTER 4

Increasing the Yield of MCL-PHA from Nonanoic Acid

by Co-feeding Glucose During the PHA Accumulation

Stage in Two-stage Fed-batch Fermentations of

Pseudomonas-putida KT2440

Zhiyong Sun, Juliana A. Ramday, Martin Guay, Bruce A. Ramsay

Originally published online March 12th, 2007 in Journal of Biotechnology

70

4.1 Abstract

A method was developed to increase the yield of MCL-PHA from nonanoic acid in the

PHA accumulation phase. Pseudomonas putida KT2440 was grown on glucose until

ammonium-limitation was imposed. In the second (accumulation) stage, either glucose,

nonanoic acid, or a mixture of these carbon and energy sources was supplied. Since the

medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) subunits produced are

unique from each carbon source, their relative contribution to PHA yield could be

calculated. Y(C7+C9)/NA was 0.254 mol mol-1 during PHA synthesis from nonanoic acid.

Y(C8+C10)/G was only 0.057 mol mol-1 during PHA synthesis from glucose. When nonanoic

acid and glucose were fed together, Y(C7+C9)/NA almost doubled to 0.450 mol mol-1 while

Y(C8+C10)/G decreased to 0.011 mol mol-1. These results demonstrate that substantial

savings can be obtained by feeding glucose with substrates that are good for PHA

production but much more expensive than glucose.

4.2 Introduction

Pseudomonas putida can synthesize medium-chain-length PHAs (MCL-PHAs) from a

variety of carbon sources (Huisman et al. 1989; Huijberts et al. 1992). Its metabolism has

been well studied and depends on whether aliphatics or carbohydrates are supplied

(Eggink et al. 1992). Consequently, MCL-PHA production processes have been

71

developed using either aliphatics (Kim et al. 1997; Kim 2002) or carbohydrates (Diniz et

al. 2004) by employing two-stage, fed-batch fermentations with a nutrient-limited second

stage for MCL-PHA accumulation. However, how a combination of these two types of

carbon sources affect the efficiency of MCL-PHA synthesis is not known. Since

aliphatics are generally much more expensive than common industrial carbohydrates, and

since substrate costs make up a large proportion of the total production cost, the aim of

this study was to better understand the relative contributions of these two types of carbon

sources to MCL-PHA synthesis. This knowledge may aid in the development of more

economical MCL-PHA fermentation processes.

Pseudomonas putida KT2440 was used because it is one of the most studied putida

strains (Timmis 2002). To separate PHA synthesis from growth and because it’s a

commonly employed approach to producing useful amounts of PHA, this study employed

two-stage fed-batch fermentations and focused only on PHA synthesis during the

nutrient-limited, non-growth (second) stage. The key to understand the contribution of

each type of carbon source is to identify the corresponding PHA subunit yield from that

carbon source. Nonanoic acid (NA) and glucose (G) were chosen as the representatives of

aliphatics and carbohydrates, respectively, because it has been clearly shown that only

odd carbon number monomers can be formed from nonanoic acid (Du and Yu 2002; Sun

72

et al. 2007/Chapter 5) and only even-numbered carbon subunits from glucose (Huijberts

et al. 1992; Sanchez et al. 2003).


Culture (P. putida KT2440, also known as ATCC 47054) conservation, media

composition for inoculum and first stage (growth stage) of the fermentation, nutrients

(glucose, nitrogen, and phosphorus) analyses as well as fermentation conditions were

previously described (Sun et al. 2006/Chapter 3). PHA content (Sun et al. 2007/Chapter

5) and nonanoic acid concentration (Ramsay et al. 1991) were determined by gas

chromatography.

During the growth stage of all three two-stage fed-batch fermentations glucose was

fed based on cumulative glucose consumption estimated from CO2 data (Sun et al.

2006/Chapter 3). NH4OH was replaced by 2M KOH at an estimated cell concentration of

40 g l-1 such that NH4+ would be depleted at a cell concentration about 45-50 g l-1. Three

fermentations were performed identically for the first stage, but carbon source(s) feeding

strategy of the second stage (PHA accumulation stage) differed as following.

G-only PHA synthesis: only glucose was fed as the PHA synthesis substrate according to

Eq. 1.

73

2 /

t PHAt PHA G t PHA

CO G PHA

CCPS CGCY

−− − −

−

∆ = = (1)

where ∆St -PHA-G is the amount of glucose fed, CGCt-PHA the estimated cumulative

glucose consumption since PHA-time zero, CCPt-PHA is the cumulative CO2 production

since PHA-time zero, which is the beginning of the PHA accumulation stage. YCO2/G-PHA

is the yield of CO2 from glucose consumption during non-growth stage, which was

obtained as 1.07 g g-1 from previous study.

NA-only PHA synthesis: only nonanoic acid was fed as the PHA substrate at linear rates

according to Eq. 2, following a pulse addition of 3 g l-1 at the beginning of the second

stage.

( )0t PHA NA PHA NA PHA PHAS F t t− − −∆ = ⋅ − (2)

where ∆St -PHA-NA is the amount of nonanoic acid fed, FPHA-NA is the linear feeding

rate, which was 0.47 g NA l-1 h-1 during the first 13 h of the PHA stage then increased to

1.01 g NA l-1 h-1 till the end of the fermentation, and tPHA0 is the time point when a new

feeding rate is applied.

NA+G co-feeding PHA synthesis: both carbon sources were fed simultaneously during

the PHA accumulation stage. Nonanoic acid was fed at a linear rate of 0.47 g NA l-1 h-1

74

according to Eq. 2, following a pulse addition of 3 g l-1, and glucose was fed according to

Eq. 1.

4.4 Results and discussion

The yield of CO2 from glucose during the growth phase of P. putida KT2440 has been

shown to be about 0.63 g g-1 (Sun et al. 2006/Chapter 3). Although the yield during

non-growth PHA accumulation using glucose is also constant (data not shown), it is much

larger (1.07 g g-1). By feeding glucose according to the estimation of its consumption, the

glucose concentration was maintained between 5 g l-1 and 20 g l-1, well below the

inhibitory level but above a growth-limiting level during the growth phase of all

fermentations. The feeding of nonanoic acid at the chosen linear rates resulted in

nonanoic acid concentration below the 3 g l-1 level, above which growth inhibition

occurs. (Sun et al. 2007/Chapter 5).

Only 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) were found as

significant subunits of the PHA synthesized during G-only fermentation (Figure 4-1a);

while only 3-hydroxyheptanoate (C7) and 3-hydroxynonanoate (C9) formed during

NA-only fermentation (Figure 4-1b). When the two carbon sources were fed together, all

of these subunits were detected (Figure 4-1c). Based on these results and on the known

synthetic pathways, it can be assumed that C8 and C10 components were solely derived

75

from glucose, while C7 and C9 were solely from nonanoic acid. Therefore, the

relationships between carbon consumption and corresponding PHA components yield can

be plotted (Figure 4-2).

Figure 4-1 PHA subunits produced during the nitrogen-limited stage of each fermentation. Nitrogen-limitation began at about the same time as the first data point shown in each figure. (a) G-only PHA synthesis. (b) NA-only PHA synthesis. (c) NA+G co-feeding PHA synthesis. The PHA subunits are 3-HHp, 3-hydroxyheptanoate, 3-HO, 3-hydroxyoctanoate, 3-HN, 3-hydroxynonanoate, 3-HD, 3-hydroxydecanoate.

Figure 4-2 Relationship between total carbon of nonanoic acid (NA) or glucose (G) consumed and total carbon accumulated into the corresponding PHA subunits. Closed squares are from G-only PHA synthesis, open squares from NA-only PHA synthesis, and half-closed squares from NA+G co-feeding.

The slopes of the regression curves reflect the separate overall yields, which are

Y(C7+C9)/NA (the C7 and C9 yield from nonanoic acid) and Y(C8+C10)/G (the C8 and C10

yield from glucose). Y(C7+C9)/NA was 0.254 mol mol-1 during NA-only PHA synthesis,

Culture time (h)

12 13 14 15 16 17 18 19

PH

A s

ubun

its (g

l-1 )

0.00.20.40.60.81.01.21.41.61.8

3-HO (C8)3-HD (C10)

Culture time (h)

10 20 30 40 500123456789

103-HHp (C7)3-HN (C9)

Culture time (h)

10 20 30 40 50012345678

(a) (b) (c)

Total glucose carbon consumed (mmol)0 1000 2000 3000 4000 5000To

tal c

arbo

n in

(C8+

C10

) sub

units

(mm

ol)

0

40

80

120

160

200G-onlyNA+G co-feed

Total nonanoic acid carbon consumed (mmol)

0 400 800 1200 1600 2000Tota

l car

bon

of (C

7+C

9) s

ubun

its (m

mol

)

0

100

200

300

400

500

NA-only

76

which is very close to Durner’s result (YC-PHA/C= 0.24 g g-1) when culturing P. oleovorans

(now classified as a P. putida strain) on nonanoate (Durner et al. 2001). In contrast,

Y(C8+C10)/G was only 0.057 mol mol-1 under G-only PHA synthesis. These yields

demonstrate that nonanoic acid (or aliphatics in general) is a much more efficient carbon

source than glucose (carbohydrates in general) for MCL-PHA synthesis. Furthermore,

under NA+G co-feeding conditions, Y(C7+C9)/NA almost doubled to 0.450 mol mol-1 while

Y(C8+C10)/G decreased to 0.011 mol mol-1. These data demonstrate that more nonanoic acid

can be diverted to PHA synthesis simply by co-feeding glucose. Although glucose

addition would also affect the subunit composition and thus the thermo-mechanical

properties, glucose is presently much cheaper than nonanoic acid. Such a co-feeding

strategy could significantly reduce the cost of production of certain types of MCL-PHA.

4.5 References


Du GC, Yu J (2002) Metabolic analysis on fatty acid utilization by Pseudomonas oleovorans: mcl-poly(3-hydroxyalkanoates) synthesis versus beta-oxidation. Process Biochemistry 38:325-332


77

Eggink G, Dewaard P, Huijberts GNM (1992) The role of fatty-acid biosynthesis and degradation in the supply of substrates for poly(3-hydroxyalkanoate) formation in Pseudomonas putida. FEMS Microbiol Rev 103:159-163


Huisman GW, de Leeuw O, Eggink G, Witholt B (1989) Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl Environ Microbiol 55:1949-1954




Sanchez RJ, Schripsema J, da Silva LF, Taciro MK, Pradella JGC, Gomez JGC (2003) Medium-chain-length polyhydroxyalkanoic acids (PHA(mcl)) produced by Pseudomonas putida IPT 046 from renewable sources. European Polymer Journal 39: 1385-1394

Sun Z, Ramsay JA, Guay M, Ramsay BA (2006) Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440. Appl Microbiol Biotechnol 71:423-431

Sun Z, Ramsay JA, Guay M, Ramsay BA (2007) Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440. Appl Microbiol Biotechnol 74:69-77

Timmis KN (2002) Pseudomonas putida: a cosmopolitan opportunist par excellence Environ Microbiol 4:779-781

78

CHAPTER 5

Carbon-limited Fed-batch Production of

Medium-chain-length Polyhydroxyalkanoates from

Nonanoic Acid by Pseudomonas putida KT2440


Originally published February, 2007 in Applied Microbiology and Biotechnology, 74 (1): 69-77

79

5.1 Abstract

Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h-1 but

accumulated almost no poly(3-hydroxyalkanoates) (PHA). Subsequent

nitrogen-limitation on nonanoic acid resulted in the accumulation of only 27%

medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited

growth (µ= 0.15 h-1) produced 70 g l-1 biomass containing 75% PHA. At a higher

exponential feed rate (µ= 0.25 h-1), the overall productivity was increased but less

biomass (56 g l-1) was produced due to higher oxygen demand, and the biomass contained

less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic

acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective

method of producing MCL-PHA. Nitrogen limitation is unnecessary.

5.2 Introduction

Poly(3-hydroxyalkanoates) (PHAs) have attracted extensive commercial interest due to

their inherent biocompatibility and biodegradability (Zinn et al. 2001). In particular,

medium-chain-length PHAs (MCL-PHAs), which contain 6 to 14 carbons in their

repeating units, show great promise as thermoelastomers for biomedical applications,

such as drug delivery (Pouton and Akhtar 1996) and tissue engineering (Williams et al.

1999; Chen and Wu 2005). Efficient production of MCL-PHAs is a prerequisite for

80

extensive study of these polyesters. Although it was observed that 8-carbon and 9-carbon

alkanes or alkanoates result in the highest MCL-PHA accumulation (Brandl et al. 1988;

Gross et al. 1989; Kang et al. 2001), and octanoic acid was used as the carbon source in

pH-stat processes with nitrogen limitation to obtain 47 g l-1 biomass containing 55% PHA

(Dufresne and Samain 1998) and 63 g l-1 containing 62% PHA (Kim 2002), almost no

research into the development of production processes using nonanoic acid as the

principle carbon source was reported. While octanoic acid is derived from coconut and

palm kernal oils, nonanoic acid can be produced from oleic, linoleic and erucic and other

carboxylic acids produced in temperate zone plants such as canola. For a variety of

reasons, nonanoic acid has potential as a commodity feedstock for ‘biorefinery’ processes

in industrialized countries.

Nitrogen (N) or phosphorus (P) limitation stimulates rapid PHA synthesis in most of

the well-studied SCL (short-chain-length)-PHA-synthesizing bacteria, such as Ralstonia

eutropha. Therefore most SCL-PHA production processes employ a stage of rapid cell

growth followed by a PHA accumulation stage, which is almost always N-limited or

P-limited. Possibly due to an assumption that MCL-PHA production physiology is similar

to that of most SCL-PHA accumulating bacteria, almost all publications dealing with

MCL-PHA production have incorporated N or P limitation (Preusting et al. 1993;

Huijberts and Eggink 1996; Dufresne and Samain 1998; Jung et al. 2001; Kim 2002).

81

Other than continuous plug-flow processes, which are impractical at a commercial scale,

fed-batch fermentation is theoretically and practically the best method of achieving a high

density of biomass containing the highest possible amount of PHA. High PHA content is

critical to the reduction of PHA separation cost in commercial processes. Since the rate of

substrate demand is difficult to measure or predict, design and control of a N-limited or

P-limited accumulation phase in a fed-batch process is difficult when it involves the

feeding of inhibitory substrates such as short-chain-length and medium-chain-length

carboxylic acids. While a pH-stat approach is feasible, simple exponential feeding of

substrate is more reliable and potentially more productive. However, exponential feeding

is only possible when N or P limitation is not imposed.

The metabolic regulation of SCL-PHA has been well studied (Senior and Dawes

1971; Steinbüchel and Lütke-Eversloh 2003), but less is known about the factors

controlling MCL-PHA synthesis. While the rate of MCL-PHA production in P.

resinovorans is greatly stimulated by N-limitation (Ramsay et al. 1992), this did not occur

in P. oleovorans GPo1 ATCC 29347 (Ramsay et al. 1991) now recognized to be a strain

of P. putida (Diard et al. 2002). Significant MCL-PHA accumulation during the

exponential growth phase of P. putida KT2442 (Huisman et al. 1992), P. putida U

(Carnicero et al. 1997) and P. putida GPo1 (Durner et al. 2001) has been reported,

indicating little need for N or P limitation. We found that high MCL-PHA content

82

(>50%) occurred under carbon-limited conditions in a chemostat study of P. putida

KT2440 using nonanoic acid as carbon source (unpublished data). P. putida strains are

known to produce large amounts of MCL-PHA (Lee et al. 2000; Diniz et al. 2004), and

strain KT2440 (ATCC 47054) has no plasmid load (Timmis 2002). Based on these

observations, the aim of the present study was to compare a single-stage carbon-limited

process with a two-stage process incorporating an N-limited stage for the production of

MCL-PHAs by P. putida KT2440. Production from nonanoic acid and glucose was also

compared.


5.3.1 Microorganism and growth medium

P. putida KT2440 (ATCC 47054) was maintained in lyophiles and on nutrient agar plates

at 4 ºC. For the fermentation using glucose as sole substrate during the growth phase, the

inoculum medium, initial and feeding medium for the growth phase of the fermentation

was described previously (Sun et al. 2006/Chapter 3). For all other fermentations the

inoculum medium contained per liter: 4.70 g (NH4)2SO4, 0.80 g MgSO4·7H2O, 12.00 g

Na2HPO4·7H2O, 2.70 g KH2PO4, 3.00 g nutrient broth. The initial culture medium

contained per liter: 4.70 g (NH4)2SO4, 0.80 g MgSO4·7H2O, 18.00 g Na2HPO4·7H2O, 4.05

g KH2PO4, 1-3 g nonanoic acid and 10 ml trace element solution. Each liter of trace

element solution contained 10 g FeSO4·7H2O, 3 g CaCl2·2H2O, 2.2 g ZnSO4·7H2O, 0.5 g

83

MnSO4·4H2O, 0.3 g H3BO3, 0.2 g CoCl2·6H2O, 0.15 g Na2MoO4·2H2O, 0.02 g

NiCl2·6H2O and 1.00 g CuSO4·5H2O. Nitrogen was supplied using 14% (w/v) NH4OH to

control the pH during growth and was replaced by 2 M KOH during N-limited stages.

Nonanoic acid (96%, Sigma Aldrich) was filter-sterilized. To avoid precipitation, all the

needed MgSO4·7H2O was not added to the initial medium. Additional MgSO4·7H2O was

fed at a ratio of 0.033 g MgSO4·7H2O g-1 nonanoic acid addition assuming a YX/Mg of 240

g g-1 (Sun et al. 2006/Chapter 3) and a YX/NA of 0.80 g g-1.


Inoculum culture was cultivated in 500 ml shake flasks (100 ml medium) at 30±1ºC and

200 rpm overnight. Fed-batch fermentations were all carried out at 31±1 ºC with a 3.5 l

initial working volume in a Minifors 5 l stirred tank bioreactor (Infors HT, Bottmingen,

Switzerland). Data acquisition (dissolved oxygen, outlet gas CO2, carbon source and base

addition, and pH) and control was conducted with LabVIEW 6.1 (National Instruments).

pH was controlled at 6.85±0.05. Dissolved oxygen was measured with an Ingold

polarographic probe and maintained at or above 40% air saturation (except where

indicated) by adjusting the agitation speed up to 1,200 rpm and by automatically adjusting

the mixture of air and pure oxygen flow via mass flow controllers while maintaining total

gas flow at 1 vvm. Exit gas CO2 content was measured with an infrared CO2 monitor

84

(Guardian Plus, Topac Inc. Hingham, MA, USA). Feeding of nonanoic acid with

peristaltic pumps was automatically controlled based on the mass of the reservoirs.

5.3.3 Substrate feeding and control methods

5.3.3.1 Nutrient-unlimited growth on glucose followed by an N-limited accumulation phase on nonanoic acid (two-stage fed-batch)

A continuous feeding strategy based on glucose estimation was applied to first obtain

about 40 g l-1 of biomass, as previously described (Sun et al. 2006/Chapter 3).

N-limitation was then imposed by replacing ammonium hydroxide with KOH. During

this N-limited stage, a pulse of 3 g l-1 nonanoic acid was first added, followed by linear

feeding at 0.47 g NA l-1 h-1 until 17 h, then 1.01 g NA l-1 h-1 until the end of the

fermentation.

5.3.3.2 Single-stage, nonanoic acid-limited fed-batch

The initial nonanoic acid concentration was 1 g l-1. Additional substrate was fed at the

rate shown in Eq. 1.

0

/ /

tt

X S X S

tX XS eY Y

µ⋅∆ = = ⋅ (1)

where ∆St is total nonanoic acid required to produce biomass Xt at time t, YX/S is the yield

of biomass from nonanoic acid (0.80 g g-1), X0 is the initial biomass, obtained by

measuring the OD650nm of the inoculum culture and converted to cell concentration in the

bioreactor, and µ is the desired specific growth rate.

85

5.3.3.3 Nonanoic acid-limited growth followed by an N-limited accumulation phase on nonanoic acid (two-stage fed-batch)

With 3 g l-1 of initial nonanoic acid, exponential feeding was applied according to Eq. 1

with a target µ= 0.25 h-1. Shortly before reaching the maximum oxygen transfer capacity,

nitrogen limitation was imposed by replacing ammonium hydroxide with KOH. During

this N-limited stage, nonanoic acid was fed at a rate of 1.31 g NA l-1 h-1 until 41.5 h, then

1.82 g NA l-1 h-1 until the end of the fermentation.


Cell dry weight was determined after lyophilization of biomass obtained by centrifugation

of 5 ml culture broth at 17,000 x g for 15 min and washing twice with 5 ml distilled

water. The supernatant of the sample and the distilled water wash were saved for analysis

of the nonanoic acid and key nutrients. Nonanoic acid concentration was determined by

methylation followed by GC analysis with decanoic acid as internal standard (Ramsay et

al. 1991). Phosphate was measured via reduction of phosphomolybdate to molybdene

blue (Clesceri et al. 1999). Ammonium was determined by the phenol-hypochlorite

method (Weatherburn 1967). PHA content and composition was determined by

modification of the Braunegg et al. (1978) and Lageveen et al. (1988) methodologies.

Lyophilized cell samples were suspended with 2 ml of methanol containing 15% (v/v)

H2SO4 and 0.2% (w/v) benzoic acid (internal standard) and 2 ml of chloroform.

Methanolysis was carried out in a 100 oC water bath for 3.5 h with periodic vigorous

86

mixing. After cooling to room temperature, 2 ml distilled water was added and vortexed

for 1 min. The resulting emulsion was left overnight for phase separation. The organic

phase was used for GC analysis. The PHA standard for this assay was prepared by

repeated cycles of solvent extraction followed by precipitation in cold methanol. Its

composition was determined by NMR. Residual biomass (Xr) was defined as the total cell

concentration minus the PHA concentration. The specific PHA synthesis rate based on

the amount of residual biomass (qPHA(Xr)) was defined as (dP/dt)/Xr (g PHA g of Xr -1 h-1).

The specific PHA synthesis rate based on the amount of PHA (qPHA(PHA)) was defined as

(dP/dt)/PHA (g PHA g PHA-1 h-1). They were calculated using the slope of the PHA

concentration curve and the measured Xr and PHA concentration values, respectively, at

that point.

5.4 Results Growth on glucose followed by nonanoic acid linear feeding under N-limitation

In SCL-PHA fermentations with bacteria such as Ralstonia eutropha or Burkholderia

cepacia, glucose is typically fed to produce dense biomass (growth phase). N or P is then

allowed to fall to a growth-limiting value which stimulates PHA synthesis. At the point of

N or P limitation or slightly before, co-substrates such as propionic acid are fed to

produce the desired copolymer composition during accumulation phase (Ramsay et al.

1990). High-cell-density cultivation of P. putida KT2440 on glucose had already been

87

established (Sun et al. 2006/Chapter 3). Based on this knowledge, 40 g l-1 of biomass was

produced using glucose as the sole source of carbon and energy. Almost no PHA

synthesis was detected in P. putida grown on glucose without N or P limitation (Figure

5-1). The specific growth rate throughout this phase was 0.48 h-1. Although some PHA

was initially produced during the N-limited stage, the nonanoic acid feed rate of 0.47 g

NA l-1 h-1 proved to be insufficient. Increasing the feed rate to 1.01 g NA l-1 h-1 enabled

the cells to accumulate more PHA. The nonanoic acid concentration in the reactor

remained below 2 g l-1 during this period. Eventually 26.8% PHA was accumulated in

46.1 g l-1 biomass giving a cumulative PHA productivity of 0.25 g NA l-1 h-1.

Figure 5-1 Growth of P. putida KT2440 on glucose followed by N-limited growth on nonanoic acid (NA). The division between growth phase and N-limited phase is indicated by the dashed line. The nonanoic acid feeding rate (FNA) was increased from 0.47 g NA l-1 h-1 to 1.01 g NA l-1 h-1 at 27 h during the N-limited phase, as indicated by the dash-dot line.

Culture time (h)

0 10 20 30 40 50

Bio

mas

s (g

l-1)

0

10

20

30

40

50

PH

A (%

)

0

5

10

15

20

25

30

NH

4+ an

d N

A co

ncen

tratio

n (g

l-1)

0

2

4

6

8

10

q PHA

(Xr)

(g g

-1 h

-1)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Biomass% PHANH4

+

NAqPHA (Xr)

FNA=0.47 g l-1 h-1

FNA=1.01 g l-1 h-1

88

5.4.1 Single-stage, exponential feeding of nonanoic acid (target µ= 0.15 h-1)

To further assess whether N or P limitation is required for substantial MCL-PHA

synthesis in P. putida, a nonanoic acid single-stage fed-batch process was conducted. To

avoid overfeeding, the µ in the feeding equation (Eq. 1) was set at a conservative value of

0.15 h-1. The nonanoic acid concentration was maintained well below 1 g l-1 until 41 h

(Figure 5-2). The exponential rates of consumption and growth indicate that nonanoic

acid was the only limiting nutrient until the end of the fermentation. Control of pH with

ammonium hydroxide allowed the ammonium concentration to be maintained between 1

and 2 g l-1 during the entire fermentation. Phosphate concentration was not controlled but

it never decreased to a level that limited the growth rate. The total cell concentration

increased up to 70.2 g l-1 and the measured µ, based on dry biomass samples, was the

predicted value of 0.15 h-1. The inoculum culture (grown on nutrient broth) contained no

measurable MCL-PHA but PHA content increased throughout the fermentation to a

maximum of 75.4%, giving a cumulative PHA productivity of 1.11 g PHA l-1 h-1. Mainly

hydroxynonanoate (C9) and hydroxyheptanoate (C7) PHA repeating units were detected.

Also about 1 molar percent of a combination of hydroxyhexanoate (C6),

hydroxyoctanoate (C8) and hydroxydecanoate (C10) was detected. The ratio of C9/C7

was about 2.4:1 (mol/mol) throughout the fermentation. Although agitation was maximal

and 1 vvm of pure oxygen was supplied to the fermentor, after 41 h the dissolved oxygen

concentration decreased to zero. Oxygen limited cell growth, and consequently caused

89

nonanoic acid to rapidly accumulate in the medium (to above 3 g l-1). This resulted in

growth inhibition, cell lysis, and excessive foaming.

Figure 5-2 Carbon-limited growth of P. putida KT2440 on nonanoic acid (NA) at µ= 0.15 h-1.

5.4.2 Single-stage, exponential feeding of nonanoic acid (target µ= 0.25 h-1)

To improve productivity and evaluate the effects of a higher growth rate on PHA

accumulation, the single-stage carbon-limited fermentation was repeated but at a higher

target µ (0.25 h-1) in the feeding equation (Eq.1). Key results were compared to those

obtained at µ= 0.15 h-1 (Figure 5-3). Very similar patterns were observed in the two

fermentations despite the difference in feed rate. The final total biomass, total PHA, and

% PHA of the fermentation at µ= 0.25 h-1 were 56.0 g l-1, 37.4 g l-1, and 66.9%,

Culture time (h)

0 10 20 30 40 50

Biom

ass

(g l-1

) and

PH

A (%

)

0

10

20

30

40

50

60

70

80

NH

4+ , PO

4+ , and

NA

con

cent

ratio

n (g

l-1)

0

1

2

3

4

5

6

7

8

Biomass% PHA NH4

+

PO4+

NA

90

respectively, which were lower than the final values at the lower µ (Figure 5-3a).

However, the final cumulative PHA productivity (Figure 5-3b) increased to 1.44 g PHA

l-1 h-1 at µ= 0.25 h-1 because the total cultivation time decreased significantly. The specific

rate of PHA synthesis based on residual biomass (qPHA (Xr)) increased steadily at both feed

rates up to 0.48 g g-1 h-1 at µ= 0.25 h-1 and 0.53 g g-1 h-1 at µ= 0.15 h-1.

5.4.3 Single-stage, exponential feeding of nonanoic acid (target µ= 0.25 h-1) followed by N-limited culture on nonanoic acid

Single-stage, exponential feeding of nonanoic acid resulted in high PHA productivity and

content but perhaps N-limitation could further increase PHA synthesis. Therefore an

experiment was conducted in which N-limitation was imposed after exponential feeding

(target µ= 0.25 h-1) of nonanoic acid (Figure 5-4). Cell growth and PHA synthesis during

the growth stage were very similar to the single-stage fed-batch where both were fed to

achieve µ= 0.25 h-1 but nitrogen limitation was imposed at 27 h by replacing ammonium

hydroxide with KOH. Linear feeding after this point allowed the nonanoic acid

concentration to be maintained just below the 3 g l-1 level. However, although the total

amount of biomass increased, there was little increase in the percentage of PHA in the

biomass produced during the N-limited stage. The final PHA concentration was 52.4 g l-1,

corresponding to an overall (i.e. cumulative) productivity of 0.91 g PHA l-1 h-1.

91

Figure 5-3 Effect of growth rate on PHA synthesis in P. putida KT2440 under nonanoic acid limitation. Closed and open symbols represent data from the µ= 0.15 h-1 and the µ= 0.25 h-1 fermentations, respectively. qPHA(Xr) is the specific PHA synthesis rate (g PHA g Xr

-1 h-1).

Bio

mas

s an

d P

HA

conc

entra

tion

(g l-1

)

0

10

20

30

40

50

60

70

PH

A (%

)

0

20

40

60

80

100Biomass at µ=0.15 h-1

Biomass at µ=0.25 h-1

PHA at µ=0.15 h-1

PHA at µ=0.25 h-1

%PHA at µ=0.15 h-1 %PHA at µ=0.25 h-1

Culture time (h)

0 10 20 30 40

Cum

ulat

ive

PH

A p

rodu

ctiv

ity (g

l-1 h

-1)

0.0

.5

1.0

1.5

2.0

q P

HA

(Xr) (g

g-1

h-1

)0.0

.1

.2

.3

.4

.5Cumulative productivity at µ=0.15 h-1

Cumulative productivity at µ=0.25 h-1

qPHA (Xr) at µ=0.15 h-1

qPHA (Xr) at µ=0.25 h-1

(a)

(b)

92

Figure 5-4 Two-stage, fed-batch using nonanoic acid (NA) as the sole carbon source. Exponential feeding (µ= 0.25 h-1) was used during the growth phase with all other nutrients in excess. The division between the growth and N-limited phases is indicated by the dashed line. The nonanoic acid feed rate was increased from 1.31 g NA l-1 h-1 to 1.82 g NA l-1 h-1 at 41.5 h during the N-limited phase as indicated by the dash-dot line.

5.4.4 Biomass and PHA yield

Total biomass, PHA, and residual biomass (Xr) data from both single-stage

(carbon-limited) fed-batch fermentations were plotted against total nonanoic acid

consumption (Figure 5-5). There was a linear relationship between total biomass and

nonanoic acid consumption demonstrating a yield of 0.83 g biomass g-1 nonanoic acid

(r2= 0.995) throughout both fermentations. In contrast, the yield of PHA and residual

biomass varied during the growth. The yield of PHA increased gradually throughout the

growth phase as more carbon was directed to PHA synthesis and less towards residual

Culture time (h)

0 10 20 30 40 50 60

Bio

mas

s (g

l-1) a

nd P

HA

(%)

0

20

40

60

80

100

NH

4+ , and

NA

conc

entra

tion

(g l-1

)

0

1

2

3

4

5

6

7

8

Biomass%PHANH4

+

NA

FNA=1.31 g l-1 h-1 FNA=1.82 g l-1 h-1

93

biomass. The overall PHA yield was about 0.60 g g-1 nonanoic acid while overall yield of

residual biomass was 0.24 g g-1 nonanoic acid.

Figure 5-5 Yield of total biomass, PHA, and residual biomass (Xr) from nonanoic acid (NA). Closed and open symbols represent data from μ= 0.15 h-1 and the μ= 0.25 h-1 fermentations, respectively. The slopes of the curves indicate yields from nonanoic acid.

5.5 Discussion

5.5.1 Process design implications

Relatively little PHA was accumulated whether the N-limited PHA accumulation phase

on nonanoic acid was preceded by growth on glucose or on nonanoic acid. Although

better control of nonanoate concentration during N-limited production would increase

PHA production, simple exponential feeding of nonanoic acid with no N or P limitation

Total NA consumption (g)

0 50 100 150 200 250 300

Biom

ass,

PH

A, a

nd X

r (g)

0

50

100

150

200

X at µ=0.15 h-1

X at µ=0.25 h-1

PHA at µ=0.15 h-1

PHA at µ=0.25 h-1

Xr at µ=0.15 h-1

Xr at µ=0.25 h-1

94

was clearly more productive, based on results presented in this paper. Similar results are

likely with octanoic acid, which is also carboxylic acid with one carbon less than

nonanoic acid. In shake flasks, nonanoic acid concentrations higher than 3 g l-1 inhibit

growth of P. putida KT2440. This has led process development researchers to work with

less toxic substrates such as oleic acid. Although remarkable progress has been made in

the production of PHA from oleic acid (Huijberts and Eggink 1996; Lee et al. 2000), its

use limits the type of PHA that can be made and can lead to downstream processing

difficulties. The standard approach to PHA production exemplified by the Biopol process

(Byrom 1990) consists of dense biomass production followed by an accumulation phase.

The Biopol process is relatively easy to control because the toxic co-substrate (propionic

acid) used in the accumulation phase is typically mixed at low concentration with a high

concentration of glucose. Use of a single toxic substrate such as nonanoic acid in an

accumulation phase requires more sophisticated control. Fortunately a provoked

accumulation phase (second phase in two-stage fermentation) does not appear to be

required or even beneficial to MCL-PHA synthesis in P. putida KT2440. The Biomer

process (Hänggi 1990) currently used for PHB production employs a single-stage

fermentation with no accumulation phase. The organism used in the Biomer process, A.

latus, exhibits a growth-associated PHA production pattern (Braunegg and Bogensberger

1985). Co-substrates may be fed to produce copolymers in such a single-stage process

(Ramsay et al. 1990). Although the kinetics of PHA synthesis in P. putida KT2440 are

95

not directly linked to growth, we propose a similar single-stage process in which

nonanoic acid or related substrates are fed exponentially. Co-substrates may be mixed

with the major substrate to produce the desired polymer subunit composition.

Since a lower specific growth rate demands less oxygen transfer, a higher final PHA

content (75%) was achieved than when µ was controlled at 0.25 h-1 (67%). However, the

cumulative PHA productivity decreased from 1.44 g PHA l-1 h-1 to 1.11 g PHA l-1 h-1

when operating at a lower µ due to the longer culturing time. Thus there is a trade-off

between the amount of PHA in the biomass and fermentation productivity. An optimal

approach can be calculated based on economic considerations such as substrate and

separation costs, as well as the oxygen transfer and mixing properties of the fermentor to

be employed.

5.5.2 Yield

Yields of total biomass and PHA from nonanoic acid were 0.83 g g-1 and 0.60 g g-1,

respectively (Figure 5-5). These are relatively high when compared to 0.40 g PHA g

octanoate-1 under glucose co-feeding conditions (Kim et al. 1997), or 0.63 g PHA g

octane-1 under optimized PHA accumulating conditions in the second stage of a

continuous process (Hazenberg and Witholt 1997). The consistency of the yield values

(Figure 5-5) and C9/C7 ratio of fermentations at different growth rates indicate that this is

a highly repeatable process.

96

There appears to be a low maintenance energy requirement in this strain because the

biomass yield values were essentially the same at both growth rates. Much more PHA is

being synthesized during latter parts of the fermentation yet the yield remained the same

(Figure 5-5). This indicates that the true yields for YX/S and YP/S are very similar. Since

the % PHA, PHA concentration and qPHA(Xr) increased continuously at both growth rates

until oxygen transfer limited production, higher final PHA content and productivity could

be achieved in a bioreactor system with better oxygen transfer capacity. Extended

cultivation using linear feeding of nonanoic acid may further increase PHA accumulation.

5.5.3 Kinetics of MCL-PHA synthesis

Although nutrient (mainly N and P) limitation was commonly applied to MCL-PHA

production processes, some controversial results have been reported. Limitation of N or P

was shown to be stimulatory to the synthesis of PHA in P. oleovorans ATCC 29347

growing on n-alkanes (Lageveen et al. 1988), P. resinovorans and P. putida BM01 on

octanoate (Ramsay et al. 1992; Kim et al. 1997) and P. putida KT2442 on oleic acid

(Huijberts and Eggink 1996; Lee et al. 2000). In contrast, it was also reported that nutrient

limitation is unnecessary for the production of significant amounts of PHA in P. putida

KT2442 (Huisman et al. 1992) , P. oleovorans ATCC 29347 (Ramsay et al. 1991; Durner

et al. 2001 ) or P. putida U (Carnicero et al. 1997) on octanoate. In the present study, high

PHA accumulation was achieved during nonanoic acid limited growth with no other

97

apparent nutrient limitation. The link between MCL-PHA accumulation and growth

limitation by key nutrients such as nitrogen or phosphate seems to differ depending on the

strain, carbon sources, the cultivation conditions, or possibly a combination of these

factors.

There are many possible explanations for the production of substantial amounts of

PHA during carbon-limited growth. Some deficiency in the TCA cycle, blockage in

ß-oxidation, a detoxification mechanism or, as in the case of Azotobacter vinelandii UWD

an ineffective NADH oxidase (Page and Knosp 1989) may divert substrate and energy

toward PHA synthesis to cause this effect. Although it was not our purpose to study the

mechanisms governing PHA production in this organism, data analysis suggests that

unlike A. latus (Braunegg and Bogensberger 1985), the kinetics of MCL-PHA synthesis

during the growth of P. putida KT2440 are not strictly linked to growth kinetics. Rather

than remaining constant, the PHA content increased from close to zero in the inoculum to

above 70%. The continually increasing qPHA (Xr) (Figure 5-3b) demonstrated that the

growing cells were synthesizing PHA at a rate higher than the growth rate of the other

biomass components (residual biomass). After a certain point in the fermentation process,

the specific rate of PHA synthesis remains constant only if calculated based on the

amount of PHA in the cells (Figure 5-6). This indicates that the rate of PHA synthesis is

controlled by a property of the granules themselves, possibly related to phasin proteins

98

found on the surface of PHA granule. Much more kinetic, physiological, and biochemical

study is required to understand the mechanisms of MCL-PHA synthesis during growth of

P. putida KT2440 and other MCL-PHA accumulating bacteria.

Figure 5-6 The specific rate of PHA synthesis (per gram of PHA) approaches a constant value as the concentration of PHA in the biomass increases.

Acknowledgement This research was supported by the Natural Science and Engineering

Research Council of Canada and a grant from BIOCAP Canada.

5.6 References

Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonas oleovorans as a source of poly(beta-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54:1977-1982

% PHA

0 10 20 30 40 50 60 70 80

q PH

A (P

HA

) (g

g-1 h

-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

qPHA (PHA) at µ=0.15 h-1

qPHA (PHA) at µ=0.25 h-1

99

Braunegg G, Bogensberger B (1985) About kinetics of growth and accumulation of poly-D(-)-3-hydroxybutric acid in Alcaligenes latus strains. Acta Biotechnol 5:339-345

Braunegg G, Sonnleitner B, Lafferty RM (1978) Rapid gas-chromatographic method for determination of poly-beta-hydroxybutyric acid in microbial biomass. Eur J Appl Microb Biotechnol 6:29-37

Byrom D (1990) Industrial production of copolymer from Alcaligenes eutrophus. In: Dawes EA (eds) Novel biodegradable microbial polymers, Kluwer Academic Publishers, Netherlands, pp 113-117.

Carnicero D, FernandezValverde M, Canedo LM, Schleissner C, Luengo JM (1997) Octanoic acid uptake in Pseudomonas putida U. FEMS Microbiol Lett 149:51-58


Clesceri LS, Greenberg AE, Eaton AD (1999) Standard methods for the examination of water and wastewater 20th edition. American Public Health Association, Washington, DC.

Diard S, Carlier JP, Ageron E, Grimont PAD, Langlois V, Guerin P, Bouvet OMM (2002) Accumulation of poly(3-hydroxybutyrate) from octanoate, in different Pseudomonas belonging to the rRNA homology group I. Syst Appl Microbiol 25:183-188


Dufresne A, Samain E (1998) Preparation and characterization of a poly(beta-hydroxyoctanoate) latex produced by Pseudomonas oleovorans. Macromolecules 31:6426-6433


100

Gross RA, Demello C, Lenz RW, Brandl H, Fuller RC (1989) Biosynthesis and characterization of poly(beta-hydroxyalkanoates) produced by Pseudomonas oleovorans. Macromolecules 22:1106-1115

Hänggi, UJ (1990) Pilot scale production of PHA with A. latus In: Dawes EA (eds), Novel biodegradable microbial polymers, Kluwer Academic Publishers, Netherlands, pp 65-70

Hazenberg W, Witholt B (1997) Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic considerations. Appl Microbiol Biotechnol 48:588-596

Huijberts GNM, Eggink G (1996) Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures. Appl Microbiol Biotechnol 46:233-239

Huisman GW, Wonink E, De Koning G, Preusting H, Witholt B (1992) Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains. Appl Microbiol Biotechnol 38:1-5

Jung K, Hazenberg W, Prieto M, Witholt B (2001) Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates). Biotechnol Bioeng 72:19-24

Kang HO, Chung CW, Kim HW, Kim YB, Rhee YH (2001) Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanoates by Pseudomonas sp. DSY-82. Antonie Van Leeuwenhoek 80:185-191



Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans - effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924-2932

101


Page WJ, Knosp O (1989) Hyperproduction of poly-beta-hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl Environ Microbiol 55:1334-1339


Preusting H, Vanhouten R, Hoefs A, Vanlangenberghe EK, Favrebulle O, Witholt B (1993) High-cell-density cultivation of Pseudomonas oleovorans - growth and production of poly (3-hydroxyalkanoates) in 2-liquid phase batch and fed-batch systems. Biotechnol Bioeng 41:550-556

Ramsay BA, Ramsay JA, Lomaliza K, Chavarie C, Bataille P (1990) Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid copolymers. Appl Environ Microbiol 56:2093-2098.


Ramsay BA, Saracovan I, Ramsay JA, Marchessault RH (1992) Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans. Appl Environ Microbiol 58:744-746

Senior PJ, Dawes EA (1971) Poly-beta-hydroxybutyrate biosynthesis and regulation of glucose metabolism in Azotobacter berjerinchii. Biochem J 125(1): 55-66


Sun Z, Ramsay JA, Guay M, Ramsay BA (2006) Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440. Appl Microbiol Biotechnol 71 (4): 423-431

Timmis KN (2002) Pseudomonas putida: a cosmopolitan opportunist par excellence. Environ Microbiol 4:779-781

102

Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971-974

Williams SF, Martin DP, Horowitz DM, Peoples OP (1999) PHA applications: addressing the price performance issue: I. Tissue engineering. Int J Biol Macromol 25:111-121

Zinn M, Witholt B, Egli T (2001) Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53:5-21

103

CHAPTER 6

Fed-batch Production of Medium-chain-length

Polyhydroxyalkanoates by Pseudomonas putida KT2440

using Nonanoic Acid and Glucose Co-feeding


Originally submitted June, 2007 to Journal of Biotechnology

104

6.1 Abstract

Medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced by

carbon-limited, single-stage fed-batch fermentations of Pseudomonas putida KT2440

using a nonanoic acid (NA) and glucose (G) co-feeding strategy, in order to enhance the

yield of PHA from nonanoic acid. With NA:G= 1:1 in the feed, an exponential (µ= 0.25

h-1) feeding strategy followed by linear substrate feeding was used to achieve a dry

weight biomass of 70.7 g l-1 containing 56% PHA. This resulted in an overall PHA

productivity of 1.44 g PHA l-1 h-1, equal to that obtained by nonanoic acid alone

fermentation at µ= 0.25 h-1, while the final overall yield of nonanoic acid to PHA was

increased by 25% (0.69 g PHA g NA-1 versus 0.55 g g-1). Further increase of the glucose

fraction in the feed (NA:G= 1:1.5) slightly increased the yield (0.71 g PHA g NA-1) but

decreased PHA content (48%) and productivity (1.16 g PHA l-1 h-1). The addition of

glucose to the feed did not change the PHA composition, although the ratio of

3-OH-nonanoate to 3-OH-heptanoate was slightly increased, probably due to a decrease

in the β-oxidation of nonanoate.

6.2 Introduction

Polyhydroxyalkanoates (PHA) are biocompatible, biodegradable polyesters synthesized

by many microorganisms. PHAs are categorized as short-chain-length (SCL-PHA)

105

containing 3 to 5 carbons in their repeating units, medium-chain-length (MCL-PHA)

containing 6 or more monomeric carbons, and SCL-MCL, which contain both SCL and

MCL repeating units. SCL-PHAs, such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

(PHB/V), and SCL- MCL-PHAs, such as

poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHB/HHx) can now be produced on an

industrial scale (Chen et al. 2001; Lee et al. 1999). However, the production of

MCL-PHAs remains at the lab scale due to the nature of the carbon sources,

microorganisms, and the degree of process automation, etc. (Sun et al. 2007b/Chapter 2).

For the development of potential applications of MCL-PHAs, as well as future industrial

development, efficient and economical production systems are required. MCL-PHA

fermentation process development has been recently reviewed by Sun et al.

(2007b/Chapter 2).

The economics of PHA production is determined by the volumetric PHA

productivity, final content in the biomass, carbon substrate cost and PHA yield, as well as

the degree of complexity and automation of the fermentation process itself. Substrate cost

and the yield of PHA from substrate have a substantial impact on the overall cost of

production, and this effect tends to increase with process scale (Choi and Lee, 1997).

Although both aliphatic substrates and carbohydrates have been used in lab scale

production of MCL-PHA by strains of P. putida (Diniz et al. 2004; Kim, 2002), aliphatic

106

substrates typically result in MCL-PHA with a wider variety of monomeric composition,

which is dependent on the substrates fed (Hartmann et al. 2006). Since substrates, such as

octanoate and nonanoate, are much more expensive than carbohydrates, efforts have been

made to use glucose and octanoate consecutively in two-step, fed-batch fermentations to

increase the overall PHA yield from octanoate (Kim et al. 1997). In our recent two-stage,

fed-batch fermentation study, we investigated the relative contribution of glucose and

nonanoic acid to cell maintenance and MCL-PHA synthesis during the nitrogen-limited,

second stage (Sun et al. 2007c/Chapter 4). We found that the PHA yield from nonanoic

acid was almost doubled when glucose was fed as a co-substrate.

In this study, which was based on a recently reported single-stage carbon-limited

fed-batch MCL-PHA fermentation process (Sun et al. 2007a/Chapter 5), we investigated

the possibility of using glucose as a co-substrate in a more cost-effective process for the

production of MCL-PHA from nonanoic acid.



Pseudomonas putida KT2440 (ATCC 47054) was maintained on nutrient agar plates at 4

ºC prior to inoculation. The inoculum medium contained per liter: 4.70 g (NH4)2SO4, 0.80

g MgSO4·7H2O, 12.00 g Na2HPO4·7H2O, 2.70 g KH2PO4, 9 g glucose, 1 g nutrient broth.

The initial fermentation medium contained per liter: 4.70 g (NH4)2SO4, 0.80 g

107

MgSO4·7H2O, 18.00 g Na2HPO4·7H2O, 4.05 g KH2PO4, 1 g carbon substrates in total and

10 ml trace element solution. Each liter of trace element solution contained 10 g

FeSO4·7H2O, 3 g CaCl2·2H2O, 2.2 g ZnSO4·7H2O, 0.5 g MnSO4·4H2O, 0.3 g H3BO3, 0.2

g CoCl2·6H2O, 0.15 g Na2MoO4·2H2O, 0.02 g NiCl2·6H2O and 1.00 g CuSO4·5H2O.

Nonanoic acid (98%, Spectrum Chemicals) was fed in its pure form. Glucose (99.5%,

Sigma-Aldrich) solution of 640 g l-1 was fed separately as it is immiscible with nonanoic

acid. Ammonium was supplied by using 14% (w/v) NH4OH solution for pH control,

throughout the fermentation. To avoid initial precipitation of medium components,

additional MgSO4·7H2O was mixed in the glucose feeding solution at 33 g l-1 based on

YX/Mg of 240 g g-1 (Sun et al. 2006/Chapter 3).


The inoculum was cultivated in two 500 ml shake flasks (150 ml medium each) at 30±1ºC

and 200 rpm for about 12 h. Fed-batch fermentations were carried out at 28±1ºC with 3.0

l initial working volume in a Minifors 5 l stirred tank bioreactor (Infors HT, Bottmingen,

Switzerland). Data acquisition (dissolved oxygen, outlet gas CO2 content, substrate and

base addition, and pH) and control was conducted with LabVIEW6.1 (National

Instruments). pH was controlled at 6.85±0.05. Dissolved oxygen was measured with an

Ingold polarographic probe and maintained at or above 40% air saturation (except where

indicated) by adjusting the agitation speed up to 1,200 rpm and by automatically adjusting

108

the mixture of air and pure oxygen flow via mass flow controllers while maintaining the

total gas flow at 1 vvm. Exit gas CO2 (%) was measured with an infrared CO2 monitor

(Guardian Plus, Topac Inc. Hingham, MA, USA) as direct indication of culture activity.

Feeding of nonanoic acid and glucose with peristaltic pumps was automatically and

separately controlled based on the mass of the reservoirs. Antifoam 204 (Sigma Aldrich)

was injected manually through a sterile septum whenever necessary.


A total of 1 g l-1 of nonanoic acid (NA) and glucose (G) was added to the initial medium.

The total carbon requirement was calculated according to the exponential growth

equation (Eq. 1).

( )/ /

1t 0t

X S X S

tX XS eY Y

µ⋅∆∆ = = ⋅ − (1)

Where / / /X S X NA NA X G GY Y r Y r= ⋅ + ⋅ (2)

The amount of nonanoic acid and glucose required were calculated separately

according to pre-defined ratio, and fed accordingly.

t NA t NAS S r−∆ = ∆ ⋅ (3)

t G t GS S r−∆ = ∆ ⋅ (4)

109

In the equations above, ΔSt (g) is the total carbon source required to produce biomass

ΔXt (g) at cultivation time t (h); YX/S, YX/NA, and YX/G are the yield of biomass from total

carbon, nonanoic acid, and glucose, respectively; X0 (g) is the initial biomass, obtained by

measuring the optical density of the inoculum; μ (h-1) is the desired specific growth rate;

ΔSt-NA (or ΔSt-G, g) and rNA (or rG, %) are the total nonanoic acid (or glucose) required at

cultivation time t and the proportion of the mass of nonanoic acid (or glucose) in the feed,

respectively.

Two fermentations with different nonanoic acid to glucose ratio were conducted in

this study. During the NA:G= 1:1 fermentation, at 24.9 h when the estimated cell

concentration was about 50 g l-1, the substrate feed rate (F, g l-1 h-1) was set at 17 g l-1 h-1

until the end of the fermentation (27.3 h). The NA:G= 1:1.5 fermentation was operated

using the exponential feeding strategy (Eq. 1) until 26.45 h.


Cell dry weight was determined with lyophilized biomass, which was obtained by

centrifugation of 5 ml culture broth at 10,000 x g for 15 min and washed with distilled

water. The supernatant of the centrifuged broth was saved for analysis of the major

nutrients, nonanoic acid (NA), and glucose (G). Phosphate, ammonium and glucose

concentrations were determined as described previously (Sun et al. 2006/Chapter 3). NA

concentration was determined by methylation reaction followed by GC analysis (Ramsay

110

et al. 1991), using benzoic acid as the internal standard. Samples for GC analysis of

cellular PHA content and composition were prepared as described previously (Sun et al.

2007a/Chapter 5). A gas chromatography (CP-3800, Varian Inc.) with FID was used in all

GC analysis. For PHA analysis the GC parameters were: injector temperature 250 ºC,

detector temperature 275 ºC, 1 μl injection, and a split ratio 10. The oven temperature

profile was: 90 ºC, 0.5 min, 6 ºC min-1 to 96 ºC, 7 ºC min-1 to 131 ºC, 20 ºC min-1 to 181

ºC, 5 min. The MCL-PHA standard for the GC analysis was prepared as described by

Jiang et al. (2006). The monomeric components were confirmed by GC-MS and

composition determined by nuclear magnetic resonance (NMR). The SCL-PHA standard

was Biopol, which was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (81 and 19 mol %

of HB and HV, respectively), originally purchased from Imperial Chemical Industries

(ICI). All analyses were done in duplicate with the average of the results presented in this

paper.

6.4 Results

6.4.1 Nonanoic acid and glucose co-feeding for MCL-PHA production

As previously reported (Sun et al. 2007a/Chapter 5), P. putida KT2440 is able to

synthesize substantial amount of MCL-PHA using nonanoic acid as the only carbon

source during carbon-limited, exponential growth, without nitrogen nor phosphorus

limitation. To lower the total substrate cost, nonanoic acid and glucose were used as

111

co-substrates first at a 1:1 (w/w) ratio ( NAr = Gr = 50%). Since YX/NA= 0.83 g g-1 and YX/G=

0.41 g g-1 (Sun et al. 2007a/Chapter 5; Sun et al. 2006/Chapter 3), YX/S was calculated as

0.62 g g-1 for the substrate feeding equation (Eq 1). X0 was set at 0.30 g (0.10 g l-1) based

on the amount of biomass measured in the inoculum. The desired specific growth rate

was 0.25 h-1. Ammonium and phosphate concentration was analysed and shown always in

excess during the fermentation (data not shown), similar to previous nonanoic acid

fed-batch studies (Sun et al. 2007a/Chapter 5). Nonanoic acid and glucose concentrations

confirm that growth was carbon limited throughout the fermentation (Figure 6-1).

Biomass first increased exponentially to 51 g l-1 due to exponential feeding, then

increased linearly up to 71 g l-1 due to linear feeding. The culture initially contained very

little PHA since the inoculum was grown on glucose and nutrient broth. The PHA content

increased to 56% at the end of cultivation. Very soon after the start of linear feeding,

dissolved oxygen dropped to zero although 1 vvm of pure oxygen was supplied. Feeding

was maintained for another two hours before excessive foaming terminated the

fermentation. This fermentation resulted in 39 g l-1 PHA, which gave a volumetric

productivity of 1.44 g PHA l-1 h-1, with total nonanoic acid and glucose consumption of

178 g and 177 g, respectively.

112

Figure 6-1 Single-stage MCL-PHA production by P. putida KT2440 using nonanoic acid (NA) and glucose (G) co-feeding strategy with NA:G= 1:1 (w/w). Exponential feeding stopped and linear feeding (constant feed rate of 17 g l-1 h-1) began at 24.9 h. Only DO data around the occurance of oxygen limitation (25 h) are shown, before which DO was always maintained around 40% air saturation.

To further increase the yield of PHA from nonanoic acid, the ratio of nonanoic acid

to glucose was decreased. At NA:G= 1:1.5 (w/w), the feeding equation values of rNA=

40% and rG= 60%, YX/S was lowered to 0.58 g g-1, again based on YX/NA= 0.83 g g-1 and

YX/G= 0.41 g g-1. X0 was set as 0.29 g (0.097 g l-1) based on the biomass of the inoculum

while the desired specific growth rate remained as 0.25 h-1. A slightly higher yield of

PHA from nonanoic acid was achieved (Figure 6-4b), while the final biomass, PHA

content, and PHA productivity were all lower than the NA:G= 1:1 fermentation (Figure

6-2). Key results of both fermentations are summarized in Table 6-1. Data for MCL-PHA

Culture time (h)

5 10 15 20 25 30Cel

l and

PH

A c

once

ntra

tion

(g l-1

) and

PH

A c

onte

nt (%

)

0

10

20

30

40

50

60

70

NA

and

G c

once

ntra

tion

(g l-1

)

0

2

4

6

8

10

Dis

solv

ed O

xyge

n (%

air

satu

ratio

n)

0

50

100

150

200

BiomassPHA% PHANAGDO

113

production from nonanoic acid with no glucose are taken from a previous study, at a

specific growth rate of 0.25 h-1 (Sun et al. 2007a/Chapter 5).

Figure 6-2 Single-stage MCL-PHA production by P. putida KT2440 using nonanoic acid (NA) and glucose (G) co-feeding strategy with NA:G= 1:1.5 (w/w). Exponential feeding was applied throughout the fermentation.

6.4.2 Monomeric compositions during co-substrate cultivation

Analysis of the NA:G= 1:1.5 fermentation showed that co-feeding this amount of glucose

had no significant effect on the monomeric composition (Figure 6-3b). The major

components were still 3-OH-nonanoate (about 65%), 3-OH-heptanoate (about 33%) and

3-OH-valerate (about 1.5%). There was only about 1% of 3-OH-octanoate,

3-OH-hexanoate and 3-OH-decanoate combined. A similar percentage of these C-even

repeating units were detected when only nonanoic acid was fed (Sun et al. 2007a/Chapter

Culture time (h)

5 10 15 20 25 30Cel

l and

PH

A c

once

ntra

tion

(g l-1

) and

PH

A c

onte

nt (%

)

0

10

20

30

40

50

60

70

NA

and

G c

once

ntra

tion

(g l-1

)

0

2

4

6

8

10

BiomassPHA% PHANAG

114

5). Therefore these were not likely derived from glucose. The ratio of C9 to C7 repeating

units was slightly increased by co-feeding glucose when compared to nonanoic acid alone

(from 1.90 mol mol-1 to 2.10 mol mol-1) (Figure 6-3a).

Figure 6-3 (a) 3-OH-nonanoate (C9) to 3-OH-heptanoate (C7) ratio from NA:G= 1:1.5 co-feeding fermentation and NA sole feeding fermentation. (b) PHA composition throughout the NA:G= 1:1.5 fermentation. C5, 3-OH-valerate; C7, 3-OH-heptanoate; C9, 3-OH-nonanoate; C-even, total of mainly 3-OH-hexanoate, 3-OH-octanoate and 3-OH-decanoate. m is the slope of the corresponding regression line.

6.4.3 Biomass and PHA Yield

When a simple exponential substrate feeding strategy, such as Eq. (1), is applied, it is

assumed that the biomass yield (YX/S) is accurate and constant. For the NA:G= 1:1 and

NA:G= 1:1.5 fermentations, the YX/C were calculated to be 0.62 g g-1 and 0.58 g g-1,

respectively, based on previous research. After plotting the total biomass versus total

carbon substrates consumption obtained from the two glucose-nonanoic acid co-feeding

fermentations (Figure 6-4a), it is clear that the yield was fairly constant throughout the

C7 molar concentration (mmol l-1)0 20 40 60 80 100

C9

mol

ar c

once

ntra

tion

(mm

ol l-1

)

0

20

40

60

80

100

120

140

160

180

NA:G=1:1.5m=2.10 mol mol-1

NA onlym=1.90 mol mol-1

Culture time (h)10 15 20 25

Mon

omer

ic c

ompo

sitio

n (m

ol %

)

0

20

40

60

80

100

C5C7C9C-even

(a) (b)

115

fermentation. The slopes of the regression curves are very close to what was predicted

based on YX/NA= 0.83 g g-1 and YX/G= 0.41 g g-1. Therefore, Eq. 2 proved to be a

reasonable way to estimate the biomass yield from the carbon substrates under

co-substrate conditions, when the yield from each single substrate has been

predetermined.

Total PHA accumulation at each sampling point was also plotted against cumulative

nonanoic acid consumption, using data from three fermentations (Figure 6-4b). Linear

regression was applied to each series of data and forced through origin, and the slope of

each regression curve was considered as the average nonanoic acid to PHA yield ( /average

PHA NAY

). Comparing the results from the NA only feeding fermentation and the NA:G= 1:1

fermentation, /average

PHA NAY increased by 25% (0.53 g g-1 to 0.66 g g-1). Further increase in

glucose feeding portion (NA:G= 1:1.5) resulted in slightly higher /average

PHA NAY , which was

0.69 g g-1 and about 30% increase from using nonanoic acid alone.

116

Figure 6-4 (a) Substrate to biomass yield. (b) nonanoic acid (NA) to PHA yield plots. m is the slope of the corresponding linear regression line. All linear regression curves were forced through origin.

NA consumption (g)

0 50100

150200

250

PH

A a

ccum

ulat

ion

(g)

0

20

40

60

80

100

120

140NA onlym=0.53 g g-1

NA:G=1:1m=0.66 g g-1

NA:G=1:1.5m=0.69 g g-1

Total carbon substrates consumption (g)

0100

200300

400

Tota

l bio

mas

s (g

)

0

50

100

150

200

250NA:G=1:1m=0.63 g g-1

NA:G=1:1.5m=0.59 g g-1

(a) (b)

117

Table 6-1 Summary of fermentation results.

Fermentation Final X (g l-1)

Final PHA (g l-1)

Final PHA content (%)

PHA overall productivity (g l-1 h-1)

Total NA consumption (g)

Total G consumption (g)

Average YPHA/NA (g g-1)

Final Overall YPHA/NA (g g-1)

Reference

NA only 56.0 37.4 67 1.44 231 0 0.53 0.55 Sun et al. 2007a

NA:G=1:1 70.7 39.4 56 1.44 178 177 0.66 0.69 This work

NA:G=1:1.5 64.2 30.8 48 1.16 137 217 0.69 0.71 This work

Table 6-2 Summary of literature MCL-PHA production and yield

Micro- organism

Fermentation Notes X (g l-1)



Major YPHA/C (g g-1)

Major substrate Supplementary substrate

Reference

P. putida IFO14464 Two-stage batch

2nd stage N limited 4.8 18 0.03 0.17 Octanoate N/A (HORI et al. 1994)

P. putida BM01 Two-stage fed-batch

Octanoate as sole substrate 41.7 60 0.71 0.28 Octanoate N/A (Kim et al. 1997)

P. putida BM01 Two-stage fed-batch

1st stage glucose, 2nd stage octanoate, 2nd stage N limited

54.8 65.5 0.92 0.40 Octanoate Glucose (Kim et al. 1997)

P. putida IPT046 Two-stage fed-batch

2nd stage P limited 50 63 0.80 0.19 Glucose

Fructose (Diniz et al. 2004)

P. putida GPo1 Two-stage continuous

2nd-stage N limited, estimated yield

18 63 1.06 0.63 Octane N/A (Hazenberg and Witholt 1997)

P. putida KT2442 Continuous N limited, estimated yield 30 23 0.69 0.15 Oleic acid N/A (Huijberts and Eggink 1996)

P. putida KT2440 Single-stage C limited 56 67 1.44 0.55 Nonanoic acid N/A (Sun et al. 2007a)

P. putida KT2440 Single-stage C limited, co-feeding 71 56 1.44 0.69 Nonanoic acid Glucose This work

118

6.5 Disucssion

6.5.1 High yield and PHA productivity by co-substrate carbon-limited fermentation

Based on computer simulation of industrial scale PHA production processes, substrate

accounts for about 30% of the total MCL-PHA production cost if an MCL carboxylic acid

such as octanoate is used (deKoning et al. 1997) and, at least in the case of SCL-PHA,

this increases as the scale of production increases (Choi and Lee, 1997). Therefore, it is

highly desirable to use a low cost carbon source, and/or increase the carbon substrate to

PHA yield for more economical MCL-PHA production. The present study demonstrates

that these objectives can be achieved by using glucose to supplement nonanoic acid

feeding in single-stage carbon-limited fed-batch MCL-PHA fermentations. Assuming the

product contains 65 molar percent of 3-OH-nanonoate and 35 percent of

3-OH-heptanoate, the maximum yield of MCL-PHA from nonanoic acid is 0.93 g g-1.

Using nonanoic acid as the sole substrate, 56 g l-1 biomass containing 67% MCL-PHA

could be obtained by the carbon-limited single-stage fed-batch fermentation (Sun et al.

2007a/Chapter 5). However, the /average

PHA NAY was only 0.53 g g-1 (Figure 6-4b), as a large

portion of the nonanoic acid was used for synthesis of residual biomass (total biomass

minus PHA). As the NA:G= 1:1 fermentation results demonstrated (Figure 6-1), nonanoic

acid and glucose could be consumed simultaneously by P. putida, and the /average

PHA NAY was

enhanced by 25% (0.53 to 0.66 g g-1) by the co-feeding of glucose. An important

119

difference between NA+G co-feeding during a nitrogen-limited second stage (the

standard method of producing PHA) and co-feeding during exponential growth, is the

effect of glucose on the PHA composition. While during the nitrogen-limited stage

glucose yielded 3-OH-decanoate and 3-OH-octanoate accounting for 10 molar percent of

the total PHA synthesized (Sun et al. 2007c/Chapter 4), it yielded an insignificant amount

of C-even (i.e. glucose derived) repeating units during C-limited exponential fed batch.

Similar results have been reported in other studies (Diniz et al. 2004; Kim et al. 1996; Sun

et al. 2007a/Chapter 5), where little PHA could be synthesized during the growth phase of

various strains of P. putida when glucose was the sole substrate. Therefore, when the

second energy source (glucose) was supplied, the β-oxidation pathway, which is the

energy generating pathway when fatty acid was sole substrate, was inhibited, leading to

more nonanoic acid being diverted to MCL-PHA synthesis. As a consequence, the C9 to

C7 ratio during the co-feeding also slightly increased when compared to the ratio

obtained when only NA is fed (Figure 6-3a).

When the nonanoic acid to glucose ratio was further decreased (NA:G= 1:1.5

fermentation), it was hoped that glucose would provide enough carbon source for all Xr

production, leading to the theoretical maximum nonanoic acid to PHA conversion (0.93 g

PHA g-1 NA). However, the /average

PHA NAY increased only slightly (0.69 g g-1, Figure 6-4b),

and only 47% PHA was accumulated at the end of the fermentation. Apparently, simply

120

supplying glucose as the second substrate cannot completely inhibit β-oxidation and

allow the diversion of all nonanoic acid to PHA. The decreased nonanoic acid to glucose

ratio likely resulted in an insufficient supply of nonanoic acid for PHA synthesis,

probably due to a constant flux of acetyl-CoA from β-oxidation toward cell growth, and

led to a lower final PHA content and lower final PHA productivity (Table 6-1). Thus, the

NA:G= 1:1 co-substrate ratio resulted in better production of MCL-PHA with similar

composition (in terms of C9 and C7 molar portion among the total) and productivity (1.44

g PHA l-1 h-1) when compared to fermentations using a only nonanoic acid (Sun et al.

2007a/Chapter 5), but with significant enhancement in /average

PHA NAY .

The purpose of linear (i.e. constant rate) feeding after biomass reached above 50 g l-1

during the NA:G= 1:1 fermentation was to avoid oxygen limitation and achieve higher

biomass, hence the rate of oxygen consumption should be constant when the rate of

biomass synthesis (dX/dt) was constant. Indeed, a slightly higher final biomass (71 g l-1

versus 64 g l-1) was achieved when the result of this fermentation is compared to those

fermentations where only exponential feeding (NA feeding and NA:G= 1:1.5 feeding

fermentations) was employed. However, in all cases, oxygen limitation eventually led to

foaming resulting in the termination of the fermentation. Foaming seems to be less of a

problem for MCL-PHA fermentations using oleic acid (Lee et al. 2000), probably due to

the antifoaming properties of oleic acid.

121

6.5.2 Yield potential of MCL-PHA synthesis

MCL-PHA synthesis yield data from the literature are summarized in Table 6-2. Clearly,

single-stage, carbon-limited co-substrate fermentation resulted in the highest substrate to

PHA yield. If /overall

PHA NAY is defined as PHA synthesized per gram nonanoic acid consumed

at a given point of the fermentation, the relation between /overall

PHA NAY and PHA content

during fed-batch fermentations may be studied as in Figure 6-5. It is obvious that the

/overall

PHA NAY increases with PHA content during a fermentation. To study the theoretical

maximum /overall

PHA NAY , the experimental /overall

PHA NAY data in Figure 6-5 are extrapolated with

respect to the PHA content. For nonanoic acid, single substrate feeding, the maximum

yield (0.93 g PHA g-1 NA) can be achieved only at 100% PHA content, while for NA+G

co-feeding cultivation the maximum yield can theoretically be achieved at 70% PHA,

which should be much easier to obtain. A similar relation between overall PHA yield and

the PHA content was calculated by Hazenberg and Witholt (1997). The above analysis, as

well as the simplicity of the process itself, indicates that this NA+G co-feeding process is

the most practical way to achieve cost-effective MCL-PHA production.


Research Council of Canada (NSERC) and BIOCAP Canada.

122

Figure 6-5 The relationship between PHA content and the cumulative nonanoic acid to PHA yield (

/overall

PHA NAY ). /overall

PHA NAY is the cumulative PHA yield from nonanoic acid at a given sampling point.

6.6 References

Chen GQ, Zhang G, Park SJ, Lee SY (2001) Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Appl Microbiol Biotechnol 57:50-55

Choi JI, Lee SY (1997) Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng 17:335-342

deKoning GJM, Kellerhals M, vanMeurs C, Witholt B (1997) A process for the recovery of poly(hydroxyalkanoates) from pseudomonads. 2. process development and economic evaluation. Bioprocess Eng 17:15-21


PHA content (%)

0 20 40 60 80 100

Yie

ldov

eral

l PH

A/N

A (g

g-1

)

0.0

.2

.4

.6

.8

1.0

NA:G=1:1.5NA:G=1:1NA only

123


Hazenberg W, Witholt B (1997) Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: Economic considerations. Appl Microbiol Biotechnol 48:588-596

Hori K, Soga K, Doi Y (1994) Effects of culture conditions on molecular-weights of poly(3-hydroxyalkanoates) produced by Pseudomonas putida from octanoate. Biotechnol Lett 16:709-714

Huijberts GNM, and Eggink G (1996) Production of poly(3-hydroxyalkanoates) by Pseudomonas putida KT2442 in continuous cultures. Appl Microbiol Biotechnol 46:233-239

Jiang X, Ramsay JA, Ramsay BA (2006) Acetone extraction of mcl-PHA from Pseudomonas putida KT2440. J Microbiol Methods 67:212-219

Kim BS (2002) Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans Biotechnol Lett 24:125-130

Kim GJ, Lee IY, Choi DK, Yoon SC, Park YH (1996) High cell density cultivation of Pseudomonas putida BM01 using glucose. Journal of Microbiology and Biotechnology 6:221-224


Lee SY, Choi JI ,Wong HH (1999) Recent advances in polyhydroxyalkanoate production by bacterial fermentation: Mini-review. Int J Biol Macromol 25:31-36



124


Sun Z, Ramsay JA, Guay M, Ramsay BA (2007a) Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440. Appl Microbiol Biotechnol 74:69-77

Sun Z, Ramsay JA, Guay M, Ramsay BA (2007b) Fermentation process development for the production of medium-chain-length poly-3-hydroxyalkanoates. Appl Microbiol Biotechnol 75:475-485

Sun Z, Ramsay JA, Guay M, Ramsay BA (2007c) Increasing the yield of MCL-PHA from nonanoic acid by co-feeding glucuose during the PHA accumulation stage in two-stage fed-batch fermentations of Pseudomonas putida KT2440. J Bacteriol DOI:101016/jbiotec200702023

125

CHAPTER 7

Fed-batch Production of Unsaturated

Medium-chain-length Polyhydroxyalkanoates with

Controlled Composition by

Pseudomonas putida KT2440


Originally submitted June, 2007 to Applied Microbiology and Biotechnology

126

7.1 Abstract

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at

a productivity range of 0.63-1.09 g PHA l-1 h-1 with final PHA content ranging from 42.6%

to 55.8%, by single-stage, carbon-limited fed-batch fermentations of Pseudomonas putida

KT2440, using exponential feeding of nonanoic acid (NA) and 10-undecenoic acid

(UDA=) mixture. Different specific growth rate (0.14 h-1 vs. 0.23 h-1) with similar

substrate composition (NA:UDA== 5:1) had little effect on the final achievable PHA

content and relative composition. Co-feeding of the two carbon sources resulted in PHA

content plateau (about 55% when NA:UDA== 5.07:1, and 42% when NA:UDA== 2.47:1),

which was not found from previous nonanoic acid only fermentations. The relative molar

fraction of all 3-OH-alkanoates of the PHA product was fairly constant throughout each

fermentation. Linear relationship between the unsaturation in PHA product and the

unsaturated carbon source fraction in the feed was demonstrated, which makes it possible

to produce unsaturated MCL-PHAs with controlled polymeric compositions.

7.2 Introduction

Medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHAs) have attracted extensive

interest, not only because of their biocompatibility and biodegradability, but also their

diversity. The large variability of chemical structure and material properties (Hazer and

127

Steinbüchel 2007) make them candidates for many applications, such as drug delivery

(Pouton and Akhtar 1996) and tissue engineering (Chen and Wu 2005). Most MCL-PHAs

consist solely of repeating units of 3-hydroxyalkanoates, but MCL-PHAs containing

functional groups in the side chain, often referred to as functionalized PHAs (Hazer and

Steinbüchel 2007), can also be synthesized. These functional groups, such as

carbon-carbon double bonds (Fritzsche et al. 1990), triple bonds (Kim et al. 1998),

halogens (Kim et al. 1996), and phenyl groups (Hazer et al. 1996), are usually

incorporated by appropriate microorganisms grown on suitable carbon source(s)

containing the corresponding functional group(s). To date, however, few such

functionalized PHAs have been produced at even a gram scale possibly due to the toxicity

of the functional carbon substrates, inefficiency in substrate utilization by the cultures

employed, and/or unavailability of such substrates, as many of them require dedicated

chemical synthesis. Among various functionalized PHAs, those containing olefinic

groups in the side chains, i.e. unsaturated PHAs, are likely one of the most useful

functional PHAs (Hazer and Steinbüchel 2007) due to the possibility of chemical

modifications on or via the unsaturated sites, such as chlorination (Arkin et al. 2000),

cross-linking (Dufresne et al. 2001), carboxylation (Kurth et al. 2002) and epoxidation

(Park et al. 1998). Unsaturated PHAs are typically soft, sticky materials. Since the

melting and the glass transition temperature vary according to the fraction of unsaturated

components (Kim et al. 1995), and since different applications have different

128

requirements, the degree of unsaturation needs to be finely controlled in order to obtain

materials with desired properties.

Most studies on the synthesis of MCL-PHA employed strains of Pseudomonas (Sun

et al. 2007b/Chapter 2). Edible oils (Ashby and Foglia 1998) and carbohydrates (Sanchez

et al. 2003) have been shown to support the synthesis of MCL-PHAs with unsaturated

fractions, but the degree of unsaturation is difficult to be controlled using those carbon

sources. However, when mixtures of saturated and unsaturated aliphatic acids were used

as the carbon sources, a clear relationship between fraction of the unsaturated carbon

source in the medium and the unsaturated fraction in the PHA was demonstrated (Kim et

al. 1995; Kellerhals et al. 1999; Hartmann et al. 2006). Unfortunately, the highest

volumetric productivity of unsaturated PHAs reported was only 0.20 g PHA l-1 h-1

(Kellerhals et al. 1999, Table 7-1), which is much less than that of saturated MCL-PHAs

(Sun et al. 2007b/Chapter 2).

We have recently reported efficient MCL-PHA production by single-stage,

carbon-limited, fed-batch fermentation using a nonanoic acid exponential feeding strategy

(Sun et al. 2007a/Chapter 5). It was clearly demonstrated that nitrogen limitation was not

necessary for MCL-PHA synthesis in Pseudomonas putida KT2440. In the current study,

such a fed-batch technique was applied to the production of unsaturated MCL-PHAs and

129

to control the monomeric composition. The effect of specific growth rate on the PHA

composition was also investigated.



Pseudomonas putida KT2440 (ATCC 47054) was maintained in lyophiles and on nutrient

agar plates at 4 ºC. For all fermentations, 9 g l-1 of glucose plus 1 g l-1 of nutrient broth

was used as the carbon sources in the inoculum medium. The initial fermentation medium

contained about 0.66 g l-1 of the mixture of nonanoic acid (NA, 98%, Spectrum) and

10-undecenoic acid (UDA=, 97%, Sigma Aldrich) with a desired molar ratio (specified in

the results section). Such substrate mixture was prepared prior to sterilization. Other

medium components are reported by Sun et al. (2007a/Chapter 5). Nitrogen was supplied

by using a 14% (w/v) NH4OH solution for pH control. To avoid initial precipitation of

medium components, additional MgSO4·7H2O was fed at a ratio of 0.033 g MgSO4·7H2O

g-1 carbon mixture added, assuming a YX/Mg of 240 g g-1 (Sun et al. 2006/Chapter 3) and a

YX/S of 0.80 g g-1.


Inocula were cultivated in 500-ml shake flasks (150 ml medium) at 30±1ºC and 200 rpm

for about 12 h. Fed-batch fermentations were carried out at 28±1ºC with 3.0 l initial

working volume in a Minifors 5 l stirred tank bioreactor (Infors HT, Bottmingen,

130

Switzerland). Data acquisition, pH control, dissolved oxygen control, and exit gas CO2

measurement were previously described (Sun et al. 2007a/Chapter 5). Substrate feeding

using peristaltic pumps was automatically controlled based on the mass of the reservoir

containing the substrate mixture. Antifoam 204 (Sigma Aldrich) was added drop wise

manually through a sterile septa using an injection needle whenever required.


Three carbon-limited fed-batch fermentations, each at a controlled specific growth rate

(μ), were carried out with different molar ratios of nonanoic acid to undecenoic acid as

follows: (a) NA:UDA== 4.98:1 (molar ratio) and μ= 0.15 h-1; (b) NA:UDA == 5.07:1 and

μ= 0.25 h-1; (c) NA:UDA== 2.47:1 and μ= 0.25 h-1. The initial substrate concentration was

about 0.66 g l-1. To achieve the desired specific growth rate, substrate was then fed

continuously to the culture according to the feed Eq. (1):

( )0

/ /

1tt

X C X C

tX XS eY Y

µ⋅∆∆ = = ⋅ − (1)

Where ΔSt (g) is the total substrate required to produce biomass ΔXt (g) at cultivation

time t (h); YX/C is the yield of biomass from substrate, assumed to be constant at 0.80 g g-1

based on a previous study (Sun et al. 2007a/Chapter 5); X0 (g) is the initial biomass,

obtained by measuring the optical density of the inoculum; and μ (h-1) is the desired

specific growth rate.

131


Cell dry weight of the lyophilized biomass was determined after centrifugation of 5 ml

culture broth at 10,000 x g for 15 min and washing with distilled water. The supernatant

of the centrifuged broth was saved for analysis of nonanoic acid (NA), undecenoic acid

(UDA=), and other major nutrients. Phosphate and ammonium concentration were

determined colorimetrically (Sun et al. 2007a/Chapter 5). Nonanoic acid and undecenoic

acid concentration were determined by methylation followed by GC analysis (Ramsay et

al. 1991), using benzoic acid as the internal standard. The procedures of methylation

reaction for GC analysis of the cellular PHA content and composition were described in

Sun et al. (2007a/Chapter 5). A gas chromatography (CP-3800, Varian Inc.) equipped with

flame-ionization-detector was used in all GC analysis. The GC parameters were: injector

temperature 250 ºC, detector temperature 275 ºC, 1 μl injection, split ratio 10. The oven

temperature profile was: 90 ºC, 0.5 min, 6 ºC min-1 to 96 ºC, 7 ºC min-1 to 131 ºC, 20 ºC

min-1 to 181 ºC, 5 min. The MCL-PHA standard for the GC analysis was prepared as

described by Jiang et al. (2006). The monomeric components were confirmed by GC-MS

and composition determined by nuclear magnetic resonance (NMR). The SCL-PHA

standard was Biopol, which was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (81 and

19 mol % of HB and HV, respectively), originally purchased from Imperial Chemical

Industries (ICI). All analysis was done in duplicates with the average of the results

presented in this paper.

132

7.4 Results

7.4.1 Production of unsaturated MCL-PHAs by exponential feeding of nonanoic acid and 10-undecenoic acid

A fed-batch fermentation (fermentation a) was conducted by exponentially feeding

nonanoic acid (NA) and undecenoic acid (UDA=) at a molar ratio of 4.98:1. The desired μ

was set at 0.15 h-1 in the feed equation (Eq. 1). As seen in Figure 7-1, the overall biomass

increased exponentially, and the actual μ was determined to be 0.14 h-1 by nonlinear curve

fitting (dashed line in Figure 7-1). Shortly after inoculation, the culture became

carbon-limited, and remained carbon-limited throughout the balance of the fermentation

until the 42 h point. At this time, cell growth became limited by oxygen, causing both

nonanoic acid and undecenoic acid to accumulate rapidly in the fermentation broth. The

other major nutrients, ammonium and phosphorus, were kept at concentrations very

similar to those of a previous study (Sun et al. 2007a/Chapter 5). The inoculum culture

(grown mainly on glucose) contained negligible PHA, but the PHA content increased to

55.6% of cell dry weight at the end of the fermentation, giving a cumulative unsaturated

MCL-PHA productivity of 0.71 g PHA l-1 h-1 (Table 7-1). The PHA content achieved a

plateau when the cell concentration reached above 20 g l-1.

133

Figure 7-1 Fermentation a, exponential feeding of nonanoic acid (NA) and 10-undecenoic acid (UDA=) (molar ratio 4.98:1) with desired μ= 0.15 h-1. The dissolved oxygen data are shown after 30 h, before when it was kept around 40% air saturation. Curve fitting (dashed line) was done by nonlinear regression function of Sigmaplot 9.01.

Fermentations with higher desired μ (0.25 h-1) were also conducted with different

nonanoic acid to undecenoic acid ratio (fermentations b and c, with NA:UDA== 5.07:1

and 2.47:1, respectively) (Figure 7-2), to determine the effect of growth rate and substrate

mixture on the PHA synthesis. The cell growth curves from both fermentations were very

similar, indicating that the ratio of nonanoic acid and undecenoic acid had little effect on

biomass yield. Using a similar nonanoic acid to undecenoic acid ratio (fermentation b),

the PHA content also plateaued at around 55%, very similar to that of the μ= 0.14 h-1

fermentation, despite the much higher specific growth rate (0.23 h-1 vs. 0.14 h-1). The

PHA overall productivity also increased to 1.09 g PHA l-1 h-1 due to the shortened

Culture time (h)

0 10 20 30 40 50

Biom

ass

(g l-1

), PH

A (g

l-1) a

nd P

HA

cont

ent (

%)

0

10

20

30

40

50

60

NA

and

UD

A= con

cent

ratio

n (g

l-1)

0

2

4

6

8

10

Dis

solv

e ox

ygen

(% a

ir sa

tura

tion)

0

50

100

150

200Biomass% PHA PHANAUND=

Curve fitDO %

134

fermentation time. However in fermentation c, the PHA content achieved the plateau only

at around 42%. The final results of the three fermentations are summarized in Table 7-1,

with some literature results as comparisons.

Figure 7-2 Biomass, PHA concentration and PHA content of fermentations b and c, with the same μ (0.25 h-1) and different NA to UDA= ratio (5.07:1 and 2.47:1, respectively).

Table 7-1 Fermentation results summary.

Fermentation Actual NA:UDA=

(molar ratio)

Actual μ (h-1)

Final X (g l-1)

Final PHA (g l-1)



Reference

(a) 4.98:1 0.14 54.10 30.07 55.59 0.71 This work

(b) 5.07:1 0.23 48.12 26.86 55.82 1.09 This work

(c) 2.47:1 0.24 33.58 14.31 42.61 0.63 This work Closed-loop fed-batch

Varied Varied 30 10 33 0.20 Kellerhals et al.

(1999)

Continuous 1:1 0.1-0.3 1.11-1.44 0.13-0.47 12-37 0.04-0.07 Hartmann et al.

(2006)

Culture time (h)

5 10 15 20 25

Bio

mas

s (g

l-1),

PH

A (g

l-1) a

nd P

HA

con

tent

(%)

0

10

20

30

40

50

60

Biomass (5.07:1)Biomass (2.47:1)% PHA (5.07:1) % PHA (2.47:1) PHA (5.07:1) PHA (2.47:1)

135

7.4.2 Monomeric compositions

Throughout the fermentations, MCL-PHA containing various 3-OH-alkanoates with

saturated or unsaturated side chains was synthesized. The relative molar fraction of

saturated and unsaturated components remained fairly stable throughout the cultivation

(Figure 7-3b, 3c). The approximate molar fractions of all detectable components are

shown in Table 7-2. Despite the difference in nonanoic acid to undecenoic acid ratio in

the feed or the actual cell growth rate, 3-OH-nonanoate was always the most abundant

among the saturated components, while 3-OH-nonenoate was the most abundant among

the unsaturated components. Interestingly, even carbon number 3-OH-alkanoates

(3-OH-octanoate, 3-OH-decanoate, 3-OH-hexanoate, and 3-OH-butyrate) with a total of

about 1 mol % were also detected, but only later in the fermentations.

Table 7-2 Molar fraction of all 3-OH-alkanoates detected in the MCL-PHA product from different fermentations.

Fermentation Actual NA:UDA=

(molar ratio)

C5 C7 C9 C11 C7:1 C9:1 C11:1 C-even Saturated: unsaturated (molar ratio)

(a), μ= 0.14 h-1 4.98:1 1.5 29.5 53.0 0.5 1.0 7.2 6.3 1.0 5.82

(b), μ= 0.23 h-1 5.07:1 1.6 30.0 52.5 0.5 1.0 7.2 6.3 0.9 5.73

(c), μ= 0.24 h-1 2.47:1 3.0 25.0 44.5 0.6 2.2 13.3 10.8 0.6 2.76

C5, 3-OH-valerate, C7, 3-OH-heptanoate, C7:1, 3-OH-heptenoate, C9, 3-OH-nonanoate, C9:1, 3-OH-nonenoate, C11, 3-OH-undecanoate, C11:1, 3-OH-undecenoate, C-even, total of 3-OH-octanoate, 3-OH-decanoate, 3-OH-hexanoate, and 3-OH-butyrate (descending in relative fration).

Since the fractions of all components were relatively constant, the total molar amount

of saturated repeating units versus the total molar of unsaturated ones from three

fermentations were plotted in Figure 7-3a. The small amount of even carbon number

136

3-OH-alkanoates was not included in the saturated vs. unsaturated plots. The linear

relationships demonstrate that the ratio of saturated to unsaturated components of the

MCL-PHA remained constant throughout each fermentation (Table 7-2). They were

always slightly higher than the corresponding ratios of nonanoic acid to undecenoic acid

in the feed.

Figure 7-3 (a) Linear relationship between total amount of saturated components and total amount of unsaturated components from three fermentations. (b) Relative monomeric composition of the MCL-PHA synthesized during fermentation b. (c) Relative monomeric composition of the MCL-PHA synthesized during fermentation c. The dash-dot line in (b) and (c) divides saturated and unsaturated components. C5, 3-OH-valerate, C7, 3-OH-heptanoate, C7:1, 3-OH-heptenoate, C9, 3-OH-nonanoate, C9:1, 3-OH-nonenoate, C11, 3-OH-undecanoate, C11:1, 3-OH-undecenoate. The small amount of even carbon number 3-OH-alkanoates is not included these plots.

PH

A m

onom

eric

com

posi

tion

(mol

%)

0%

20%

40%

60%

80%

100%

Culture time (h)

5 10 15 20 250%

20%

40%

60%

80%

100%

C5C7 C9 C11 C7:1 C9:1 C11:1

Total of unsaturated components (mmol)

0 5 10 15 20 25 30 35

Tota

l of s

atur

ated

com

pone

nts

(mm

ol)

0

20

40

60

80

100

120

140

160

1804.98:1 at µ=0.14 h-1

Slope=5.82

5.07:1 at µ=0.23 h-1

Slope=5.73

2.47:1 at µ=0.24 h-1

Slope=2.76

(a)

(b)

(c)

137

7.5 Discussion

7.5.1 Process design for unsaturated MCL-PHAs production

Although a highly promising material, unsaturated MCL-PHAs have not been produced at

a scale comparable to saturated MCL-PHAs; and this could hamper the development of

applications for these biopolyesters. In a closed-loop fed-batch process, unsaturated

MCL-PHAs were produced up to 33% of the dry biomass (0.20 g PHAs l-1 h-1) by

co-feeding of Na-octanoate and Na-undecenoate (Kellerhals et al. 1999) (Table 7-1).

Since feeding of the two substrates was controlled individually, the unsaturated fraction

varied throughout the cultivation, which is not ideal for the production of such

MCL-PHAs with controlled properties. In another study (Hartmann et al. 2006),

chemostat cultivation was found to be a suitable method to produce unsaturated

MCL-PHAs with defined monomeric composition, while batch processes resulted in a

slight change in the composition throughout the cultivation. However, the PHA

productivity of such chemostat process was only within the 0.04-0.07 g PHA l-1 h-1 range.

Using a single stage, exponential feeding fed-batch fermentation process (Sun et al.

2007a/Chapter 5), we produced unsaturated MCL-PHAs at a productivity range of

0.63-1.09 g PHA l-1 h-1 with a final PHA content ranging from 42.6% to 55.8%, by

feeding a mixture of saturated alkanoic acid (nonanoic acid), and unsaturated alkanoic

acid (10-undecenoic acid). Both carbon sources are among the most common substrates

138

used to produce unsaturated MCL-PHAs (Sun et al. 2007b/Chapter 2), and are relatively

inexpensive compared to other aliphatic carbon sources. Furthermore, these substrates

mix very well with each other and are all miscible in the aqueous media, making the

sterilization and feeding process much easier. The productivity and final PHA content are

much higher than what has been reported in the literature, and the process does not

involve many manual operations or expensive instrumentation. These features render this

process relatively attractive for industrial application.

7.5.2 Effect of specific growth rate and varied substrate composition on PHA synthesis

Under very different specific growth rate (0.14 h-1 vs. 0.23 h-1) with similar substrate

composition (NA:UDA= of 4.98:1 vs. 5.07:1), the final achievable PHA content and

relative composition show little significant difference (Table 7-2).

However, the increasing fraction of unsaturated carbon substrate (undecenoic acid in

this case) seems to have big impact on the final achievable PHA content. Reviewing the

results of our previous report on saturated MCL-PHA production by nonanoic acid alone

(Sun et al. 2007a/Chapter 5), the PHA content did not stop increasing before oxygen

transfer limitation was encountered. It achieved 67% at the end of a µ= 0.25 h-1

fermentation and can potentially reach as high as 75% if there was sufficient oxygen

transfer. However, during fermentation b of this study, when undecenoic acid was about

139

17% molar fraction in the substrate, a plateau of about 55% was attained before the end of

the cultivation. When undecenoic acid fraction was further increased to 28% in

fermentation c, the plateau occurred at only about 42%. This indicates that undecenoic

acid is not a substrate as favorable as nonanoic acid to P. putida KT2440 for PHA

synthesis, and maybe also inhibit cell growth. Similar observations were also reported by

Kim et al. (1995).

7.5.3 Unsaturated MCL-PHAs with controllable compositions

As demonstrated in Figure 7-3, the ratio of saturated to unsaturated components of the

PHAs stayed fairly constant throughout the fermentation, regardless of specific growth

rate or substrate composition. It had been concluded (Hartmann et al. 2006) that batch

cultivation was not ideal for achieving desired monomeric composition as a slight change

over time was observed. This may have been due to unbalanced growth in batch

cultivation, as the concentration of nutrients change with time. In fed-batch cultivation,

such as what we presented in this study, a more balance growth and PHA synthesis is

more likely to occur, which leads to a constant composition of the resulting PHAs.

When plotting the ratio of saturated to unsaturated components versus the ratio of

saturated to unsaturated carbon source from two fermentations (b and c, Table 7-2), a

straight line through the origin can be generated (r2= 0.99) (Figure 7-4). The slightly

higher ratio of saturated components (compared to the ratio of saturated substrate in the

140

feed) indicates that nonanoic acid is slightly more favorable for PHA synthesis. This

curve may be used to predict the monomeric composition during production of

unsaturated MCL-PHAs. Since this ratio is very constant during the entire cultivation,

unsaturated MCL-PHAs with a desired monomeric composition may be produced using

P. putida KT2440 by adjusting the composition of the substrate according to Figure 7-4.

The current process can also be considered as a model process, and likely be applied to

produce other types of functionalized MCL-PHAs on a larger scale than those that have

been previously reported.

Figure 7-4 Relationship between unsaturation of the MCL-PHA product and the substrate for fermentations at desired µ= 0.25h-1 (fermentations b and c). The r2 is 0.99 for the regression curve.


Research Council of Canada (NSERC).

Sat/Unsat ratio in the substrate

0 1 2 3 4 5 6

Sat

/Uns

at ra

tio in

PH

A

0

1

2

3

4

5

6

141

7.6 References

Arkin AH, Hazer B, Borcakli M (2000) Chlorination of poly(3-hydroxyalkanoates) containing unsaturated side chains. Macromolecules 33:3219-3223

Ashby RD, Foglia TA (1998) Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates. Appl Microbiol Biotechnol 49:431-437


Dufresne A, Reche L, Marchessault RH, Lacroix M (2001) Gamma-ray crosslinking of poly (3-hydroxyoctanoate-co-undecenoate). Int J Biol Macromol 29:73-82

Fritzsche K, Lenz RW, Fuller RC (1990) Production of unsaturated polyesters by Pseudomonas oleovorans. Int J Biol Macromol 12:85-91


Hazer B, Lenz RW, Fuller RC (1996) Bacterial production of poly-3-hydroxyalkanoates containing arylalkyl substituent groups. Polymer 37:5951-5957

Hazer B, Steinbüchel A (2007) Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications. Appl Microbiol Biotechnol 74:1-12

Jiang X, Ramsay JA, Ramsay BA (2006) Acetone extraction of mcl-PHA from Pseudomonas putida KT2440. J Microbiol Methods 67:212-219

Kellerhals MB, Kessler B, Witholt B (1999) Closed-loop control of bacterial high-cell-density fed-batch cultures: Production of mcl-PHAs by Pseudomonas putida KT2442 under single-substrate and cofeeding conditions. Biotechnol Bioeng 65:306-315

Kim DY, Kim YB, Rhee YH (1998) Bacterial poly(3-hydroxyalkanoates) bearing carbon-carbon triple bonds. Macromolecules 31:4760-4763

Kim O, Gross RA, Hammar WJ, Newmark RA (1996) Microbial synthesis of poly(beta-hydroxyalkanoates) containing fluorinated side-chain substituents. Macromolecules 29:4572-4581

142

Kim YB, Lenz RW, Fuller RC (1995) Poly-3-hydroxyalkanoates containing unsaturated repeating units produced by Pseudomonas oleovorans. J Polym Sci Pol Chem 33:1367-1374

Kurth N, Renard E, Brachet F, Robic D, Guerin P, Bourbouze R (2002) Poly(3-hydroxyoctanoate) containing pendant carboxylic groups for the preparation of nanoparticles aimed at drug transport and release. Polymer 43:1095-1101

Park WH, Lenz RW, Goodwin S (1998) Epoxidation of bacterial polyesters with unsaturated side chains. I. Production and epoxidation of polyesters from 10-undecenoic acid. Macromolecules 31:1480-1486



Sanchez RJ, Schripsema J, da Silva LF, Taciro MK, Pradella JGC, Gomez JGC (2003) Medium-chain-length polyhydroxyalkanoic acids (PHA(mcl)) produced by Pseudomonas putida IPT 046 from renewable sources. Eur Polym J 39:1385-1394

Sun Z, Ramsay JA, Guay M, Ramsay BA (2007a) Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440. Appl Microbiol Biotechnol 74:69-77

Sun Z, Ramsay JA, Guay M, Ramsay BA (2007b) Fermentation process development for the production of medium-chain-length poly-3-hydroxyalkanoates. Appl Microbiol Biotechnol 75: 475-485


143

CHAPTER 8 Conclusions

8.1 Summary and contributions

8.1.1 High-cell-density cultivation of P. putida KT2440

High-cell-density is often required to improve biomass and product formation during

microbial fermentations. With industrial applications in mind, such fermentation

processes should be automated, easily controlled, and have high biomass productivity.

The fermentation processes developed for P. putida KT2440 cultivation using four

different automated substrate feeding strategies (Chapter 3) fulfill all these requirements.

The highest biomass productivity (4.3 g X l-1 h-1) was achieved by continuous feeding

based on real-time glucose consumption estimation, since during such cultivation the

substrate requirements of the culture can be continuously and fully satisfied. This

substrate feeding strategy is ideal for the growth stage (first stage) of a conventional

two-stage PHA production process in order to achieve high productivity with a short

cultivation time (Chapter 4). The exponential substrate feeding strategy proved to be an

efficient and effective method to achieve a desired specific growth rate (µ, must be lower

than the µmax of the microorganism). This feeding strategy is ideal to achieve

carbon-limited and controlled growth, so that the toxic effects of substrate(s) can be

virtually eliminated and the effect of specific growth rate on product formation can be

revealed (Chapter 5, 6, 7). The high-cell-density cultivation strategies can not only benefit

144

the cultivation of P. putida and the production of MCL-PHA, but may also be used as

valuable reference for the production of other microorganisms and microbial products.

8.1.2 Carbon-limited, single-stage MCL-PHA production

Despite the fact that most literature studies of MCL-PHA production employed either

nitrogen or phosphorus limitation, carbon-limited (N and P in excess), single-stage,

exponential fed fermentations were found to be highly suitable for MCL-PHA production

by P. putida KT2440 (Chapter 5). Partially due to its high toxicity, nonanoic acid has

never been used for the production of MCL-PHA except in shake-flasks. In this study, the

toxicity was virtually eliminated by carbon-limitation so that no complicated

instrumentation was required for process control. Full automation can be easily achieved

by this approach. A PHA content of 64% was obtained by the end of a µ= 0.25 h-1

fermentation, while 75% PHA (highest MCL-PHA content ever shown in a fermentation

process) was achieved when the oxygen demand was lowered by lowering µ (0.15 h-1).

Therefore, MCL-PHA synthesis and content increase is likely to be limited only by

oxygen transfer when using nonanoic acid alone. Such a process has the potential of even

higher overall PHA productivity and content when fermentation systems with better

oxygen transfer capacity are available. For a given system, the choice of specific growth

rate has to consider the trade-off between achievable final PHA content and final PHA

productivity such that the overall PHA productivity is satisfactory while final PHA

145

content is high enough for efficient separation of PHA from biomass. Since P. putida

KT2440 has a wide substrate range, similar aliphatic substrates, such octanoic acid,

heptanoic acid, and decanoic acid, are also expected to be suitable for the exponential

feeding process. Therefore MCL-PHAs with different monomeric compositions can be

produced.

8.1.3 Co-substrate feeding strategy for functionalized MCL-PHA production

Functionalized MCL-PHAs are usually more difficult to produce due to a low yield of

product from the substrate carrying the functional group, and the lack of availability of

such substrates. Using nonanoic acid with 10-undecenoic acid as the co-substrate

(Chapter 7), not only was unsaturated MCL-PHA be produced at a productivity (1.09 l-1

h-1), much higher than literature reports, but the composition of the PHA product was

controlled by the controlling the substrate ratio. Such a strategy can be applied to the

production of other types of functionalized MCL-PHAs with controlled compositions,

with other combinations of major substrates (nonanoic acid or other MCL carboxylic

acids) and co-substrates carrying functional groups.

8.1.4 Yield enhancement and substrate cost reduction

Carbohydrates are among the least expensive substrates for microbial fermentations. In

the synthesis of MCL-PHA by P. putida KT2440, glucose yielded substantial biomass but

insignificant PHA. To take advantage of the low cost of glucose and the high PHA yield

146

efficiency of nonanoic acid, co-feeding strategies were developed in a conventional

two-stage MCL-PHA production process (Chapter 4) and a carbon-limited, single-stage

MCL-PHA production process (Chapter 6). Both studies demonstrated that a substantial

nonanoic acid to PHA yield enhancement can be achieved by using glucose as the

co-substrate. In the two-stage production process (Chapter 4), the YPHA/NA was almost

doubled while the composition of the PHA product changed only slightly comparing to

feeding only nonanoic acid in the same process. In the single-stage production process

(Chapter 6), the YPHA/NA was increased up to 30% with no significant change in the PHA

composition and an overall PHA productivity (1.44 g PHA l-1 h-1) was achieved (Chapter

5). Since co-feeding of glucose does not alter the composition of the PHA product, such a

co-feeding strategy can be applied when other aliphatic substrates are to be used for

MCL-PHA synthesis in order to enhance the yield of those substrates and lower the total

substrate cost. The more expensive the substrate is (10-undecnoic acid for example), the

more benefit it will be to use carbohydrate as a co-substrate.

8.2 Recommendations for future work

To further optimize the carbon-limited, single stage MCL-PHA production process

developed in this study, the mechanisms governing the MCL-PHA synthesis in P. putida

KT2440 should be studied in depth. Based on the fermentation results presented in this

study (Chapter 5), it is clear that the mechanisms are different from those of typical

147

SCL-PHA-synthesizing microorganisms. Two types of SCL-PHA synthesis are well

known, non-growth associated and growth associated. Non-growth associated

SCL-PHA-synthesizing microorganisms, such as Ralstonia eutropha, accumulate PHA

only (or mostly) during a non-growth phase when one or more nutrients (e.g. nitrogen or

phosphorus) becomes limiting while carbon source is in excess. Growth-associated

SCL-PHA-synthesizing microorganisms, such as Alcaligenes latus, synthesize PHA

during growth phase with a constant PHA content and specific PHA synthesis rate.

However, P. putida KT2440 synthesizes MCL-PHA at a rate that is higher than the

specific growth rate of the residual biomass, resulting in increasing PHA content

throughout the growth phase. A deficiency in the TCA cycle, blockage in β-oxidation, a

substrate detoxicification mechanism, and properties of the PHA granules should all be

investigated as possible mechanisms controlling such PHA synthesis.

The oxygen transfer capacity of the bioreactor was found to limit the achievable PHA

productivity. Approaches to increase the oxygen transfer of a given fermentation system

are therefore necessary, such as pressurized bioreactors or by employing an oxygen

carrier compound such as dodecane. Alternatively, other modes of cultivation should be

considered. Continuous fermentation running at high-cell-density may be a feasible way

of achieving higher volumetric PHA productivity. For example, if a steady state can be

reached at cell concentration of 20 g l-1 containing 50% PHA at a dilution rate of 0.2 h-1,

148

the volumetric productivity would be 2 g PHA l-1 h-1. Optimization of such a continuous

process may result in even higher PHA content and productivity.

Unfortunately, continuous culture is rarely used for industrial scale production since

contamination is virtually impossible to eliminate. The possibility of using a decaying

exponential fed-batch process should, therefore, also be examined. In this approach the

controlled specific growth rate is gradually lowered throughout the fermentation by

carbon-limitation until the oxygen transfer capacity of the bioreactor is nearly attained. At

this point the feed rate can be maintained at a constant value or gradually lowered (kLa is

known to decrease with culture density). In theory, such a feeding regime should give the

maximum productivity obtainable in fed-batch mode fermentation. A preliminary result

(not presented in this thesis) demonstrated that the final achievable biomass can be as

high as 90 g l-1, a near 30% increase comparing to the highest biomass concentration (70

g l-1) presented in this thesis.

MCL-PHA-Yong-Thesis

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